Search gst AND (fusion OR fused OR tagged OR tag) and (sf9 OR insect OR baculovirus) Field: All Fields, Limits: Publication Date from 1994/01/01 to 2003/01/01 Entrez pubmed Results Items 1 - 132 of 132 1: Chen Y et al. Phage display of functional h...[PMID: 14599359] Related Articles, Books, LinkOut PMID- 14599359 OWN - NLM STAT- MEDLINE DA - 20031105 DCOM- 20031204 LR - 20041117 PUBM- Print IS - 1087-0571 VI - 7 IP - 5 DP - 2002 Oct TI - Phage display of functional human TNF-alpha converting enzyme catalytic domain: a rapid method for the production of stabilized proteolytic proteins for assay development and high-throughput screening. PG - 433-40 AB - The catalytic domain of human tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-alpha to generate the mature TNF-alpha in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-alpha-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 degrees C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 degrees C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS. AD - Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ, USA. FAU - Chen, Yangde AU - Chen Y FAU - Diener, Katrina AU - Diener K FAU - Patel, Indravadan R AU - Patel IR FAU - Kawooya, John K AU - Kawooya JK FAU - Martin, Gary A AU - Martin GA FAU - Yamdagni, Preeti AU - Yamdagni P FAU - Zhang, Xin AU - Zhang X FAU - Sandrasalphaa, Anthony AU - Sandrasalphaa A FAU - Sahasrabudhe, Sudhir AU - Sahasrabudhe S FAU - Busch, Steven J AU - Busch SJ LA - eng PT - Journal Article PL - United States TA - J Biomol Screen JID - 9612112 RN - 0 (Enzyme Inhibitors) RN - 0 (Peptide Library) RN - 0 (Proteins) RN - 0 (Recombinant Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.4.24 (Metalloendopeptidases) RN - EC 3.4.24.- (TNF-alpha converting enzyme) SB - IM MH - Baculoviridae/genetics MH - Biological Assay/*methods MH - Catalytic Domain MH - Combinatorial Chemistry Techniques/methods MH - Drug Evaluation, Preclinical/methods MH - Enzyme Inhibitors/pharmacology MH - Enzyme Stability MH - Escherichia coli/genetics MH - Glutathione Transferase/genetics/metabolism MH - Humans MH - Metalloendopeptidases/antagonists & inhibitors/genetics/*metabolism MH - *Peptide Library MH - Protein Engineering/methods MH - Proteins/genetics/*metabolism MH - Recombinant Proteins/genetics/metabolism EDAT- 2003/11/06 05:00 MHDA- 2003/12/05 05:00 AID - 10.1177/108705702237675 [doi] PST - ppublish SO - J Biomol Screen 2002 Oct;7(5):433-40. NR -------------------------------------------------------------------------------- 2: Cappelli E et al. Drosophila S3 ribosomal prote...[PMID: 12874813] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 12874813 OWN - NLM STAT- MEDLINE DA - 20030722 DCOM- 20030909 LR - 20041117 PUBM- Print IS - 0893-6692 VI - 42 IP - 1 DP - 2003 TI - Drosophila S3 ribosomal protein accelerates repair of 8-oxoguanine performed by human and mouse cell extracts. PG - 50-8 AB - The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3' blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and beta-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells. CI - Copyright 2003 Wiley-Liss, Inc. AD - DNA Repair Unit, Mutagenesis Laboratory - Istituto Nazionale Ricerca Cancro, Genova, Italy. FAU - Cappelli, Enrico AU - Cappelli E FAU - D'Osualdo, Andrea AU - D'Osualdo A FAU - Bogliolo, Massimo AU - Bogliolo M FAU - Kelley, Mark R AU - Kelley MR FAU - Frosina, Guido AU - Frosina G LA - eng PT - Journal Article PL - United States TA - Environ Mol Mutagen JID - 8800109 RN - 0 (Cell Extracts) RN - 0 (Ribosomal Proteins) RN - 0 (ribosomal protein S3) RN - 118-00-3 (Guanosine) RN - 3868-31-3 (8-hydroxyguanosine) RN - 9007-49-2 (DNA) RN - EC 3.2.2.- (N-Glycosyl Hydrolases) RN - EC 3.2.2.23 (DNA-Formamidopyrimidine Glycosylase) SB - IM MH - Animals MH - Cell Extracts/chemistry/*pharmacology MH - DNA/drug effects MH - DNA Damage/*drug effects MH - DNA Repair/*drug effects MH - DNA-Formamidopyrimidine Glycosylase MH - Drosophila melanogaster/*physiology MH - Fibroblasts/chemistry/drug effects/metabolism MH - Guanosine/*analogs & derivatives/*genetics/metabolism MH - Humans MH - Mice MH - N-Glycosyl Hydrolases/toxicity MH - Research Support, Non-U.S. Gov't MH - Ribosomal Proteins/isolation & purification/*pharmacology EDAT- 2003/07/23 05:00 MHDA- 2003/09/10 05:00 AID - 10.1002/em.10166 [doi] PST - ppublish SO - Environ Mol Mutagen 2003;42(1):50-8. NR -------------------------------------------------------------------------------- 3: Zhou P et al. [Molecular cloning and expres...[PMID: 12548987] Related Articles, Books, LinkOut PMID- 12548987 OWN - NLM STAT- MEDLINE DA - 20030128 DCOM- 20040810 LR - 20041117 PUBM- Print IS - 0001-6209 VI - 40 IP - 3 DP - 2000 Jun TI - [Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine] PG - 243-51 AB - A 1.1 bp fragment of E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain, a standard virulent strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. The nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84.7%, 92.6% and 95.2% respectively, and the amino acid sequence 89.4%, 92.6% and 94.6%, respectively, we subcloned 1.1 bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST-E2 and GST-GFP-E2 fusion protein respectively by homologous recombination in sf-9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS-7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclonal antibody the pcE2 DNA was directly injected into BALB/c mice intramuscular(i.m.) and the CSFV E2-specific antibodies was measured by enzyme-linked immunosorbent assay(ELISA) the ELISA results indicated the E2-specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA vaccine against CSFV. AD - College of Life Sciences, Peking University, Beijing 100871. FAU - Zhou, P AU - Zhou P FAU - Lu, Y AU - Lu Y FAU - Chen, J AU - Chen J FAU - Zhai, Z AU - Zhai Z FAU - Ding, M AU - Ding M LA - chi PT - Journal Article PL - China TA - Wei Sheng Wu Xue Bao JID - 21610860R RN - 0 (Vaccines, DNA) RN - 0 (Viral Envelope Proteins) RN - 0 (Viral Vaccines) RN - 0 (glycoprotein E2, classical swine fever virus) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - COS Cells MH - Cell Line MH - Classical swine fever virus/*immunology MH - Cloning, Molecular MH - English Abstract MH - Gene Expression MH - Mice MH - Mice, Inbred BALB C MH - Molecular Sequence Data MH - Research Support, Non-U.S. Gov't MH - Sequence Homology MH - Spodoptera MH - Vaccines, DNA/*immunology MH - Viral Envelope Proteins/biosynthesis/*genetics/*immunology MH - Viral Vaccines/*immunology EDAT- 2003/01/29 04:00 MHDA- 2004/08/11 05:00 PST - ppublish SO - Wei Sheng Wu Xue Bao 2000 Jun;40(3):243-51. DR -------------------------------------------------------------------------------- 4: Zhao W et al. Interaction of casein kinase ...[PMID: 12379220] Related Articles, Gene, HomoloGene, Compound, Substance, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 12379220 OWN - NLM STAT- MEDLINE DA - 20021015 DCOM- 20021122 LR - 20041203 PUBM- Print IS - 0006-291X VI - 298 IP - 1 DP - 2002 Oct 18 TI - Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster. PG - 60-6 AB - The ubiquitous eukaryotic protein kinase CKII (casein kinase II) has been found to interact with a number of cellular proteins, either through the catalytic subunit or the regulatory subunit. Using the yeast two-hybrid screening method, we found that the catalytic subunit of Drosophila melanogaster CKII (DmCKII) interacts with Drosophila ribosomal protein L22 (rpL22). This interaction was also observed in vitro with a glutathione-S-transferase (GST)-rpL22 fusion protein. The predicted full-length Drosophila rpL22 protein has an N-terminal extension rich in alanine, lysine, and proline that appears to be unique to Drosophila. Deletion mapping revealed that the conserved core of rpL22 is responsible for the interaction with CKII. Moreover, purified DmCKII can phosphorylate a GST-L22 fusion protein at the C-terminal end, suggesting that this protein may be a substrate of CKII in Drosophila. AD - Department of Biochemistry and Molecular Biology, Life Sciences Building, The University of Georgia, Athens, GA 30602-7229, USA. FAU - Zhao, Wenfan AU - Zhao W FAU - Bidwai, Ashok P AU - Bidwai AP FAU - Glover, Claiborne V C AU - Glover CV LA - eng SI - GENBANK/U42587 GR - GM33237/GM/NIGMS PT - Journal Article PL - United States TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (Drosophila Proteins) RN - 0 (RNA-Binding Proteins) RN - 0 (Ribosomal Proteins) RN - 0 (RpL22 protein, Drosophila) RN - EC 2.7.1.37 (Casein Kinase II) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Animals MH - Binding Sites MH - Casein Kinase II MH - *Drosophila Proteins MH - Drosophila melanogaster/*enzymology/metabolism MH - Molecular Sequence Data MH - Phosphorylation MH - Protein-Serine-Threonine Kinases/*metabolism MH - RNA-Binding Proteins/chemistry/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - *Ribosomal Proteins MH - Two-Hybrid System Techniques EDAT- 2002/10/16 04:00 MHDA- 2002/11/26 04:00 AID - S0006291X02023963 [pii] PST - ppublish SO - Biochem Biophys Res Commun 2002 Oct 18;298(1):60-6. NR -------------------------------------------------------------------------------- 5: Sehrawat S et al. Temporal expression profile o...[PMID: 12234668] Related Articles, Nucleotide, Protein, Books, LinkOut PMID- 12234668 OWN - NLM STAT- MEDLINE DA - 20020917 DCOM- 20030220 LR - 20041117 PUBM- Print IS - 0378-1119 VI - 294 IP - 1-2 DP - 2002 Jul 10 TI - Temporal expression profile of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus. PG - 67-75 AB - Temporal expression profile of lef4, the gene encoding late gene expression factor 4 (LEF4) from the baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), has been analysed. lef4 behaved like an early gene and the transcripts were detectable from 6 h post infection (hpi) which reached maximal levels by 18-24 hpi, and declined considerably at later times. The LEF4 open reading frame was bacterially expressed as a glutathione S-transferase (GST) fusion protein which was solubilized from the inclusion bodies and purified by adsorption to the affinity matrix, GST-Sepharose. Using polyclonal antibodies raised against the bacterially expressed protein, the temporal profile of LEF4 synthesis in BmNPV-infected BmN cells was analysed. The LEF4 protein levels were also higher at 24 hpi compared to 12 or 36 hpi, correlating with the RNA patterns. The protein was predominantly localized to the nucleus of the infected BmN cell and only a small portion was present in the cytosolic fraction. Preliminary studies with antisense lef4 expression revealed substantial reduction in expression from the viral polyhedrin promoter without significantly affecting the viral DNA replication. AD - Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. FAU - Sehrawat, Seema AU - Sehrawat S FAU - Gopinathan, Karumathil P AU - Gopinathan KP LA - eng SI - GENBANK/AY033655 PT - Journal Article PL - Netherlands TA - Gene JID - 7706761 RN - 0 (DNA, Antisense) RN - 0 (DNA, Viral) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) RN - 0 (late gene expression factor 4, Bombyx mori nucleopolyhedrovirus) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Animals MH - Bombyx/cytology/*virology MH - Cell Line MH - Cloning, Molecular MH - DNA, Antisense/genetics MH - DNA, Viral/chemistry/genetics MH - Fluorescent Antibody Technique, Indirect MH - Gene Expression Regulation, Viral MH - Glutathione Transferase/genetics/metabolism MH - Molecular Sequence Data MH - Nucleopolyhedrovirus/*genetics/growth & development MH - Promoter Regions (Genetics)/genetics MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Time Factors MH - Transcription, Genetic MH - Transfection MH - Viral Proteins/*genetics/metabolism EDAT- 2002/09/18 10:00 MHDA- 2003/02/21 04:00 AID - S0378111902007692 [pii] PST - ppublish SO - Gene 2002 Jul 10;294(1-2):67-75. NR -------------------------------------------------------------------------------- 6: Duhe RJ et al. Characterization of the in vi...[PMID: 12190118] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 12190118 OWN - NLM STAT- MEDLINE DA - 20020822 DCOM- 20030410 LR - 20041217 PUBM- Print IS - 0300-8177 VI - 236 IP - 1-2 DP - 2002 Jul TI - Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein. PG - 23-35 AB - The biochemical and biophysical characteristics of Janus protein-tyrosine kinases (JAKs), which are essential early mediators of cytokine-initiated signal propagation, are virtually undefined. To facilitate the in vitro analysis of JAK-mediated catalysis, we substantially purified a soluble recombinant JAK2 and developed a novel means of quantifying JAK-catalyzed product formation. Glutathione-S-transferase fusion proteins containing active and inactive forms of rat Janus kinase 2 (GST:rJAK2 and GST:rJAK2(CA795)) were highly purified via affinity chromatography. A microtiterplate-based ELISA was used to measure tyrosine phosphorylation of a streptavidin-immobilized biotinylated STAT1-derived peptide. The ELISA data indicated that only about 1% of the enzyme was involved in exogenous substrate phosphorylation. Other immobilized peptides served as apparent substrates with varying efficacy. Traditional radioisotopic autokinase assays demonstrated that the activity of the purified fusion protein was inhibited by a variety of tyrphostin inhibitors. Non-radiolabeled adenine nucleotides, but not guanine nucleotides, inhibited the radioisotopic autokinase assay. These observations verify that the catalytic activity of JAK2 is highly regulated, and are consistent with the suggestion that JAK2 may require additional accessory proteins, such as a potential upstream regulatory kinase, for full catalytic activity. AD - Intramural Research Support Program, SAIC-Frederick, MD, USA. RDUHE@pharmacology.umsmed.edu FAU - Duhe, Roy J AU - Duhe RJ FAU - Clark, Emily A AU - Clark EA FAU - Farrar, William L AU - Farrar WL LA - eng GR - NO1-CO-5600/CO/NCI PT - Journal Article PL - Netherlands TA - Mol Cell Biochem JID - 0364456 RN - 0 (DNA, Complementary) RN - 0 (DNA-Binding Proteins) RN - 0 (Enzyme Inhibitors) RN - 0 (Peptides) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Stat1 protein) RN - 0 (Trans-Activators) RN - 0 (Tyrphostins) RN - 55520-40-6 (Tyrosine) RN - 73-24-5 (Adenine) RN - 73-40-5 (Guanine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Janus kinase 2) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) SB - IM MH - Adenine/chemistry MH - Animals MH - Biotinylation MH - Catalysis MH - Cell Line MH - Chromatography, Affinity MH - DNA, Complementary/metabolism MH - DNA-Binding Proteins/metabolism MH - Dose-Response Relationship, Drug MH - Electrophoresis, Polyacrylamide Gel MH - Enzyme Inhibitors/pharmacology MH - Enzyme-Linked Immunosorbent Assay MH - Glutathione Transferase/*metabolism MH - Guanine/chemistry MH - Insects MH - Models, Chemical MH - Peptides/chemistry MH - Phosphorylation MH - Protein-Tyrosine Kinase/chemistry/*metabolism MH - *Proto-Oncogene Proteins MH - Rats MH - Recombinant Fusion Proteins/chemistry/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Trans-Activators/metabolism MH - Tyrosine/metabolism MH - Tyrphostins/metabolism/pharmacology EDAT- 2002/08/23 10:00 MHDA- 2003/04/11 05:00 PST - ppublish SO - Mol Cell Biochem 2002 Jul;236(1-2):23-35. PR -------------------------------------------------------------------------------- 7: Dhar SK et al. A approximately 35 kDa polype...[PMID: 12186657] Related Articles, Compound via MeSH, Substance via MeSH, Free in PMC, Books, LinkOut PMID- 12186657 OWN - NLM STAT- MEDLINE DA - 20021119 DCOM- 20021216 LR - 20041117 PUBM- Print-Electronic IS - 1471-2091 VI - 3 IP - 1 DP - 2002 Aug 19 TI - A approximately 35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP. PG - 23 AB - BACKGROUND: The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. RESULTS: Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. CONCLUSIONS: We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes. AD - Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110067, India. skdhar2002@yahoo.co.in FAU - Dhar, Suman K AU - Dhar SK FAU - Mondal, Neelima AU - Mondal N FAU - Soni, Rajesh K AU - Soni RK FAU - Mukhopadhyay, Gauranga AU - Mukhopadhyay G LA - eng PT - Journal Article DEP - 20020819 PL - England TA - BMC Biochem JID - 101084098 RN - 0 (Cell Cycle Proteins) RN - 0 (DNA, Fungal) RN - 0 (DNA-Binding Proteins) RN - 0 (Peptides) RN - 0 (Proto-Oncogene Proteins c-myc) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 122544-18-7 (CDC6 protein, S cerevisiae) RN - 56-65-5 (Adenosine Triphosphate) RN - 60-00-4 (Edetic Acid) RN - 7647-14-5 (Sodium Chloride) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Adenosine Triphosphate/*pharmacology MH - Animals MH - Binding Sites/genetics MH - Cell Cycle Proteins/chemistry/genetics/metabolism MH - Cell Line MH - DNA, Fungal/genetics/metabolism MH - DNA-Binding Proteins/chemistry/genetics/*metabolism MH - Edetic Acid/pharmacology MH - Glutathione Transferase/genetics/metabolism MH - Humans MH - Molecular Weight MH - Mutation MH - Peptides/chemistry/genetics/*metabolism MH - Protein Binding/drug effects MH - Proto-Oncogene Proteins c-myc/genetics MH - Recombinant Fusion Proteins/genetics/metabolism MH - Replication Origin/*genetics MH - Research Support, Non-U.S. Gov't MH - Saccharomyces cerevisiae/genetics MH - *Saccharomyces cerevisiae Proteins MH - Sodium Chloride/pharmacology MH - Spodoptera MH - Temperature EDAT- 2002/08/21 10:00 MHDA- 2002/12/18 04:00 PHST- 2002/03/28 [received] PHST- 2002/08/19 [accepted] PHST- 2002/08/19 [aheadofprint] PST - epublish SO - BMC Biochem 2002 Aug 19;3(1):23. DR -------------------------------------------------------------------------------- 8: Yang L et al. Cloning of Etp28 Gene of Eime...[PMID: 12174264] Related Articles, Books, LinkOut PMID- 12174264 OWN - NLM STAT- Publisher DA - 20020813 PUBM- Print IS - 0582-9879 VI - 30 IP - 3 DP - 1998 TI - Cloning of Etp28 Gene of Eimeria tenella (Guangdong Strain) Sporozoites and Its Expression in Baculovirus Expression System. PG - 293-298 AB - A refractile body antigen Etp28 Gene of Eimeria tenella (Guangdong Strain) sporozoites was cloned by PCR from the synthesized first strand cDNA. It has a high homology with the previously reported Etp28 gene of Merck Strain LS18. The gene was expressed through standard procedures in the modified Autographa californica nuclear polyhedrosis virus (AcMNPV-OCC(-)) expression system and large amount of heterologous fusion protein (GST-6xHis-Etp28) was obtained. The expression product was about 21.3% of the total cellular protein of an infected cell and corresponding to approximately 0.42 mg of recombinant protein per 10(6) cells. And the immunoprotective trial showed that this candidate for recombinant vaccine conferred partial protection against chicken coccidiosis. AD - State Key Laboratory for Biological Control, Zhongshan University, Guangzhou 510275, China. LS07@zsu.edu.cn AU - Yang L AU - Zhu W AU - Wang XZ AU - Long QX AU - Xie MQ AU - Huang XQ LA - ENG PT - JOURNAL ARTICLE TA - Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) JID - 20730160R EDAT- 2002/08/14 10:00 MHDA- 2002/08/14 10:00 PST - ppublish SO - Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1998;30(3):293-298. DR -------------------------------------------------------------------------------- 9: Yang GZ et al. Production of recombinant hum...[PMID: 12144509] Related Articles, Substance via MeSH, Books, LinkOut PMID- 12144509 OWN - NLM STAT- MEDLINE DA - 20020729 DCOM- 20021121 LR - 20041117 PUBM- Print IS - 0929-8665 VI - 9 IP - 4 DP - 2002 Aug TI - Production of recombinant human calcitonin from silkworm (B. mori) larvae infected by baculovirus. PG - 323-9 AB - A synthetic modified gene encoding the human calcitonin analog (hmCT) was expressed by use of the baculovirus expression system. After injection with recombinant baculoviruses, the hmCT-GST fusion protein was produced within the silkworm larvae. The fusion protein was purified by affinity chromatography. Biological activity of hmCT for hypercalcemic effect was determined in normal rats. AD - Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Science, Shanghai, 200031, China. FAU - Yang, Guan-Zhen AU - Yang GZ FAU - Chen, Zhen-zhen AU - Chen ZZ FAU - Cui, Da-Fu AU - Cui DF FAU - Li, Bo-liang AU - Li BL FAU - Wu, Xiang-fu AU - Wu XF LA - eng PT - Journal Article PL - Netherlands TA - Protein Pept Lett JID - 9441434 RN - 0 (Recombinant Fusion Proteins) RN - 9007-12-9 (Calcitonin) SB - IM MH - Animals MH - Baculoviridae/genetics/*physiology MH - Bombyx/genetics/*physiology/virology MH - Calcitonin/genetics/*metabolism/pharmacology MH - Genes, Reporter MH - Hemolymph/chemistry MH - Humans MH - Hypercalcemia/chemically induced MH - Larva/physiology MH - Rats MH - Recombinant Fusion Proteins/genetics/metabolism/pharmacology EDAT- 2002/07/30 10:00 MHDA- 2002/11/26 04:00 PST - ppublish SO - Protein Pept Lett 2002 Aug;9(4):323-9. DR -------------------------------------------------------------------------------- 10: Oldham CE et al. The ubiquitin-interacting mot...[PMID: 12121618] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 12121618 OWN - NLM STAT- MEDLINE DA - 20020717 DCOM- 20030204 LR - 20041117 PUBM- Print IS - 0960-9822 VI - 12 IP - 13 DP - 2002 Jul 9 TI - The ubiquitin-interacting motifs target the endocytic adaptor protein epsin for ubiquitination. PG - 1112-6 AB - The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination. AD - Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. FAU - Oldham, Carla E AU - Oldham CE FAU - Mohney, Robert P AU - Mohney RP FAU - Miller, Stephanie L H AU - Miller SL FAU - Hanes, Richard N AU - Hanes RN FAU - O'Bryan, John P AU - O'Bryan JP LA - eng PT - Journal Article PL - England TA - Curr Biol JID - 9107782 RN - 0 (Carrier Proteins) RN - 0 (Neuropeptides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Ubiquitin) RN - 0 (Vesicular Transport Proteins) RN - 0 (epsin) SB - IM MH - Amino Acid Motifs MH - Animals MH - Carrier Proteins/genetics/*metabolism MH - Drosophila MH - Endocytosis MH - Neuropeptides/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Ubiquitin/*metabolism MH - *Vesicular Transport Proteins MH - Xenopus EDAT- 2002/07/18 10:00 MHDA- 2003/02/05 04:00 AID - S0960982202009004 [pii] PST - ppublish SO - Curr Biol 2002 Jul 9;12(13):1112-6. NR -------------------------------------------------------------------------------- 11: Liao WT et al. Expression and functional ana...[PMID: 12054521] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 12054521 OWN - NLM STAT- MEDLINE DA - 20020610 DCOM- 20020712 LR - 20050108 PUBM- Print IS - 0006-291X VI - 293 IP - 2 DP - 2002 May 3 TI - Expression and functional analysis of an inhibitor of apoptosis protein from Trichoplusia ni. PG - 675-9 AB - An inhibitor of the apoptosis protein (IAP) family gene from Trichoplusia ni, Tn-IAP1v, a variant of lepidopteran Tn-IAP1, was cloned by RT-PCR. There are six single nucleotide polymorphisms between the two Tn-IAP1 variants, resulting in three predicted single amino acid polymorphisms. With the GST fusion expression system, soluble recombinant Tn-IAP1v was highly expressed in Escherichia coli and then purified by affinity chromatography. Caspase inhibition assays indicated that recombinant Tn-IAP1v could specifically inhibit human caspase-9 in vitro instead of caspase-3, -7, and -8, which was further confirmed by the observation that recombinant Tn-IAP1v can directly bind caspase-9 in the protein pull-down assay. These results suggested that Tn-IAP1v might serve as an initiator caspase inhibitor in vivo in the conserved mitochondria apoptotic pathway. CI - Copyright 2002 Elsevier Science (USA). AD - Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China. FAU - Liao, Wen-Tao AU - Liao WT FAU - Yang, Yang AU - Yang Y FAU - Wu, Xiang-Fu AU - Wu XF LA - eng PT - Journal Article PL - United States TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (Cysteine Proteinase Inhibitors) RN - 0 (Cytochrome c Group) RN - 0 (Deoxyadenine Nucleotides) RN - 0 (Insect Proteins) RN - 0 (Proteins) RN - 0 (inhibitor of apoptosis proteins) RN - 1927-31-7 (2'-deoxyadenosine triphosphate) RN - EC 3.4.22.- (Caspases) RN - EC 3.4.22.- (caspase 9) SB - IM MH - Animals MH - Caspases/metabolism MH - Cell Line MH - Cloning, Molecular MH - Cysteine Proteinase Inhibitors/genetics/*metabolism/*pharmacology MH - Cytochrome c Group/antagonists & inhibitors MH - Deoxyadenine Nucleotides/antagonists & inhibitors MH - Humans MH - Insect Proteins/genetics/*metabolism/*pharmacology MH - *Lepidoptera MH - Polymorphism, Single Nucleotide MH - *Proteins EDAT- 2002/06/11 10:00 MHDA- 2002/07/13 10:01 AID - 10.1016/S0006-291X(02)00279-6 [doi] AID - S0006-291X(02)00279-6 [pii] PST - ppublish SO - Biochem Biophys Res Commun 2002 May 3;293(2):675-9. NR -------------------------------------------------------------------------------- 12: Uchiyama Y et al. Measurement of HCV RdRp activ...[PMID: 12048062] Related Articles, Books, LinkOut PMID- 12048062 OWN - NLM STAT- Publisher DA - 20020605 PUBM- Print IS - 1386-6346 VI - 23 IP - 2 DP - 2002 Jun TI - Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions. PG - 90-97 AB - The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer. The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 degrees C and a pH of 7.5. The addition of 2.5 mM Mn(2+) increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg(2+). At higher concentrations of Mn(2+), GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted. This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp. AD - Department of Molecular Biology and Medicine, RCAST#35, University of Tokyo, 4-6-1 Komaba, Meguro, 153-8904, Tokyo, Japan AU - Uchiyama Y AU - Huang Y AU - Kanamori H AU - Uchida M AU - Doi T AU - Takamizawa A AU - Hamakubo T AU - Kodama T LA - ENG PT - JOURNAL ARTICLE TA - Hepatol Res JID - 9711801 EDAT- 2002/06/06 10:00 MHDA- 2002/06/06 10:00 AID - S138663460100167X [pii] PST - ppublish SO - Hepatol Res 2002 Jun;23(2):90-97. NR -------------------------------------------------------------------------------- 13: Udomsinprasert R et al. Expression and characterizati...[PMID: 11886777] Related Articles, Compound via MeSH, Substance via MeSH, Nucleotide, Protein, Books, LinkOut PMID- 11886777 OWN - NLM STAT- MEDLINE DA - 20020311 DCOM- 20020529 LR - 20041117 PUBM- Print IS - 0965-1748 VI - 32 IP - 4 DP - 2002 Apr TI - Expression and characterization of a novel class of glutathione S-transferase from Anopheles dirus. PG - 425-33 AB - A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs. AD - Institute of Molecular Biology and Genetics, Mahidol University, Salaya campus, 73170, Nakhon Pathom, Thailand. FAU - Udomsinprasert, R AU - Udomsinprasert R FAU - Ketterman, A J AU - Ketterman AJ LA - eng SI - GENBANK/AY014405 PT - Journal Article PL - England TA - Insect Biochem Mol Biol JID - 9207282 RN - 0 (1-chloro-2,4-dinitrobenzene-glutathione conjugate) RN - 0 (DNA, Complementary) RN - 0 (Insect Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 70-18-8 (Glutathione) RN - 97-00-7 (Dinitrochlorobenzene) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Anopheles/*enzymology/genetics MH - Base Sequence MH - Cloning, Molecular MH - DNA, Complementary MH - Dinitrochlorobenzene/metabolism MH - *Gene Expression MH - Glutathione/metabolism MH - Glutathione Transferase/antagonists & inhibitors/*genetics/isolation & purification/metabolism MH - Insect Proteins/antagonists & inhibitors/*genetics/isolation & purification/metabolism MH - Molecular Sequence Data MH - Promoter Regions (Genetics) MH - Recombinant Fusion Proteins/antagonists & inhibitors/genetics/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid EDAT- 2002/03/12 10:00 MHDA- 2002/05/30 10:01 AID - S0965174801001199 [pii] PST - ppublish SO - Insect Biochem Mol Biol 2002 Apr;32(4):425-33. NR -------------------------------------------------------------------------------- 14: Kontos CD et al. The endothelial receptor tyro...[PMID: 11865050] Related Articles, Gene, HomoloGene, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Free in PMC, Books, LinkOut PMID- 11865050 OWN - NLM STAT- MEDLINE DA - 20020226 DCOM- 20020312 LR - 20050111 PUBM- Print IS - 0270-7306 VI - 22 IP - 6 DP - 2002 Mar TI - The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis. PG - 1704-13 AB - Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies. AD - Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, NC 27710, USA. cdkontos@duke.edu FAU - Kontos, Christopher D AU - Kontos CD FAU - Cha, Eugene H AU - Cha EH FAU - York, John D AU - York JD FAU - Peters, Kevin G AU - Peters KG LA - eng GR - HL 03557/HL/NHLBI GR - HL 55265/HL/NHLBI PT - Journal Article PL - United States TA - Mol Cell Biol JID - 8109087 RN - 0 (Chimeric Proteins) RN - 0 (Enzyme Inhibitors) RN - 0 (Phosphatidylinositol Phosphates) RN - 0 (Protein Subunits) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Receptors, Cell Surface) RN - 81627-83-0 (Macrophage Colony-Stimulating Factor) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) RN - EC 2.7.1.112 (Receptor, Macrophage Colony-Stimulating Factor) RN - EC 2.7.1.112 (Receptor, TIE-1) RN - EC 2.7.1.112 (Receptors, TIE) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (proto-oncogene proteins c-akt) SB - IM MH - 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors/*metabolism MH - 3T3 Cells MH - Animals MH - Apoptosis/drug effects/*physiology/radiation effects MH - Binding Sites/physiology MH - Cell Line MH - Chimeric Proteins/genetics/metabolism MH - Endothelium, Vascular/*metabolism MH - Enzyme Activation/drug effects MH - Enzyme Inhibitors/pharmacology MH - Macrophage Colony-Stimulating Factor/pharmacology MH - Mice MH - Mutagenesis, Site-Directed MH - Phosphatidylinositol Phosphates/analysis/biosynthesis MH - Phosphorylation/drug effects MH - Protein Binding/physiology MH - Protein Subunits MH - *Protein-Serine-Threonine Kinases MH - Proto-Oncogene Proteins/*metabolism MH - Receptor Protein-Tyrosine Kinases/genetics/*metabolism/pharmacology MH - Receptor, Macrophage Colony-Stimulating Factor/genetics MH - Receptor, TIE-1 MH - Receptors, Cell Surface/genetics/*metabolism MH - Receptors, TIE MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction/drug effects/physiology MH - Spodoptera MH - Ultraviolet Rays EDAT- 2002/02/28 10:00 MHDA- 2002/03/13 10:01 PST - ppublish SO - Mol Cell Biol 2002 Mar;22(6):1704-13. DR -------------------------------------------------------------------------------- 15: Ohtoshi A et al. Analysis of beta3-endonexin m...[PMID: 11810239] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 11810239 OWN - NLM STAT- MEDLINE DA - 20020125 DCOM- 20020219 LR - 20050204 PUBM- Print-Electronic IS - 1617-4615 VI - 266 IP - 4 DP - 2001 Dec TI - Analysis of beta3-endonexin mutants for their ability to interact with cyclin A. PG - 664-71 AB - We have recently identified beta(3)-endonexin as a molecule that interacts with cyclin A-associated kinase. In this study, beta(3)-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized. Beta(3)-endonexin has a cyclin binding motif, RxL, in its N-terminal region, and two SP sequences which resemble a known target site for cyclin-dependent kinases (Cdks). The R5A/L7A mutant of beta(3)-endonexin, in which the RxL motif has been changed to AxA, is unable to bind to cyclin A, as revealed by two-hybrid experiments and in vitro pull-down assays. A GST-beta(3)-endonexin fusion, but not the corresponding R5A/L7A mutant, inhibits phosphorylation of Rb protein by cyclin A/Cdk2 in vitro. A cyclin A/Cdk2 kinase complex produced in, and purified from, insect cells phosphorylated GST-beta(3)-endonexin in vitro. The S33A or S46A mutant is partially phosphorylated by cyclin A/Cdk2, whereas no phosphorylation of the S33A/S46A double mutant is detectable. This demonstrates that these two serine residues, each of which is followed by a proline residue, are target sites for phosphorylation by cyclin A-associated kinase. The R5A/L7A mutant form of beta(3)-endonexin, which is defective for binding to cyclin A, is also not phosphorylated by cyclin A/Cdk2, confirming that the phosphorylation requires binding to cyclin A in the kinase complex. The neutralizing effect of beta(3)-endonexin on the toxicity associated with the expression of full-length human cyclin A in budding yeast is correlated with its ability to bind to cyclin A. Taken together, these data suggest that beta(3)-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of beta(3)-endonexin to the kinase complex. AD - Division of Biotecture, Hope-tecture Research Institute, Hyogo, Japan. aotoshi@mail.mdanderson.org FAU - Ohtoshi, A AU - Ohtoshi A FAU - Otoshi, H AU - Otoshi H LA - eng PT - Journal Article DEP - 20011011 PL - Germany TA - Mol Genet Genomics JID - 101093320 RN - 0 (Cyclin A) RN - 0 (Proteins) RN - 0 (beta3 endonexin) RN - 56-45-1 (Serine) RN - EC 2.7.1.- (cyclin-dependent kinase 2) RN - EC 2.7.1.37 (CDC2-CDC28 Kinases) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - *CDC2-CDC28 Kinases MH - Cyclin A/antagonists & inhibitors/*metabolism MH - Cyclin-Dependent Kinases/antagonists & inhibitors/metabolism MH - Mutation MH - Phosphorylation MH - Protein Binding MH - Protein-Serine-Threonine Kinases/antagonists & inhibitors/metabolism MH - Proteins/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Saccharomycetales/metabolism MH - Serine/metabolism MH - Two-Hybrid System Techniques EDAT- 2002/01/26 10:00 MHDA- 2002/02/20 10:01 PHST- 2001/02/22 [received] PHST- 2001/08/27 [accepted] PHST- 2001/10/11 [aheadofprint] AID - 10.1007/s004380100588 [doi] PST - ppublish SO - Mol Genet Genomics 2001 Dec;266(4):664-71. Epub 2001 Oct 11. NR -------------------------------------------------------------------------------- 16: Grebe M et al. Expression of ecdysteroid rec...[PMID: 11755059] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11755059 OWN - NLM STAT- MEDLINE DA - 20011228 DCOM- 20020312 LR - 20041117 PUBM- Print IS - 0965-1748 VI - 32 IP - 2 DP - 2002 Feb TI - Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state. PG - 167-74 AB - Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A. AD - Abteilung fur Allgemeine Zoologie und Endokrinologie, Universitat Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. FAU - Grebe, Marco AU - Grebe M FAU - Spindler-Barth, Margarethe AU - Spindler-Barth M LA - eng PT - Journal Article PL - England TA - Insect Biochem Mol Biol JID - 9207282 RN - 0 (Acrylic Resins) RN - 0 (CF1 protein, insect) RN - 0 (DNA-Binding Proteins) RN - 0 (Receptors, Steroid) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - 0 (ecdysteroid receptor) RN - 0 (polyacrylamide gels) RN - 13408-56-5 (ponasterone A) RN - 5289-74-7 (Ecdysterone) SB - IM MH - Acrylic Resins MH - Animals MH - Blotting, Western/methods MH - Chironomidae MH - Chromatography, Affinity/methods MH - DNA-Binding Proteins/genetics/*isolation & purification/metabolism MH - Ecdysterone/*analogs & derivatives/metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Escherichia coli MH - *Gene Expression MH - Receptors, Steroid/genetics/*isolation & purification/metabolism MH - Recombinant Fusion Proteins/genetics/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Transcription Factors/genetics/*isolation & purification/metabolism EDAT- 2002/01/05 10:00 MHDA- 2002/03/13 10:01 AID - S0965174801000984 [pii] PST - ppublish SO - Insect Biochem Mol Biol 2002 Feb;32(2):167-74. NR -------------------------------------------------------------------------------- 17: Miyasaka T et al. Characterization of human tau...[PMID: 11722175] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11722175 OWN - NLM STAT- MEDLINE DA - 20011127 DCOM- 20020430 LR - 20041117 PUBM- Print IS - 1046-5928 VI - 23 IP - 3 DP - 2001 Dec TI - Characterization of human taurine transporter expressed in insect cells using a recombinant baculovirus. PG - 389-97 AB - A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases. CI - Copyright 2001 Elsevier Science. AD - Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8562, Japan. FAU - Miyasaka, T AU - Miyasaka T FAU - Kaminogawa, S AU - Kaminogawa S FAU - Shimizu, M AU - Shimizu M FAU - Hisatsune, T AU - Hisatsune T FAU - Reinach, P S AU - Reinach PS FAU - Miyamoto, Y AU - Miyamoto Y LA - eng PT - Journal Article PL - United States TA - Protein Expr Purif JID - 9101496 RN - 0 (Calcium-Binding Proteins) RN - 0 (Carrier Proteins) RN - 0 (DNA, Viral) RN - 0 (Genetic Vectors) RN - 0 (Membrane Glycoproteins) RN - 0 (Membrane Transport Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 107-35-7 (Taurine) RN - 11089-65-9 (Tunicamycin) RN - 139873-08-8 (Calnexin) RN - 148686-53-7 (taurine transporter) RN - 7440-23-5 (Sodium) SB - IM MH - Animals MH - Baculoviridae/*genetics MH - Biological Transport MH - Calcium-Binding Proteins/metabolism MH - Calnexin MH - Carrier Proteins/*genetics/isolation & purification/metabolism MH - Cell Line MH - DNA, Viral/genetics MH - Genetic Vectors MH - Glycosylation MH - Humans MH - Immunoblotting MH - Kinetics MH - Membrane Glycoproteins/*genetics/isolation & purification/metabolism MH - *Membrane Transport Proteins MH - Protein Folding MH - Rabbits MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Sodium/metabolism MH - Spodoptera/*genetics MH - Substrate Specificity MH - Taurine/metabolism MH - Tunicamycin/pharmacology EDAT- 2001/11/28 10:00 MHDA- 2002/05/01 10:01 AID - 10.1006/prep.2001.1505 [doi] AID - S1046592801915050 [pii] PST - ppublish SO - Protein Expr Purif 2001 Dec;23(3):389-97. DR -------------------------------------------------------------------------------- 18: Baer K et al. Dimerization-induced activati...[PMID: 11714281] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11714281 OWN - NLM STAT- MEDLINE DA - 20011120 DCOM- 20020110 LR - 20041117 PUBM- Print IS - 0006-2960 VI - 40 IP - 47 DP - 2001 Nov 27 TI - Dimerization-induced activation of soluble insulin/IGF-1 receptor kinases: an alternative mechanism of activation. PG - 14268-78 AB - To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues. AD - Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, 50674 Cologne, Germany. FAU - Baer, K AU - Baer K FAU - Al-Hasani, H AU - Al-Hasani H FAU - Parvaresch, S AU - Parvaresch S FAU - Corona, T AU - Corona T FAU - Rufer, A AU - Rufer A FAU - Nolle, V AU - Nolle V FAU - Bergschneider, E AU - Bergschneider E FAU - Klein, H W AU - Klein HW LA - eng PT - Journal Article PL - United States TA - Biochemistry JID - 0370623 RN - 0 (Phosphoproteins) RN - 0 (Recombinant Fusion Proteins) RN - 25104-18-1 (Polylysine) RN - 55520-40-6 (Tyrosine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Receptor, IGF Type 1) RN - EC 2.7.1.112 (Receptor, Insulin) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase/metabolism MH - Dimerization MH - Enzyme Activation MH - Glutathione Transferase/genetics MH - Kinetics MH - Phosphoproteins/metabolism MH - Phosphorylation/drug effects MH - Polylysine/pharmacology MH - Protein Binding MH - Receptor, IGF Type 1/genetics/*metabolism MH - Receptor, Insulin/genetics/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Solubility MH - Tyrosine/metabolism MH - src Homology Domains EDAT- 2001/11/21 10:00 MHDA- 2002/01/11 10:01 AID - bi015588g [pii] PST - ppublish SO - Biochemistry 2001 Nov 27;40(47):14268-78. DR -------------------------------------------------------------------------------- 19: D'Atri F et al. Cingulin interacts with F-act...[PMID: 11682052] Related Articles, Protein, Books, LinkOut PMID- 11682052 OWN - NLM STAT- MEDLINE DA - 20011029 DCOM- 20011207 LR - 20041203 PUBM- Print IS - 0014-5793 VI - 507 IP - 1 DP - 2001 Oct 19 TI - Cingulin interacts with F-actin in vitro. PG - 21-4 AB - Cingulin, a M(r) 140-160 kDa protein of the cytoplasmic plaque of epithelial tight junctions (TJ), interacts in vitro with TJ proteins and myosin. Here we investigated cingulin interaction with actin, using His-tagged, full-length Xenopus laevis cingulin expressed in insect cells, and glutathione S-transferase (GST) fusion proteins of fragments of cingulin expressed in bacteria. Purified full-length cingulin co-pelleted with F-actin after high speed centrifugation, and promoted the sedimentation of F-actin under low speed centrifugation, suggesting that cingulin is an actin-cross-linking protein. The actin interaction of GST fusion proteins containing fragments of Xenopus cingulin suggested that the F-actin binding site is between residues 101 and 294. AD - Department of Molecular Biology, University of Geneva, Geneva 4, Switzerland. FAU - D'Atri, F AU - D'Atri F FAU - Citi, S AU - Citi S LA - eng PT - Journal Article PL - Netherlands TA - FEBS Lett JID - 0155157 RN - 0 (Actins) RN - 0 (CGN protein, Xenopus) RN - 0 (CGN protein, human) RN - 0 (Cross-Linking Reagents) RN - 0 (Membrane Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Xenopus Proteins) SB - IM MH - Actins/*metabolism MH - Animals MH - Binding Sites MH - Cross-Linking Reagents MH - Cytoskeleton/metabolism MH - Humans MH - In Vitro MH - Membrane Proteins/chemistry/genetics/*metabolism MH - Peptide Mapping MH - Protein Binding MH - Rabbits MH - Recombinant Fusion Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Spodoptera MH - Tight Junctions/chemistry/metabolism MH - *Xenopus Proteins MH - Xenopus laevis EDAT- 2001/10/30 10:00 MHDA- 2002/01/05 10:01 AID - S0014579301029362 [pii] PST - ppublish SO - FEBS Lett 2001 Oct 19;507(1):21-4. NR -------------------------------------------------------------------------------- 20: Rebers JE et al. A conserved domain in arthrop...[PMID: 11520687] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 11520687 OWN - NLM STAT- MEDLINE DA - 20010824 DCOM- 20011213 LR - 20041117 PUBM- Print IS - 0965-1748 VI - 31 IP - 11 DP - 2001 Oct TI - A conserved domain in arthropod cuticular proteins binds chitin. PG - 1083-93 AB - Many insect cuticular proteins include a 35-36 amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin. Provocatively, it has no sequence similarity to the well-known cysteine-containing chitin-binding domain found in chitinases and some peritrophic membrane proteins. Using fusion proteins expressed in E. coli, we show that an extended form of the R&R consensus from proteins of hard cuticles is necessary and sufficient for chitin binding. Recombinant AGCP2b, a putative cuticular protein from the mosquito Anopheles gambiae, was expressed in E. coli and the purified protein shown to bind to chitin beads. A stretch of 65 amino acids from AGCP2b, including the R&R consensus, conferred chitin binding to glutathione-S-transferase (GST). Directed mutagenesis of some conserved amino acids within this extended R&R consensus from hard cuticle eliminated chitin binding. Thus arthropods have two distinct classes of chitin binding proteins, those with the chitin-binding domain found in lectins, chitinases and peritrophic membranes (cysCBD) and those with the cuticular protein chitin-binding domain (non-cysCBD). AD - Department of Biology, Northern Michigan University, Marquette, MI 49855, USA. jrebers@nmu.edu FAU - Rebers, J E AU - Rebers JE FAU - Willis, J H AU - Willis JH LA - eng PT - Journal Article PL - England TA - Insect Biochem Mol Biol JID - 9207282 RN - 0 (Insect Proteins) RN - 0 (Polysaccharides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (cuticle proteins, insects) RN - 1398-61-4 (Chitin) RN - 9004-34-6 (Cellulose) SB - IM MH - Amino Acid Sequence MH - Animals MH - Anopheles MH - Arthropods MH - Cellulose/metabolism MH - Chitin/*metabolism MH - *Consensus Sequence MH - Insect Proteins/genetics/*metabolism MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Polysaccharides/metabolism MH - Protein Binding MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't EDAT- 2001/08/25 10:00 MHDA- 2002/01/05 10:01 AID - S096517480100056X [pii] PST - ppublish SO - Insect Biochem Mol Biol 2001 Oct;31(11):1083-93. NR -------------------------------------------------------------------------------- 21: Yamanaka R et al. Ostip2, a novel oncoprotein t...[PMID: 11506702] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11506702 OWN - NLM STAT- MEDLINE DA - 20010816 DCOM- 20010920 LR - 20041117 PUBM- Print IS - 1044-5498 VI - 20 IP - 7 DP - 2001 Jul TI - Ostip2, a novel oncoprotein that associates with the Rho exchange factor Ost. PG - 383-90 AB - The ost protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases, RhoA and Cdc42. The N-terminal domain of Ost (Ost-N) appears to negatively regulate the oncogenic activity of the protein, as deletion of this domain drastically increases its transforming activity in NIH 3T3 cells. Using a yeast two-hybrid system, we identified five genes encoding proteins that can interact with Ost-N. One of them, designated OSTIP2 (Ost interacting protein 2), encoded a previously uncharacterized protein. The OSTIP2 product is highly expressed in skeletal muscle as a 1.2-kb transcript. Full-length OSTIP2 cDNA contained an ORF of 193 amino acids. Transcription-coupled translation of OSTIP2 cDNA in reticulocyte lysates revealed a protein product of 20 kDa, which corresponded to the predicted size of the protein. Bacterially expressed glutathione S-transferase (GST)-Ostip2 fusion protein efficiently associated in vitro with baculovirus-expressed Ost. Interestingly, expression of Ostip2 in NIH 3T3 cells efficiently induced foci of morphologically transformed cells. Moreover, inoculation of athymic (nude) mice with OSTIP2 transfectants strongly induced tumor formation. These results suggest that Ostip2 is a novel oncoprotein that can interact with the Rho exchange factor Ost. AD - Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. FAU - Yamanaka, R AU - Yamanaka R FAU - Blumenthal, R AU - Blumenthal R FAU - Lorenzi, M V AU - Lorenzi MV FAU - Tatsumoto, T AU - Tatsumoto T FAU - Miki, T AU - Miki T LA - eng PT - Journal Article PL - United States TA - DNA Cell Biol JID - 9004522 RN - 0 (DNA, Complementary) RN - 0 (Guanine Nucleotide Exchange Factors) RN - 0 (Oncogene Proteins) RN - 0 (Ost-interacting protein 2, mouse) RN - 0 (Proto-Oncogene Proteins) RN - EC 3.6.1.- (rhoA GTP-Binding Protein) SB - IM MH - 3T3 Cells MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - *Cell Transformation, Neoplastic MH - Chromosome Mapping MH - DNA, Complementary MH - Guanine Nucleotide Exchange Factors/*metabolism MH - Hela Cells MH - Humans MH - Mice MH - Mice, Nude MH - Molecular Sequence Data MH - Oncogene Proteins/*genetics/metabolism MH - Proto-Oncogene Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Saccharomyces cerevisiae MH - Two-Hybrid System Techniques MH - rhoA GTP-Binding Protein/metabolism EDAT- 2001/08/17 10:00 MHDA- 2001/09/21 10:01 AID - 10.1089/104454901750361442 [doi] PST - ppublish SO - DNA Cell Biol 2001 Jul;20(7):383-90. NR -------------------------------------------------------------------------------- 22: Hukuhara T et al. A bacterially produced virus ...[PMID: 11500090] Related Articles, Books, LinkOut PMID- 11500090 OWN - NLM STAT- MEDLINE DA - 20010813 DCOM- 20011011 LR - 20031114 PUBM- Print IS - 0022-2011 VI - 78 IP - 1 DP - 2001 Jul TI - A bacterially produced virus enhancing factor from an entomopoxvirus enhances nucleopolyhedrovirus infection in armyworm larvae. PG - 25-30 AB - Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F. CI - Copyright 2001 Academic Press. AD - College of Bioresource Sciences, Nihon University, Kameino, Fujisawa, 252-8510, Japan. tosi@brs.nihon-u.ac.jp FAU - Hukuhara, T AU - Hukuhara T FAU - Hayakawa, T AU - Hayakawa T FAU - Wijonarko, A AU - Wijonarko A LA - eng PT - Journal Article PL - United States TA - J Invertebr Pathol JID - 0014067 RN - 0 (enhancing factor) RN - EC 3.1.1.- (Phospholipases A) SB - IM MH - Animals MH - Entomopoxvirinae/*metabolism MH - Escherichia coli/*metabolism MH - Larva/virology MH - Nucleopolyhedrovirus/*physiology MH - Phospholipases A/*physiology MH - Spodoptera/*growth & development/*virology MH - Virus Diseases/*physiopathology EDAT- 2001/08/14 10:00 MHDA- 2001/10/12 10:01 AID - 10.1006/jipa.2001.5036 [doi] AID - S0022201101950360 [pii] PST - ppublish SO - J Invertebr Pathol 2001 Jul;78(1):25-30. NR -------------------------------------------------------------------------------- 23: Jirajaroenrat K et al. Heterologous expression and c...[PMID: 11439246] Related Articles, Compound via MeSH, Substance via MeSH, Nucleotide, Protein, Books, LinkOut PMID- 11439246 OWN - NLM STAT- MEDLINE DA - 20010705 DCOM- 20010920 LR - 20041117 PUBM- Print IS - 0965-1748 VI - 31 IP - 9 DP - 2001 Jul 26 TI - Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene. PG - 867-75 AB - Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three cDNAs share >67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2, adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide. adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus. AD - Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, 73170, Nakorn Pathom, Thailand. FAU - Jirajaroenrat, K AU - Jirajaroenrat K FAU - Pongjaroenkit, S AU - Pongjaroenkit S FAU - Krittanai, C AU - Krittanai C FAU - Prapanthadara, L AU - Prapanthadara L FAU - Ketterman, A J AU - Ketterman AJ LA - eng SI - GENBANK/AF273038 SI - GENBANK/AF273039 SI - GENBANK/AF273040 SI - GENBANK/AF273041 PT - Journal Article PL - England TA - Insect Biochem Mol Biol JID - 9207282 RN - 0 (DNA, Complementary) RN - 0 (Pyrethrins) RN - 0 (Recombinant Fusion Proteins) RN - 52645-53-1 (Permethrin) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - *Alternative Splicing MH - Amino Acid Sequence MH - Animals MH - Anopheles/*enzymology/genetics MH - Base Sequence MH - Cloning, Molecular MH - DNA, Complementary MH - Escherichia coli MH - Gene Expression MH - Glutathione Transferase/antagonists & inhibitors/*genetics MH - Kinetics MH - Molecular Sequence Data MH - Permethrin MH - Pyrethrins/pharmacology MH - Recombinant Fusion Proteins/genetics MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - Substrate Specificity EDAT- 2001/07/06 10:00 MHDA- 2001/09/21 10:01 AID - S0965174801000327 [pii] PST - ppublish SO - Insect Biochem Mol Biol 2001 Jul 26;31(9):867-75. NR -------------------------------------------------------------------------------- 24: Kato K et al. Epstein-Barr virus-encoded pr...[PMID: 11369891] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 11369891 OWN - NLM STAT- MEDLINE DA - 20010522 DCOM- 20010628 LR - 20041117 PUBM- Print IS - 0022-1317 VI - 82 IP - Pt 6 DP - 2001 Jun TI - Epstein-Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1delta (EF-1delta): EF-1delta is universally modified by conserved protein kinases of herpesviruses in mammalian cells. PG - 1457-63 AB - Translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1delta modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein-Barr virus was purified as a fusion protein that was labelled with [gamma-(32)P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1delta was increased both in Sf9 cells after infection with baculoviruses expressing GST-BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1delta hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1delta modification in mammalian cells. AD - Department of Tumor Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. FAU - Kato, K AU - Kato K FAU - Kawaguchi, Y AU - Kawaguchi Y FAU - Tanaka, M AU - Tanaka M FAU - Igarashi, M AU - Igarashi M FAU - Yokoyama, A AU - Yokoyama A FAU - Matsuda, G AU - Matsuda G FAU - Kanamori, M AU - Kanamori M FAU - Nakajima, K AU - Nakajima K FAU - Nishimura, Y AU - Nishimura Y FAU - Shimojima, M AU - Shimojima M FAU - Phung, H T AU - Phung HT FAU - Takahashi, E AU - Takahashi E FAU - Hirai, K AU - Hirai K LA - eng PT - Journal Article PL - England TA - J Gen Virol JID - 0077340 RN - 0 (Peptide Elongation Factor 1) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - EC 2.7.1.- (BGLF4 protein, Epstein-Barr virus) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.1.3 (Phosphoric Monoester Hydrolases) SB - IM MH - Adenosine Triphosphate/metabolism MH - Animals MH - Baculoviridae MH - Blotting, Western MH - COS Cells MH - Cell Line MH - Herpesvirus 4, Human/*enzymology/genetics MH - Humans MH - Peptide Elongation Factor 1/*metabolism MH - Phosphoric Monoester Hydrolases/metabolism MH - Phosphorylation MH - Protein Processing, Post-Translational MH - Protein-Serine-Threonine Kinases/genetics/isolation & purification/*metabolism MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Spodoptera MH - Transfection MH - *Viral Proteins EDAT- 2001/05/23 10:00 MHDA- 2001/06/29 10:01 PST - ppublish SO - J Gen Virol 2001 Jun;82(Pt 6):1457-63. DR -------------------------------------------------------------------------------- 25: Klinger M et al. Suramin and the suramin analo...[PMID: 11311147] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11311147 OWN - NLM STAT- MEDLINE DA - 20010420 DCOM- 20010531 LR - 20041117 PUBM- Print IS - 0264-6021 VI - 355 IP - Pt 3 DP - 2001 May 1 TI - Suramin and the suramin analogue NF307 discriminate among calmodulin-binding sites. PG - 827-33 AB - Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM, respectively) and blocked the cross-linking of [(125)I]calmodulin to some, but not all, target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless, suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs. AD - Institute of Pharmacology, University of Vienna, Wahringer Strasse 13a, A-1090 Vienna, Austria. FAU - Klinger, M AU - Klinger M FAU - Bofill-Cardona, E AU - Bofill-Cardona E FAU - Mayer, B AU - Mayer B FAU - Nanoff, C AU - Nanoff C FAU - Freissmuth, M AU - Freissmuth M FAU - Hohenegger, M AU - Hohenegger M LA - eng PT - Journal Article PL - England TA - Biochem J JID - 2984726R RN - 0 (Calmodulin) RN - 0 (Cross-Linking Reagents) RN - 0 (Iodine Radioisotopes) RN - 0 (NF 307) RN - 0 (Naphthalenesulfonates) RN - 0 (Oligopeptides) RN - 145-63-1 (Suramin) SB - IM MH - Allosteric Regulation MH - Amino Acid Motifs MH - Animals MH - Binding Sites MH - Brain/*drug effects/metabolism MH - Calmodulin/drug effects/*metabolism MH - Cell Membrane/drug effects/metabolism MH - Chromatography, Affinity MH - Cross-Linking Reagents/metabolism MH - In Vitro MH - Iodine Radioisotopes MH - Muscle, Skeletal/drug effects/metabolism MH - Naphthalenesulfonates/*pharmacology MH - Oligopeptides/*pharmacology MH - Rabbits MH - Research Support, Non-U.S. Gov't MH - Sarcoplasmic Reticulum/*drug effects/metabolism MH - Suramin/*pharmacology MH - Swine EDAT- 2001/04/20 10:00 MHDA- 2001/06/02 10:01 PST - ppublish SO - Biochem J 2001 May 1;355(Pt 3):827-33. NR -------------------------------------------------------------------------------- 26: Homann S et al. Expression and purification o...[PMID: 11223144] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 11223144 OWN - NLM STAT- MEDLINE DA - 20010306 DCOM- 20010521 LR - 20041117 PUBM- Print IS - 0168-1656 VI - 86 IP - 1 DP - 2001 Mar 9 TI - Expression and purification of human recombinant GST-FGF receptor-1. PG - 51-8 AB - When tumors undergo the angiogenic switch, cell growth and tissue invasion is facilitated by the formation of new capillaries from preexisting blood vessels, a process known as angiogenesis. Growth factors such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and fibroblast growth factor (FGF) trigger the process of angiogenesis. Here we describe a protocol for the expression and one-step purification of human recombinant GST-FGF receptor type 1 (FGFR-1) from Sf9 cells. This protocol allows generating an active kinase as indicated by its reactivity with a monoclonal antibody to phosphorylated tyrosine. The purified enzyme displays a specific activity of 1.2 x 10(4) pmol mg(-1) min(-1), which is in the range of activities reported for homogeneously purified recombinant kinases. We have employed a number of compounds to show that the GST-FGFR-1 preparation is suitable to the identification of tyrosine kinase inhibitors. Considering that inhibitors of angiogenesis may represent an attractive tool in therapeutic strategies targeting invasive metastatic tumors the results presented here, along with available data on the structure of the ATP-binding pocket of FGFR-1, should facilitate the rational design of specific FGFR-1 inhibitory compounds. AD - Department of Oncology, Novartis Pharma AG, K-125.4.01, Klybeckstrasse 141, CH-4057, Basel, Switzerland. FAU - Homann, S AU - Homann S FAU - Schacher, B AU - Schacher B FAU - Zumstein-Mecker, S AU - Zumstein-Mecker S FAU - Fabbro, D AU - Fabbro D FAU - Bold, G AU - Bold G FAU - Ferrari, S AU - Ferrari S LA - eng PT - Journal Article PL - Netherlands TA - J Biotechnol JID - 8411927 RN - 0 (Antibodies, Monoclonal) RN - 0 (Enzyme Inhibitors) RN - 0 (Receptors, Fibroblast Growth Factor) RN - 0 (Recombinant Fusion Proteins) RN - 104781-85-3 (Fibroblast Growth Factor 1) RN - 21820-51-9 (Phosphotyrosine) RN - 56-65-5 (Adenosine Triphosphate) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) RN - EC 2.7.1.112 (fibroblast growth factor receptor 1) SB - IM MH - Adenosine Triphosphate/metabolism MH - Animals MH - Antibodies, Monoclonal MH - Baculoviridae/genetics MH - Binding Sites MH - Enzyme Inhibitors/pharmacology MH - Enzyme Stability MH - Fibroblast Growth Factor 1 MH - Freezing MH - *Gene Expression MH - Glutathione Transferase/*genetics/metabolism MH - Humans MH - Kinetics MH - Phosphorylation MH - Phosphotyrosine/immunology/metabolism MH - Receptor Protein-Tyrosine Kinases/antagonists & inhibitors/*genetics/metabolism MH - Receptors, Fibroblast Growth Factor/antagonists & inhibitors/*genetics/metabolism MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Spodoptera/metabolism MH - Substrate Specificity EDAT- 2001/02/27 10:00 MHDA- 2001/05/25 10:01 AID - S0168165600003990 [pii] PST - ppublish SO - J Biotechnol 2001 Mar 9;86(1):51-8. DR -------------------------------------------------------------------------------- 27: Shirato H et al. Identification and characteri...[PMID: 11076525] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 11076525 OWN - NLM STAT- MEDLINE DA - 20001130 DCOM- 20001228 LR - 20041117 PUBM- Print IS - 0006-2960 VI - 39 IP - 45 DP - 2000 Nov 14 TI - Identification and characterization of a novel protein inhibitor of type 1 protein phosphatase. PG - 13848-55 AB - We have isolated human cDNA for a novel type 1 protein phosphatase (PP1) inhibitory protein, named inhibitor-4 (I-4), from a cDNA library of germ cell tumors. I-4, composed of 202 amino acids, is 44% identical to a PP1 inhibitor, inhibitor-2 (I-2). I-4 conserves functionally important structure of I-2 and exhibited similar biochemical properties. I-4 inhibited activity of the catalytic subunit of PP1 (PP1C), specifically with an IC(50) of 0.2 nM, more potently than I-2 with an IC(50) of 2 nM. I-4 weakly inhibited the activity of myosin-associated phosphates (PP1M). However, the level of inhibition of PP1M was increased during preincubation of PP1M with I-4, suggesting that the inhibition is caused by interaction of I-4 with PP1C in such a manner that it competes with the M subunit of PP1M. Gel overlay experiments showed that I-4 binds PP1C directly. Three I-4 peptides containing the N-terminal residues 1-123, 1-131, and 1-142 all showed strong binding ability to PP1C but did not show PP1 inhibitory activity, whereas an I-2 peptide (residues 1-134), lacking the corresponding C-terminal residues, potently inhibited PP1C activity as previously reported. Removal of the 18 N-terminal amino acid residues from I-4 dramatically reduced the PP1 binding activity with a correlated loss of inhibitory activity, whereas removal of the 10 N-terminal residues had only a little effect. The two peptides GST-I-4(19-131) and GST-I-4(132-202) showed ability to bind to PP1C, albeit very weakly. These results strongly suggest a multiple-point interaction between I-4 and PP1C, which is thought to cause the inhibition of I-4 which is stronger than the inhibition of I-2. AD - Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. FAU - Shirato, H AU - Shirato H FAU - Shima, H AU - Shima H FAU - Sakashita, G AU - Sakashita G FAU - Nakano, T AU - Nakano T FAU - Ito, M AU - Ito M FAU - Lee, E Y AU - Lee EY FAU - Kikuchi, K AU - Kikuchi K LA - eng SI - GENBANK/AB044137 PT - Journal Article PL - UNITED STATES TA - Biochemistry JID - 0370623 RN - 0 (Enzyme Inhibitors) RN - 0 (Holoenzymes) RN - 0 (Isoenzymes) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (protein phosphatase inhibitor-4) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.1.3.16 (Phosphoprotein Phosphatase) RN - EC 3.1.3.53 (Myosin-Light-Chain Phosphatase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Binding, Competitive/genetics MH - Catalytic Domain/genetics MH - Cloning, Molecular MH - Comparative Study MH - Drosophila melanogaster MH - Enzyme Inhibitors/*chemistry/*isolation & purification/pharmacology MH - Glutathione Transferase/genetics MH - Holoenzymes/metabolism MH - Humans MH - Isoenzymes/antagonists & inhibitors/genetics MH - Molecular Sequence Data MH - Myosin-Light-Chain Phosphatase MH - Phosphoprotein Phosphatase/*antagonists & inhibitors/genetics/metabolism MH - Protein Binding/genetics MH - Proteins/*chemistry/genetics/*isolation & purification/physiology MH - Rats MH - Recombinant Fusion Proteins/antagonists & inhibitors/pharmacology MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Sequence Analysis, Protein MH - Sequence Deletion EDAT- 2000/11/15 11:00 MHDA- 2001/02/28 10:01 AID - bi001326n [pii] PST - ppublish SO - Biochemistry 2000 Nov 14;39(45):13848-55. NR -------------------------------------------------------------------------------- 28: Iwata H et al. Expression of porcine interle...[PMID: 11073083] Related Articles, Substance via MeSH, Books, LinkOut PMID- 11073083 OWN - NLM STAT- MEDLINE DA - 20010214 DCOM- 20010405 LR - 20031114 PUBM- Print IS - 0916-7250 VI - 62 IP - 10 DP - 2000 Oct TI - Expression of porcine interleukin-2 in Escherichia coli. PG - 1101-4 AB - A mature form of porcine interleukin-2 (IL-2) protein without signal peptides was expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli using pGEX vector. Since most of GST-IL-2 fusion protein was detected in an insoluble fraction on SDS-PAGE analysis, the insoluble fusion protein was solubilized by refolding procedure using urea. The recombinant IL-2 (rIL-2) was purified by a batch method using Glutathione Sepharose 4B and factor Xa digestion and used for preparation of antisera in mice. The antisera reacted with rIL-2 expressed in baculovirus system on immunoblot analysis. In addition, the purified rIL-2 showed a high biological activity on CTLL-2 proliferative response. AD - Department of Veterinary Hygiene, Faculty ofAgriculture, Yamaguchi University, Japan. FAU - Iwata, H AU - Iwata H FAU - Yamamoto, M AU - Yamamoto M FAU - Hasegawa, A AU - Hasegawa A FAU - Kurata, K AU - Kurata K FAU - Inoue, T AU - Inoue T LA - eng PT - Journal Article PL - JAPAN TA - J Vet Med Sci JID - 9105360 RN - 0 (Genetic Vectors) RN - 0 (Interleukin-2) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Animals MH - Chromatography, Affinity/veterinary MH - Electrophoresis, Polyacrylamide Gel/veterinary MH - Escherichia coli/*metabolism MH - Gene Expression Regulation, Bacterial MH - Genetic Vectors MH - Interleukin-2/*genetics/metabolism MH - Mice MH - Protein Folding MH - Recombinant Fusion Proteins/metabolism MH - Swine/genetics/*metabolism MH - T-Lymphocytes, Cytotoxic/metabolism EDAT- 2000/11/10 11:00 MHDA- 2001/04/06 10:01 PST - ppublish SO - J Vet Med Sci 2000 Oct;62(10):1101-4. NR -------------------------------------------------------------------------------- 29: Chan-Fook C et al. Hepatitis C virus glycoprotei...[PMID: 10891408] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 10891408 OWN - NLM STAT- MEDLINE DA - 20000811 DCOM- 20000811 LR - 20050319 PUBM- Print IS - 0042-6822 VI - 273 IP - 1 DP - 2000 Jul 20 TI - Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity. PG - 60-6 AB - The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required. CI - Copyright 2000 Academic Press. AD - NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 3SR. FAU - Chan-Fook, C AU - Chan-Fook C FAU - Jiang, W R AU - Jiang WR FAU - Clarke, B E AU - Clarke BE FAU - Zitzmann, N AU - Zitzmann N FAU - Maidens, C AU - Maidens C FAU - McKeating, J A AU - McKeating JA FAU - Jones, I M AU - Jones IM LA - eng PT - Journal Article PL - UNITED STATES TA - Virology JID - 0110674 RN - 0 (Antigens, CD) RN - 0 (CD81 antigen) RN - 0 (E1 protein, hepatitis C virus) RN - 0 (Membrane Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Envelope Proteins) RN - 0 (miglustat) RN - 19130-96-2 (1-Deoxynojirimycin) RN - EC 3.2.1.20 (alpha-Glucosidases) SB - IM MH - 1-Deoxynojirimycin/analogs & derivatives/pharmacology MH - Animals MH - Antigens, CD/chemistry/genetics/*metabolism MH - Cell Line MH - Glycosylation MH - *Hepacivirus MH - Humans MH - *Membrane Proteins MH - Protein Binding MH - Protein Processing, Post-Translational MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Deletion MH - Spodoptera MH - Viral Envelope Proteins/isolation & purification/*metabolism MH - alpha-Glucosidases/antagonists & inhibitors/metabolism EDAT- 2000/07/13 11:00 MHDA- 2000/08/19 11:00 AID - 10.1006/viro.2000.0407 [doi] AID - S0042682200904070 [pii] PST - ppublish SO - Virology 2000 Jul 20;273(1):60-6. DR -------------------------------------------------------------------------------- 30: Masai H et al. Human Cdc7-related kinase com...[PMID: 10846177] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 10846177 OWN - NLM STAT- MEDLINE DA - 20001013 DCOM- 20001013 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 275 IP - 37 DP - 2000 Sep 15 TI - Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks. PG - 29042-52 AB - huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D. , Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks. AD - Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, CREST, Japan. FAU - Masai, H AU - Masai H FAU - Matsui, E AU - Matsui E FAU - You, Z AU - You Z FAU - Ishimi, Y AU - Ishimi Y FAU - Tamai, K AU - Tamai K FAU - Arai, K AU - Arai K LA - eng PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Antigens, Viral, Tumor) RN - 0 (Cell Cycle Proteins) RN - 0 (MCM2 protein, mammalian) RN - 0 (Nuclear Proteins) RN - 72-19-5 (Threonine) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Antigens, Viral, Tumor/metabolism MH - Cell Cycle Proteins/physiology MH - Cyclin-Dependent Kinases/*physiology MH - Humans MH - Nuclear Proteins/*metabolism MH - Phosphorylation MH - Protein-Serine-Threonine Kinases/*physiology MH - Threonine/*metabolism EDAT- 2000/06/10 09:00 MHDA- 2000/10/21 11:01 AID - 10.1074/jbc.M002713200 [doi] AID - M002713200 [pii] PST - ppublish SO - J Biol Chem 2000 Sep 15;275(37):29042-52. DR -------------------------------------------------------------------------------- 31: Bichet P et al. Endogenous glutathione-bindin...[PMID: 10833407] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10833407 OWN - NLM STAT- MEDLINE DA - 20000814 DCOM- 20000814 LR - 20031114 PUBM- Print IS - 1046-5928 VI - 19 IP - 1 DP - 2000 Jun TI - Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins. PG - 197-201 AB - After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions. CI - Copyright 2000 Academic Press. AD - Biotechnology Department, Hoechst Marion Roussel, Romainville, 93235, France. FAU - Bichet, P AU - Bichet P FAU - Mollat, P AU - Mollat P FAU - Capdevila, C AU - Capdevila C FAU - Sarubbi, E AU - Sarubbi E LA - eng PT - Journal Article PL - UNITED STATES TA - Protein Expr Purif JID - 9101496 RN - 0 (Recombinant Fusion Proteins) RN - 70-18-8 (Glutathione) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Animals MH - Baculoviridae/genetics MH - Cell Line MH - Chromatography, Affinity MH - Chromatography, High Pressure Liquid MH - Electrophoresis, Polyacrylamide Gel MH - Glutathione/chemistry MH - Glutathione Transferase/chemistry/*isolation & purification MH - Insects/chemistry/*enzymology MH - Protein Binding MH - Recombinant Fusion Proteins/chemistry/*isolation & purification MH - Spectrum Analysis, Mass EDAT- 2000/06/02 09:00 MHDA- 2000/08/19 11:00 AID - 10.1006/prep.2000.1239 [doi] AID - S1046592800912397 [pii] PST - ppublish SO - Protein Expr Purif 2000 Jun;19(1):197-201. DR -------------------------------------------------------------------------------- 32: Kallio-Kokko H et al. Antigenic properties and diag...[PMID: 10797384] Related Articles, Cited in PMC, Books, LinkOut PMID- 10797384 OWN - NLM STAT- MEDLINE DA - 20000609 DCOM- 20000609 LR - 20041117 PUBM- Print IS - 0146-6615 VI - 61 IP - 2 DP - 2000 Jun TI - Antigenic properties and diagnostic potential of recombinant dobrava virus nucleocapsid protein. PG - 266-74 AB - Dobrava hantavirus (DOBV) causes severe hemorrhagic fever with renal syndrome in the Balkan region and has been detected recently also in Russia, Estonia, and Germany. DOBV nucleocapsid protein (N) was produced in insect cells, using the baculovirus expression system (bac-DOBV-N), and in E. coli as a truncated (aa 1-165) glutathione-S transferase fusion protein (DOBV-dN-GST). The antigenic properties of bac-DOBV-N were found identical to native DOBV-N when examined by a panel of hantavirus-specific monoclonal antibodies. Enzyme immunoassays for detection of IgM and IgG antibodies were set up using DOBV recombinant N proteins and compared with those based on recombinant Hantaan and Puumala virus N, using panels of sera collected from DOBV, Hantaan and Puumala virus-infected patients. Full-length N protein (bac-DOBV-N) was found to be a more sensitive antigen than DOBV-dN-GST. The sensitivity values for sera from DOBV-infected patients were 100% for bac-DOBV-N and 86% for DOBV-dN-GST by IgM assays, and 98% for bac-DOBV-N and 88% for DOBV-dN-GST by IgG assays. The specificity values were 100% for bac-DOBV-N and 99% for DOBV-dN-GST by IgM assays, and 100% for both antigens by IgG assays. CI - Copyright 2000 Wiley-Liss, Inc. AD - Haartman Institute, Department of Virology, University of Helsinki, Finland. hannimari.kallio-kokko@helsinki.fi FAU - Kallio-Kokko, H AU - Kallio-Kokko H FAU - Lundkvist, A AU - Lundkvist A FAU - Plyusnin, A AU - Plyusnin A FAU - Avsic-Zupanc, T AU - Avsic-Zupanc T FAU - Vaheri, A AU - Vaheri A FAU - Vapalahti, O AU - Vapalahti O LA - eng PT - Journal Article PL - UNITED STATES TA - J Med Virol JID - 7705876 RN - 0 (Antigens, Viral) RN - 0 (Genetic Vectors) RN - 0 (Immunoglobulin G) RN - 0 (Immunoglobulin M) RN - 0 (Nucleocapsid Proteins) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Antigens, Viral/biosynthesis/*genetics/metabolism MH - Baculoviridae/genetics MH - Cloning, Molecular MH - Enzyme-Linked Immunosorbent Assay MH - Escherichia coli/genetics MH - Genetic Vectors MH - Hantavirus/chemistry/*genetics/immunology MH - Hantavirus Infections/metabolism/virology MH - Humans MH - Immunoglobulin G/analysis MH - Immunoglobulin M/analysis MH - Nucleocapsid Proteins/biosynthesis/*genetics/metabolism MH - Recombinant Fusion Proteins/biosynthesis MH - Research Support, Non-U.S. Gov't MH - Sensitivity and Specificity MH - Serology EDAT- 2000/05/08 09:00 MHDA- 2000/06/17 09:00 AID - 10.1002/(SICI)1096-9071(200006)61:2<266::AID-JMV14>3.0.CO;2-J [pii] PST - ppublish SO - J Med Virol 2000 Jun;61(2):266-74. NR -------------------------------------------------------------------------------- 33: Kawar Z et al. N-Glycan processing by a lepi...[PMID: 10764822] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10764822 OWN - NLM STAT- MEDLINE DA - 20000511 DCOM- 20000511 LR - 20041117 PUBM- Print IS - 0959-6658 VI - 10 IP - 4 DP - 2000 Apr TI - N-Glycan processing by a lepidopteran insect alpha1,2-mannosidase. PG - 347-55 AB - Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man(9)GlcNAc(2)to Man(6)Glc-NAc(2)isomer C, then more slowly converts Man(6)GlcNAc(2)isomer C to Man(5)GlcNAc(2). The slow step in the processing of Man(9)GlcNAc(2)to Man(5)GlcNAc(2)by SfManI is removal of the alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate. AD - Department of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944, USA. FAU - Kawar, Z AU - Kawar Z FAU - Romero, P A AU - Romero PA FAU - Herscovics, A AU - Herscovics A FAU - Jarvis, D L AU - Jarvis DL LA - eng GR - GM 31265/GM/NIGMS GR - GM 49734/GM/NIGMS PT - Journal Article PL - ENGLAND TA - Glycobiology JID - 9104124 RN - 0 (Genetic Vectors) RN - 0 (Polysaccharides) RN - 0 (Recombinant Fusion Proteins) RN - 31103-86-3 (Mannose) RN - 7512-17-6 (Acetylglucosamine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.2.1. (Mannosidases) RN - EC 3.2.1.24 (alpha-Mannosidase) SB - IM MH - Acetylglucosamine/metabolism MH - Animals MH - Baculoviridae/genetics MH - Carbohydrate Conformation MH - Chromatography, High Pressure Liquid MH - Genetic Vectors MH - Glutathione Transferase/genetics MH - Kinetics MH - Mannose/metabolism MH - Mannosidases/genetics/*metabolism MH - Polysaccharides/*metabolism MH - Recombinant Fusion Proteins MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera/*enzymology MH - Substrate Specificity MH - alpha-Mannosidase EDAT- 2000/04/15 09:00 MHDA- 2000/05/16 09:00 AID - cwd033 [pii] PST - ppublish SO - Glycobiology 2000 Apr;10(4):347-55. DR -------------------------------------------------------------------------------- 34: Uno T et al. Molecular cloning of a cDNA f...[PMID: 10737920] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10737920 OWN - NLM STAT- MEDLINE DA - 20000619 DCOM- 20000619 LR - 20041117 PUBM- Print IS - 0739-4462 VI - 43 IP - 4 DP - 2000 Apr TI - Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification. PG - 165-72 AB - A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch. CI - Copyright 2000 Wiley-Liss, Inc. AD - Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-Ku, Kobe, Hyogo, Japan. unotom@kobe-u.ac.jp FAU - Uno, T AU - Uno T FAU - Nakasuji, A AU - Nakasuji A FAU - Hara, W AU - Hara W FAU - Aizono, Y AU - Aizono Y LA - eng PT - Journal Article PL - UNITED STATES TA - Arch Insect Biochem Physiol JID - 8501752 RN - 0 (DNA, Complementary) RN - 0 (Insect Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 146-91-8 (Guanosine Diphosphate) RN - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate)) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.- (rho GTP-Binding Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Bombyx/genetics/metabolism MH - Cloning, Molecular MH - DNA, Complementary MH - Escherichia coli MH - Gene Expression MH - Glutathione Transferase/genetics/metabolism MH - Guanosine 5'-O-(3-Thiotriphosphate)/metabolism MH - Guanosine Diphosphate/metabolism MH - Guanosine Triphosphate/metabolism MH - Hydrolysis MH - Insect Proteins/genetics/isolation & purification/*metabolism MH - Mice MH - Molecular Sequence Data MH - Recombinant Fusion Proteins/genetics/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Homology, Amino Acid MH - rho GTP-Binding Proteins/genetics/isolation & purification/*metabolism EDAT- 2000/03/29 09:00 MHDA- 2000/06/24 11:00 AID - 10.1002/(SICI)1520-6327(200004)43:4<165::AID-ARCH2>3.0.CO;2-C [pii] PST - ppublish SO - Arch Insect Biochem Physiol 2000 Apr;43(4):165-72. NR -------------------------------------------------------------------------------- 35: Ching YP et al. Cloning of three novel neuron...[PMID: 10721722] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Domains, Nucleotide, Protein, OMIM, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 10721722 OWN - NLM STAT- MEDLINE DA - 20000403 DCOM- 20000403 LR - 20041117 PUBM- Print IS - 0378-1119 VI - 242 IP - 1-2 DP - 2000 Jan 25 TI - Cloning of three novel neuronal Cdk5 activator binding proteins. PG - 285-94 AB - Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. AD - Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon. bcyching@ust.hk FAU - Ching, Y P AU - Ching YP FAU - Qi, Z AU - Qi Z FAU - Wang, J H AU - Wang JH LA - eng PT - Journal Article PL - NETHERLANDS TA - Gene JID - 7706761 RN - 0 (CDK5RAP1 protein, human) RN - 0 (CDK5RAP2 protein, human) RN - 0 (CDK5RAP3 protein, human) RN - 0 (Carrier Proteins) RN - 0 (DNA, Complementary) RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (Nerve Tissue Proteins) RN - 0 (RNA, Messenger) RN - 0 (Recombinant Fusion Proteins) RN - 0 (neuronal Cdk5 activator (p25-p35)) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Blotting, Northern MH - Carrier Proteins/*genetics/metabolism MH - Cell Line MH - Cloning, Molecular MH - DNA, Complementary/chemistry/genetics MH - Gene Expression MH - Glutathione Transferase/genetics/metabolism MH - *Intracellular Signaling Peptides and Proteins MH - Molecular Sequence Data MH - Nerve Tissue Proteins/*genetics/*metabolism MH - Protein Binding MH - RNA, Messenger/genetics/metabolism MH - Rats MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Tissue Distribution MH - Two-Hybrid System Techniques EDAT- 2000/03/18 09:00 MHDA- 2000/08/30 11:01 AID - S0378111999004990 [pii] PST - ppublish SO - Gene 2000 Jan 25;242(1-2):285-94. NR -------------------------------------------------------------------------------- 36: Fujino T et al. Identification of oxidized pr...[PMID: 10719179] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 10719179 OWN - NLM STAT- MEDLINE DA - 20000522 DCOM- 20000522 LR - 20041117 PUBM- Print IS - 0006-3002 VI - 1478 IP - 1 DP - 2000 Mar 16 TI - Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase. PG - 102-12 AB - Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a serine protease present in human erythrocyte cytosol (Fujino et al., J. Biochem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membranes and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisopropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six peptide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl endopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N-terminal position of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that the protein was identical with human liver acylpeptide hydrolase (ACPH). OPH showed ACPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L-alanine were used as substrates. Glutathione S-transferase (GST)-tagged recombinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiological function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells. AD - School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, Japan. FAU - Fujino, T AU - Fujino T FAU - Watanabe, K AU - Watanabe K FAU - Beppu, M AU - Beppu M FAU - Kikugawa, K AU - Kikugawa K FAU - Yasuda, H AU - Yasuda H LA - eng SI - GENBANK/AF141383 PT - Journal Article PL - NETHERLANDS TA - Biochim Biophys Acta JID - 0217513 RN - 0 (Peptide Fragments) RN - 0 (Protease Inhibitors) RN - 0 (Serum Albumin, Bovine) RN - 55-91-4 (Isoflurophate) RN - EC 3.4.- (Peptide Hydrolases) RN - EC 3.4.19.1 (acylaminoacyl-peptidase) RN - EC 3.4.21 (Serine Endopeptidases) RN - EC 3.4.21.50 (lysyl endopeptidase) SB - IM MH - Amino Acid Sequence MH - Baculoviridae/genetics MH - Chromatography, High Pressure Liquid MH - Cloning, Molecular MH - Erythrocytes/*enzymology MH - Gene Expression MH - Humans MH - Isoflurophate/chemistry MH - Molecular Sequence Data MH - Oxidation-Reduction MH - Peptide Fragments/chemistry MH - Peptide Hydrolases/*chemistry/genetics MH - Protease Inhibitors/chemistry MH - Research Support, Non-U.S. Gov't MH - Serine Endopeptidases MH - Serum Albumin, Bovine/chemistry EDAT- 2000/03/17 09:00 MHDA- 2000/06/08 09:00 AID - S0167483800000042 [pii] PST - ppublish SO - Biochim Biophys Acta 2000 Mar 16;1478(1):102-12. DR -------------------------------------------------------------------------------- 37: Dou F et al. Preliminary study on the clea...[PMID: 10701454] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10701454 OWN - NLM STAT- MEDLINE DA - 20000403 DCOM- 20000403 LR - 20041117 PUBM- Print IS - 1082-6068 VI - 30 IP - 1 DP - 2000 Feb TI - Preliminary study on the cleavage of fusion protein GST-CMIV with palladium(II) complex. PG - 69-78 AB - A novel method for post-treatment of gene-engineered proteins is reported. A coden of Cys-His unit is introduced into the N-terminal of cecropin CMIV by using PCR. The gene is expressed in E. coli fused with GST. After purification, the fusion protein is cleaved by [Pd(en)(H2O)2]2+ at the His-Arg bond and the cecropin CMIV with antibacterial activity is obtained. The preliminary results held some promise of success for application of the palladium(II) complex as cleavage agent for the production of peptide drugs from gene-engineering fusion proteins. AD - Department of Biochemistry Nanjing University, PR China. FAU - Dou, F AU - Dou F FAU - Qiao, F AU - Qiao F FAU - Hu, J AU - Hu J FAU - Zhu, T AU - Zhu T FAU - Xu, X AU - Xu X FAU - Zhu, D AU - Zhu D FAU - Zhu, L AU - Zhu L LA - eng PT - Journal Article PL - UNITED STATES TA - Prep Biochem Biotechnol JID - 9607037 RN - 0 (Anti-Bacterial Agents) RN - 0 (Insect Proteins) RN - 0 (Peptide Fragments) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 0 (cecropin CMIV protein, Bombyx mori) RN - 7440-05-3 (Palladium) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Anti-Bacterial Agents/metabolism/pharmacology MH - Escherichia coli MH - Glutathione Transferase/genetics/metabolism MH - Insect Proteins/*chemistry/genetics/pharmacology MH - Palladium/*metabolism MH - Peptide Fragments/genetics/metabolism/pharmacology MH - Plasmids/genetics MH - Protein Engineering MH - Recombinant Fusion Proteins/metabolism/pharmacology MH - Research Support, Non-U.S. Gov't EDAT- 2000/03/04 MHDA- 2000/03/04 00:01 PST - ppublish SO - Prep Biochem Biotechnol 2000 Feb;30(1):69-78. NR -------------------------------------------------------------------------------- 38: Inouye K et al. Formation of the Ras dimer is...[PMID: 10660519] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 10660519 OWN - NLM STAT- MEDLINE DA - 20000316 DCOM- 20000316 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 275 IP - 6 DP - 2000 Feb 11 TI - Formation of the Ras dimer is essential for Raf-1 activation. PG - 3737-40 AB - Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation. AD - Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. FAU - Inouye, K AU - Inouye K FAU - Mizutani, S AU - Mizutani S FAU - Koide, H AU - Koide H FAU - Kaziro, Y AU - Kaziro Y LA - eng PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Liposomes) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.37 (Proto-Oncogene Proteins c-raf) RN - EC 3.6.1.- (ras Proteins) SB - IM MH - Cell Line MH - Cell Membrane/metabolism MH - Dimerization MH - Enzyme Activation MH - Escherichia coli MH - Glutathione Transferase/genetics MH - Humans MH - Liposomes/metabolism MH - Mutation MH - Proto-Oncogene Proteins c-raf/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - ras Proteins/*metabolism EDAT- 2000/02/08 09:00 MHDA- 2000/03/18 09:00 PST - ppublish SO - J Biol Chem 2000 Feb 11;275(6):3737-40. NR -------------------------------------------------------------------------------- 39: Kimura M et al. Involvement of multiple subun...[PMID: 10648788] Related Articles, Gene, Free in PMC, Cited in PMC, Books, LinkOut PMID- 10648788 OWN - NLM STAT- MEDLINE DA - 20000328 DCOM- 20000328 LR - 20041117 PUBM- Print IS - 1362-4962 VI - 28 IP - 4 DP - 2000 Feb 15 TI - Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II. PG - 952-9 AB - RNA polymerase II from the fission yeast Schizo-saccharomyces pombe consists of 12 species of subunits, Rpb1-Rpb12. We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors. For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S -transferase (GST) sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein. After successive affinity chromatography on glutathione and Ni(2+)columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of the GST-Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex. Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 interaction to some extent. We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit-subunit contacts. AD - Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. makimura@lab.nig.ac.jp FAU - Kimura, M AU - Kimura M FAU - Ishihama, A AU - Ishihama A LA - eng PT - Journal Article PL - ENGLAND TA - Nucleic Acids Res JID - 0411011 RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.7.- (RNA Polymerase II) SB - IM MH - Animals MH - Baculoviridae/genetics MH - Glutathione Transferase/genetics MH - Protein Binding MH - RNA Polymerase II/chemistry/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics MH - Research Support, Non-U.S. Gov't MH - Schizosaccharomyces/enzymology MH - Spodoptera EDAT- 2000/01/29 MHDA- 2000/04/01 AID - gkd204 [pii] PST - ppublish SO - Nucleic Acids Res 2000 Feb 15;28(4):952-9. DR -------------------------------------------------------------------------------- 40: Scott MJ et al. MSL1 plays a central role in ...[PMID: 10619853] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 10619853 OWN - NLM STAT- MEDLINE DA - 20000223 DCOM- 20000223 LR - 20050510 PUBM- Print IS - 0261-4189 VI - 19 IP - 1 DP - 2000 Jan 4 TI - MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila. PG - 144-55 AB - In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here we show that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF. The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of MSL1 has dominant-negative effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is co-overexpressed. MSL2 co-purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co-purify with the FLAG-tagged MSL1 C-terminal domain. Furthermore, the MSL1 C-terminal domain binds specifically to a GST-MOF fusion protein and co-immunoprecipitates with HA-tagged MSL3. The MSL1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. We conclude that a main role of MSL1 is to serve as the backbone for assembly of the MSL complex. AD - Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. M.J.Scott@massey.ac.nz FAU - Scott, M J AU - Scott MJ FAU - Pan, L L AU - Pan LL FAU - Cleland, S B AU - Cleland SB FAU - Knox, A L AU - Knox AL FAU - Heinrich, J AU - Heinrich J LA - eng PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (DNA-Binding Proteins) RN - 0 (Drosophila Proteins) RN - 0 (Dyes) RN - 0 (Indoles) RN - 0 (Nuclear Proteins) RN - 0 (Transcription Factors) RN - 0 (msl-1 protein, Drosophila) RN - 0 (msl-2 protein, Drosophila) RN - 47165-04-8 (DAPI) SB - IM MH - Amino Acid Sequence MH - Animals MH - Chromatography, Affinity MH - DNA-Binding Proteins/metabolism MH - *Dosage Compensation (Genetics) MH - *Drosophila Proteins MH - Drosophila melanogaster/*genetics MH - Dyes MH - Female MH - Heterozygote MH - Indoles MH - Male MH - Mice MH - Molecular Sequence Data MH - Nuclear Proteins/metabolism/*physiology MH - Research Support, Non-U.S. Gov't MH - Transcription Factors/metabolism/*physiology MH - X Chromosome/metabolism MH - Zinc Fingers EDAT- 2000/01/05 09:00 MHDA- 2000/02/26 09:00 AID - 10.1093/emboj/19.1.144 [doi] PST - ppublish SO - EMBO J 2000 Jan 4;19(1):144-55. NR -------------------------------------------------------------------------------- 41: Rotheneder H et al. Transcription factors of the ...[PMID: 10547281] Related Articles, Gene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 10547281 OWN - NLM STAT- MEDLINE DA - 20000111 DCOM- 20000111 LR - 20041208 PUBM- Print IS - 0022-2836 VI - 293 IP - 5 DP - 1999 Nov 12 TI - Transcription factors of the Sp1 family: interaction with E2F and regulation of the murine thymidine kinase promoter. PG - 1005-15 AB - Promoters of growth and cell cycle regulated genes frequently carry binding sites for transcription factors of the E2F and Sp1 families. We have demonstrated recently that direct interaction between Sp1 and a subgroup of the E2F factors is essential for the regulation of certain promoters. We show here that the amino acids necessary for this interaction in both cases are located within the DNA binding domain. This is in line with the assumption, that the interaction between E2F and Sp-factors contributes to promoter-specificity. Cyclin A, which binds to E2F-1 in close vicinity to Sp1 does not interfere with this interaction. Moreover we have investigated the ability of other members of the Sp1 family to interact with E2F-1 and to regulate the activity of the E2F and Sp1 dependent murine thymidine kinase promoter. All four factors of the Sp1 family are able to bind E2F-1 in co-immunoprecipitation and GST-pull down experiments. Mobility shift assays with oligonucleotides comprising the Sp1, or both the Sp1 and the E2F binding site suggest that Sp1 and Sp3 supply most if not all activity binding to the GC-box of the thymidine kinase promoter in murine fibroblasts. Reporter gene assays in Drosophila melanogaster SL2 cells and murine fibroblast 3T6 cells demonstrate that the thymidine kinase promoter is activated strongly by Sp1 and Sp3, weakly by Sp4, and not at all by Sp2. Co-expression of E2F-1 results in synergistic activation in 3T6 but not in SL2 cells. CI - Copyright 1999 Academic Press. AD - Vienna Biocenter, University of Vienna, Austria. jr@mol.univie.ac.at FAU - Rotheneder, H AU - Rotheneder H FAU - Geymayer, S AU - Geymayer S FAU - Haidweger, E AU - Haidweger E LA - eng PT - Journal Article PL - ENGLAND TA - J Mol Biol JID - 2985088R RN - 0 (Carrier Proteins) RN - 0 (Cell Cycle Proteins) RN - 0 (Cyclin A) RN - 0 (DNA-Binding Proteins) RN - 0 (Dp protein, Drosophila) RN - 0 (Drosophila Proteins) RN - 0 (E2F transcription factors) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Trans-Activators) RN - 0 (Transcription Factor, Sp1) RN - 0 (Transcription Factors) RN - 0 (retinoblastoma binding protein 1) RN - 0 (transcription factor DP1) RN - 9007-49-2 (DNA) RN - EC 2.7.1.21 (Thymidine Kinase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Binding Sites MH - *Carrier Proteins MH - *Cell Cycle Proteins MH - Cell Line MH - Comparative Study MH - Cyclin A/metabolism/pharmacology MH - DNA/genetics/metabolism MH - *DNA-Binding Proteins MH - *Drosophila Proteins MH - Drosophila melanogaster MH - Fibroblasts MH - Mice MH - Molecular Sequence Data MH - *Multigene Family/genetics/physiology MH - Promoter Regions (Genetics)/*genetics MH - Protein Binding/drug effects MH - Recombinant Fusion Proteins/chemistry/metabolism MH - Research Support, Non-U.S. Gov't MH - Response Elements/genetics MH - Sequence Alignment MH - Sequence Deletion MH - Thymidine Kinase/*genetics MH - *Trans-Activation (Genetics) MH - *Trans-Activators MH - Transcription Factor, Sp1/chemistry/genetics/*metabolism MH - Transcription Factors/chemistry/genetics/*metabolism EDAT- 1999/11/05 MHDA- 1999/11/05 00:01 AID - 10.1006/jmbi.1999.3213 [doi] AID - S0022283699932138 [pii] PST - ppublish SO - J Mol Biol 1999 Nov 12;293(5):1005-15. NR -------------------------------------------------------------------------------- 42: Kardassis D et al. c-Jun transactivates the prom...[PMID: 10506225] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 10506225 OWN - NLM STAT- MEDLINE DA - 19991109 DCOM- 19991109 LR - 20041211 PUBM- Print IS - 0021-9258 VI - 274 IP - 41 DP - 1999 Oct 8 TI - c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1. PG - 29572-81 AB - The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. We have recently reported that the transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. In the present study we show that the Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. By using gel electrophoretic mobility shift assays we show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun was unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene was transactivated by overexpressed c-Jun in HepG2 cells. The above data propose functional cooperation between c-Jun and Sp1. Physical interactions between the two factors were demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 was mapped within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 were demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL4-binding sites only when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, we found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, our data support a mechanism of superactivation of Sp1 by c-Jun, which is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters. AD - Department of Basic Sciences, University of Crete Medical School, Institute of Molecular Biology, Foundation of Research and Technology of Hellas, Heraklion GR-71110, Crete, Greece. kardasis@imbb.forth.gr FAU - Kardassis, D AU - Kardassis D FAU - Papakosta, P AU - Papakosta P FAU - Pardali, K AU - Pardali K FAU - Moustakas, A AU - Moustakas A LA - eng PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Cyclins) RN - 0 (DNA-Binding Proteins) RN - 0 (Fungal Proteins) RN - 0 (GAL4 protein, S cerevisiae) RN - 0 (Proto-Oncogene Proteins c-jun) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Trans-Activators) RN - 0 (Transcription Factor, Sp1) RN - 0 (Transcription Factors) RN - 0 (cyclin-dependent kinase Inhibitor p21) SB - IM MH - Animals MH - Base Sequence MH - Cyclins/*genetics MH - DNA-Binding Proteins/metabolism MH - Drosophila/genetics MH - Fungal Proteins/genetics MH - Gene Expression Regulation MH - Genes, Reporter MH - Humans MH - *Promoter Regions (Genetics) MH - Protein Binding MH - Proto-Oncogene Proteins c-jun/*metabolism MH - Recombinant Fusion Proteins MH - Research Support, Non-U.S. Gov't MH - *Saccharomyces cerevisiae Proteins MH - Trans-Activators/*pharmacology MH - Transcription Factor, Sp1/*metabolism MH - Transcription Factors/genetics MH - Tumor Cells, Cultured EDAT- 1999/10/03 MHDA- 1999/10/03 00:01 PST - ppublish SO - J Biol Chem 1999 Oct 8;274(41):29572-81. NR -------------------------------------------------------------------------------- 43: Fabbro D et al. Homogeneous purification of h...[PMID: 10497072] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10497072 OWN - NLM STAT- MEDLINE DA - 19991122 DCOM- 19991122 LR - 20050111 PUBM- Print IS - 1046-5928 VI - 17 IP - 1 DP - 1999 Oct TI - Homogeneous purification of human recombinant GST-Akt/PKB from Sf9 cells. PG - 83-8 AB - The plethora of extracellular stimuli modulating the status of a cell results in the engagement of relatively few pathways responsible for transducing signals to the interior of the cell. One such pathway is the activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositide 3'-phosphate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Akt/protein kinase B (PKB). Here we describe a protocol for the expression and one-step purification of human recombinant GST-PKB in Sf9 cells. This scheme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or structural studies. CI - Copyright 1999 Academic Press. AD - Department of Oncology, Unit Biology Basel, Novartis Pharma AG, Basel, 4057, Switzerland. FAU - Fabbro, D AU - Fabbro D FAU - Batt, D AU - Batt D FAU - Rose, P AU - Rose P FAU - Schacher, B AU - Schacher B FAU - Roberts, T M AU - Roberts TM FAU - Ferrari, S AU - Ferrari S LA - eng PT - Journal Article PL - UNITED STATES TA - Protein Expr Purif JID - 9101496 RN - 0 (Enzyme Inhibitors) RN - 0 (Histones) RN - 0 (Peptides) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 62996-74-1 (Staurosporine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (proto-oncogene proteins c-akt) SB - IM MH - Animals MH - Cell Line MH - Enzyme Inhibitors/pharmacology MH - Enzyme Stability MH - Gene Expression MH - Glutathione Transferase/genetics/isolation & purification/metabolism MH - Histones/metabolism MH - Humans MH - Nucleopolyhedrovirus/genetics MH - Peptides/metabolism MH - Phosphorylation MH - *Protein-Serine-Threonine Kinases MH - Proto-Oncogene Proteins/*genetics/*isolation & purification/metabolism MH - Recombinant Fusion Proteins/genetics/isolation & purification/metabolism MH - Spodoptera MH - Staurosporine/pharmacology MH - Substrate Specificity EDAT- 1999/09/25 MHDA- 1999/09/25 00:01 AID - 10.1006/prep.1999.1102 [doi] AID - S1046592899911026 [pii] PST - ppublish SO - Protein Expr Purif 1999 Oct;17(1):83-8. DR -------------------------------------------------------------------------------- 44: Huang J et al. c-Src association with and ph...[PMID: 10435591] Related Articles, Books, LinkOut PMID- 10435591 OWN - NLM STAT- MEDLINE DA - 19990813 DCOM- 19990813 LR - 20041117 PUBM- Print IS - 0950-9232 VI - 18 IP - 28 DP - 1999 Jul 15 TI - c-Src association with and phosphorylation of p58gag, a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor. PG - 4099-107 AB - The retroviral Gag-like protein p58gag expressed in a highly metastatic ascites rat mammary adenocarcinoma has been implicated in cell surface changes contributing to xenotransplantability. p58gag is present in the cells in a plasma membrane- and microfilament-associated signal transduction particle containing Src and is phosphorylated on tyrosine. Overlay analyses and affinity chromatography with glutathione S-transferase (GST) fusion proteins of Src homology-3 (SH3) domains showed direct binding of the Src but not the Crk SH3 domain to p58gag. This association was confirmed by co-immunoprecipitation of partially purified p58gag from ascites cell lysates with platelet Src. Further, a GST-p58gag fusion protein bound full length c-Src from either platelets or c-Src-expressing insect cells. The GST-p58gag fusion protein, but not GST, was phosphorylated by platelet or insect cell-expressed c-Src, but not by a kinase negative c-Src variant. The binding of GST-p58gag to c-Src was almost completely abolished by a 50-fold excess of the GST-SH3 domain of Src, and a parallel decrease in tyrosine phosphorylation of p58gag was observed. These results demonstrate that p58gag is tyrosine-phosphorylated as a consequence of its specific association with c-Src via its SH3 domain. These observations suggest a mechanism by which Gag proteins may contribute to retroviral maturation or pathogenesis through binding and relocalization of SH3 domain-containing proteins such as Src-like tyrosine kinases to sites of association of microfilaments with the plasma membrane. AD - Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, Florida 33101, USA. FAU - Huang, J AU - Huang J FAU - Zhang, B T AU - Zhang BT FAU - Li, Y AU - Li Y FAU - Mayer, B AU - Mayer B FAU - Carraway, K L AU - Carraway KL FAU - Carraway, C A AU - Carraway CA LA - eng GR - CA 14395/CA/NCI GR - CA72577-01A1/CA/NCI PT - Journal Article PL - ENGLAND TA - Oncogene JID - 8711562 RN - 0 (Membrane Glycoproteins) RN - 0 (Membrane Proteins) RN - 0 (Microfilament Proteins) RN - 0 (Neoplasm Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (proto-oncogene protein c-crk) RN - 156739-74-1 (protein p58) RN - EC 2.7.1.112 (Proto-Oncogene Protein pp60(c-src)) SB - IM MH - Adenocarcinoma/*metabolism MH - Animals MH - Ascites/metabolism MH - Binding, Competitive MH - Blood Platelets/enzymology MH - Female MH - Mammary Neoplasms, Experimental/*metabolism MH - Membrane Glycoproteins/*metabolism MH - Membrane Proteins/*metabolism MH - Microfilament Proteins/*metabolism MH - Models, Biological MH - Neoplasm Proteins/*metabolism MH - Neoplasm Transplantation MH - Phosphorylation MH - Protein Binding MH - *Protein Processing, Post-Translational MH - Proto-Oncogene Protein pp60(c-src)/*metabolism MH - Proto-Oncogene Proteins/metabolism MH - Rats MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Spodoptera/cytology MH - Transplantation, Heterologous MH - Tumor Cells, Cultured MH - src Homology Domains EDAT- 1999/08/06 MHDA- 1999/08/06 00:01 AID - 10.1038/sj.onc.1202779 [doi] PST - ppublish SO - Oncogene 1999 Jul 15;18(28):4099-107. PR -------------------------------------------------------------------------------- 45: Yamagiwa M et al. Activation process of diptera...[PMID: 10427035] Related Articles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 10427035 OWN - NLM STAT- MEDLINE DA - 19990915 DCOM- 19990915 LR - 20041117 PUBM- Print IS - 0099-2240 VI - 65 IP - 8 DP - 1999 Aug TI - Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp. israelensis. PG - 3464-9 AB - Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60. AD - Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. FAU - Yamagiwa, M AU - Yamagiwa M FAU - Esaki, M AU - Esaki M FAU - Otake, K AU - Otake K FAU - Inagaki, M AU - Inagaki M FAU - Komano, T AU - Komano T FAU - Amachi, T AU - Amachi T FAU - Sakai, H AU - Sakai H LA - eng PT - Journal Article PL - UNITED STATES TA - Appl Environ Microbiol JID - 7605801 RN - 0 (Bacterial Proteins) RN - 0 (Bacterial Toxins) RN - 0 (DNA Probes) RN - 0 (Endotoxins) RN - 0 (Insecticides) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 0 (insecticidal crystal protein, Bacillus Thuringiensis) SB - IM MH - Amino Acid Sequence MH - Animals MH - Bacillus thuringiensis/genetics/*metabolism MH - Bacterial Proteins/genetics/*metabolism/toxicity MH - Bacterial Toxins/genetics/metabolism/toxicity MH - Base Sequence MH - Culex/drug effects MH - DNA Probes/genetics MH - Endotoxins/genetics/*metabolism/toxicity MH - Insecticides/metabolism/toxicity MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Plasmids/genetics MH - Protein Processing, Post-Translational MH - Recombinant Fusion Proteins/genetics/metabolism/toxicity MH - Research Support, Non-U.S. Gov't EDAT- 1999/07/31 MHDA- 1999/07/31 00:01 PST - ppublish SO - Appl Environ Microbiol 1999 Aug;65(8):3464-9. NR -------------------------------------------------------------------------------- 46: Maru Y et al. BCR binds to the xeroderma pi...[PMID: 10403766] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 10403766 OWN - NLM STAT- MEDLINE DA - 19990805 DCOM- 19990805 LR - 20050223 PUBM- Print IS - 0006-291X VI - 260 IP - 2 DP - 1999 Jul 5 TI - BCR binds to the xeroderma pigmentosum group B protein. PG - 309-12 AB - The BCR gene is involved in the formation of the BCR-ABL oncogene responsible for the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that P210 BCR-ABL binds to the xeroderma pigmentosum group B protein (XPB) through the portion of BCR that is homologous to the catalytic domain of GDP-GTP exchangers such as yeast CDC24 and Dbl. In the baculovirus overexpression system which facilitates binding of coexpressed proteins, we now show that XPB binds to the intact BCR protein efficiently but not to CDC24 or Dbl, suggesting specificity of this interaction. The binding of endogenous BCR and XPB proteins was also detected in Hela cells, and this was inhibited by a blocking peptide. Full-length (1-782) XPB and its truncated form (203-782), which does not contain the nuclear localization signal, were tagged with glutathione S-transferase (GST) and were expressed in Rat1 fibroblasts. GST-XPB(203-782) was localized predominantly in the cytoplasm and bound to BCR but not to p62, one of the other components in TFIIH. GST-XPB(1-782) was largely in the nucleus and bound to p62 and BCR. Although the biological significance of the binding remains to be uncovered, BCR binds to the XPB/p62 complex. CI - Copyright 1999 Academic Press. AD - Department of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-0071, Japan. ymaru@ims.u-tokyo.ac.jp FAU - Maru, Y AU - Maru Y FAU - Kobayashi, T AU - Kobayashi T FAU - Tanaka, K AU - Tanaka K FAU - Shibuya, M AU - Shibuya M LA - eng PT - Journal Article PL - UNITED STATES TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (DNA-Binding Proteins) RN - 0 (Oncogene Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (proto-oncogene protein c-bcr) RN - 146045-44-5 (XPBC-ERCC-3 protein) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) SB - IM MH - Animals MH - Baculoviridae/genetics MH - DNA-Binding Proteins/*metabolism MH - Glutathione Transferase/genetics MH - Hela Cells MH - Humans MH - Oncogene Proteins/genetics/*metabolism MH - Protein Binding MH - *Protein-Tyrosine Kinase MH - *Proto-Oncogene Proteins MH - Rats MH - Recombinant Fusion Proteins/genetics/metabolism EDAT- 1999/07/15 MHDA- 1999/07/15 00:01 AID - 10.1006/bbrc.1999.0822 [doi] AID - S0006291X99908227 [pii] PST - ppublish SO - Biochem Biophys Res Commun 1999 Jul 5;260(2):309-12. NR -------------------------------------------------------------------------------- 47: Radkov SA et al. Epstein-Barr virus nuclear an...[PMID: 10364319] Related Articles, Compound via MeSH, Substance via MeSH, Free in PMC, Cited in PMC, Books, LinkOut PMID- 10364319 OWN - NLM STAT- MEDLINE DA - 19990723 DCOM- 19990723 LR - 20050315 PUBM- Print IS - 0022-538X VI - 73 IP - 7 DP - 1999 Jul TI - Epstein-Barr virus nuclear antigen 3C interacts with histone deacetylase to repress transcription. PG - 5688-97 AB - EBNA3C can specifically repress the expression of reporter plasmids containing EBV Cp latency-associated promoter elements. Cp is normally the main promoter for EBNA mRNA initiation, so it appears that EBNA3C contributes to a negative autoregulatory control loop. By mutational analysis it was previously established that this repression is consistent with EBNA3C being targeted to Cp by binding the cellular sequence-specific DNA-binding protein CBF1 (also known as recombination signal-binding protein [RBP]-Jkappa. Further analysis suggested that in vivo a corepressor interacts with EBNA3C in this DNA binding complex. Results presented here are all consistent with a component of such a corepressor exhibiting histone deacetylase activity. The drug trichostatin A, which specifically inhibits histone deacetylases, relieved two- to threefold the repression of Cp induced by EBNA3C in two different cell types. Moreover, repression of pTK-CAT-Cp4x by EBNA3C was specifically enhanced by cotransfection of an expression plasmid for human histone deacetylase-1 (HDAC1). Consistent with these functional assays, in vitro-translated HDAC1 bound to a glutathione S-transferase (GST) fusion protein including full-length EBNA3C, and in the reciprocal experiment EBNA3C bound to a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also revealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The region of EBNA3C involved in the interaction with HDAC1 appears to correspond to the region which is necessary for binding to CBF1/RBP-Jkappa. A direct physical interaction between EBNA3C and HDAC1 was demonstrated with recombinant proteins purified from bacterial cells, and we therefore conclude that HDAC1 and CBF1/RBP-Jkappa bind to the same or adjacent regions of EBNA3C. These data suggest that recruitment of histone deacetylase activity makes a significant contribution to the repression of transcription from Cp because EBNA3C bridges an interaction between CBF1/RBP-Jkappa and HDAC1. AD - Section of Virology and Cell Biology, Imperial College of Science, Technology and Medicine, St Mary's Campus, London W2 1PG, United Kingdom. FAU - Radkov, S A AU - Radkov SA FAU - Touitou, R AU - Touitou R FAU - Brehm, A AU - Brehm A FAU - Rowe, M AU - Rowe M FAU - West, M AU - West M FAU - Kouzarides, T AU - Kouzarides T FAU - Allday, M J AU - Allday MJ LA - eng PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (Cell Extracts) RN - 0 (DNA-Binding Proteins) RN - 0 (Enzyme Inhibitors) RN - 0 (Epstein-Barr Virus Nuclear Antigens) RN - 0 (Hydroxamic Acids) RN - 0 (Nuclear Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Repressor Proteins) RN - 143777-43-9 (immunoglobulin J recombination signal sequence binding protein) RN - 58880-19-6 (trichostatin A) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.5.1.- (Histone Deacetylases) SB - IM MH - Animals MH - Binding Sites MH - Cell Extracts MH - Cell Line MH - Cell Nucleus/metabolism MH - DNA-Binding Proteins/genetics MH - Enzyme Inhibitors/pharmacology MH - Epstein-Barr Virus Nuclear Antigens/genetics/*metabolism MH - *Gene Expression Regulation, Viral MH - Glutathione Transferase/genetics/metabolism MH - Hela Cells MH - Herpesvirus 4, Human/*metabolism MH - Histone Deacetylases/antagonists & inhibitors/genetics/*metabolism MH - Humans MH - Hydroxamic Acids/pharmacology MH - Jurkat Cells MH - *Nuclear Proteins MH - Recombinant Fusion Proteins/genetics/metabolism MH - Repressor Proteins/genetics/*metabolism MH - Research Support, Non-U.S. Gov't MH - Spodoptera/cytology MH - Transcription, Genetic MH - Tumor Cells, Cultured EDAT- 1999/06/11 MHDA- 1999/06/11 00:01 PST - ppublish SO - J Virol 1999 Jul;73(7):5688-97. NR -------------------------------------------------------------------------------- 48: Kamiie K et al. Expression of elongation fact...[PMID: 10361679] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 10361679 OWN - NLM STAT- MEDLINE DA - 19990726 DCOM- 19990726 LR - 20041117 PUBM- Print IS - 0916-8451 VI - 63 IP - 4 DP - 1999 Apr TI - Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland. PG - 666-71 AB - Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma. EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved. We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha. AD - Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University, Japan. kamiie@aomori-u.ac.jp FAU - Kamiie, K AU - Kamiie K FAU - Taira, H AU - Taira H FAU - Kobayashi, K AU - Kobayashi K FAU - Yamashita, T AU - Yamashita T FAU - Kidou, S AU - Kidou S FAU - Ejiri, S AU - Ejiri S LA - eng PT - Journal Article PL - JAPAN TA - Biosci Biotechnol Biochem JID - 9205717 RN - 0 (DNA, Complementary) RN - 0 (Guanine Nucleotides) RN - 0 (Peptide Elongation Factor 1) RN - 0 (Peptide Elongation Factors) RN - 0 (Plasmids) SB - IM MH - Amino Acid Sequence MH - Animals MH - Blotting, Northern MH - Bombyx/genetics/*metabolism MH - DNA, Complementary/biosynthesis/genetics MH - Electrophoresis, Polyacrylamide Gel MH - Escherichia coli/genetics/*metabolism MH - Exocrine Glands/*metabolism MH - Guanine Nucleotides/metabolism MH - Molecular Sequence Data MH - Peptide Elongation Factor 1 MH - Peptide Elongation Factors/*biosynthesis/genetics MH - Plasmids/*genetics EDAT- 1999/06/11 MHDA- 1999/06/11 00:01 PST - ppublish SO - Biosci Biotechnol Biochem 1999 Apr;63(4):666-71. NR -------------------------------------------------------------------------------- 49: Fujii GH et al. Transcriptional analysis of t...[PMID: 10208437] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 10208437 OWN - NLM STAT- MEDLINE DA - 19990601 DCOM- 19990601 LR - 20050111 PUBM- Print IS - 0950-9232 VI - 18 IP - 9 DP - 1999 Mar 4 TI - Transcriptional analysis of the PTEN/MMAC1 pseudogene, psiPTEN. PG - 1765-9 AB - PTEN/MMAC1 is a recently characterized tumor suppressor. A pseudogene derived from the human PTEN/MMAC1 phosphatase, psiPTEN, has been reported. Recent analysis of the pseudogene revealed conflicting results about the expression of psiPTEN. In this study, we show that the PTEN/MMAC1 pseudogene is actively transcribed in all cells and tissues examined. In some cases, pseudogene transcripts were found to represent as much as 70% of the total PTEN/MMAC1 RNA. As psiPTEN is transcribed, there is a potential for misinterpretation of PTEN/MMAC1 mutations when RT-PCR techniques are used, as well as potential for a psiPTEN-encoded translation product. Although we were unable to detect a pseudogene protein product in the cell lines examined, a baculovirus produced GST pseudogene fusion protein exhibited phosphatase activity comparable to wild type. The results of this study, taken together, indicate the potential complication of PTEN/MMAC1 molecular analysis caused by the expression of psiPTEN. AD - Department of Cellular Signaling, DNAX Research Institute, Palo Alto, California 94304, USA. FAU - Fujii, G H AU - Fujii GH FAU - Morimoto, A M AU - Morimoto AM FAU - Berson, A E AU - Berson AE FAU - Bolen, J B AU - Bolen JB LA - eng SI - GENBANK/AF023139 SI - GENBANK/U92436 PT - Journal Article PL - ENGLAND TA - Oncogene JID - 8711562 RN - 0 (DNA, Complementary) RN - 0 (Tumor Suppressor Proteins) RN - EC 3.1.3 (Phosphoric Monoester Hydrolases) RN - EC 3.1.3.48 (PTEN protein) SB - IM MH - Amino Acid Sequence MH - Base Sequence MH - DNA, Complementary MH - Gene Expression MH - *Genes, Tumor Suppressor MH - Humans MH - Molecular Sequence Data MH - Phosphoric Monoester Hydrolases/*genetics/metabolism MH - *Pseudogenes MH - Research Support, Non-U.S. Gov't MH - *Transcription, Genetic MH - Tumor Cells, Cultured MH - *Tumor Suppressor Proteins EDAT- 1999/04/20 MHDA- 1999/04/20 00:01 AID - 10.1038/sj.onc.1202492 [doi] PST - ppublish SO - Oncogene 1999 Mar 4;18(9):1765-9. DR -------------------------------------------------------------------------------- 50: Zhu L et al. Comparison of human sera reac...[PMID: 10191203] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 10191203 OWN - NLM STAT- MEDLINE DA - 19990601 DCOM- 19990601 LR - 20041117 PUBM- Print IS - 0042-6822 VI - 256 IP - 2 DP - 1999 Apr 10 TI - Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells. PG - 381-92 AB - The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies. CI - Copyright 1999 Academic Press. AD - Division of Infectious Diseases, The University of Kansas Medical Center, Kansas City, Kansas, 66160, USA. FAU - Zhu, L AU - Zhu L FAU - Wang, R AU - Wang R FAU - Sweat, A AU - Sweat A FAU - Goldstein, E AU - Goldstein E FAU - Horvat, R AU - Horvat R FAU - Chandran, B AU - Chandran B LA - eng GR - CA75911/CA/NCI GR - CA82056/CA/NCI GR - R18906/PHS PT - Journal Article PL - UNITED STATES TA - Virology JID - 0110674 RN - 0 (Antibodies, Viral) RN - 0 (Antigens, Viral) RN - 0 (Glycoproteins) RN - 0 (K8.1 protein, Human herpesvirus 8) RN - 0 (Nuclear Proteins) RN - 0 (ORF65 protein, human herpesvirus 8) RN - 0 (Phosphoproteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) RN - 0 (latent nuclear antigen (LNA)) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM SB - X MH - Adolescent MH - Adult MH - Antibodies, Viral/blood/*immunology MH - Antigens, Viral/genetics/*immunology MH - Blotting, Western MH - Cell Line MH - Comparative Study MH - Fluorescent Antibody Technique, Indirect MH - Gene Expression MH - Glutathione Transferase/genetics/immunology MH - Glycoproteins/genetics/*immunology MH - HIV Infections/immunology MH - Herpesvirus 8, Human/genetics/*immunology MH - Humans MH - Immunoblotting MH - Male MH - Middle Aged MH - Nuclear Proteins/genetics/*immunology MH - Open Reading Frames MH - *Phosphoproteins MH - Recombinant Fusion Proteins/genetics/immunology MH - Recombination, Genetic MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sarcoma, Kaposi/blood/*immunology/virology MH - Viral Proteins/genetics/*immunology MH - Virus Latency MH - Virus Replication EDAT- 1999/04/07 MHDA- 1999/04/07 00:01 AID - S0042682299996745 [pii] PST - ppublish SO - Virology 1999 Apr 10;256(2):381-92. DR -------------------------------------------------------------------------------- 51: Kefalas P et al. Chemotaxin-dependent transloc...[PMID: 10092875] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 10092875 OWN - NLM STAT- MEDLINE DA - 19990422 DCOM- 19990422 LR - 20041117 PUBM- Print IS - 0014-2956 VI - 259 IP - 3 DP - 1999 Feb TI - Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs. PG - 866-71 AB - mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane. AD - Section on Clinical Pharmacology, Imperial College School of Medicine, London, UK. FAU - Kefalas, P AU - Kefalas P FAU - Saxty, B AU - Saxty B FAU - Yadollahi-Farsani, M AU - Yadollahi-Farsani M FAU - MacDermot, J AU - MacDermot J LA - eng PT - Journal Article PL - GERMANY TA - Eur J Biochem JID - 0107600 RN - 0 (Chemotactic Factors) RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) RN - 0 (Recombinant Fusion Proteins) RN - 59880-97-6 (N-Formylmethionine Leucyl-Phenylalanine) RN - EC 2.4.2.- (ADP Ribose Transferases) RN - EC 3.1.4.3 (Phospholipase C) RN - EC 4.6.1.13 (Phosphatidylinositol Diacylglycerol-Lyase) SB - IM MH - ADP Ribose Transferases/immunology/*metabolism MH - Animals MH - Cell Membrane/enzymology MH - Chemotactic Factors/*pharmacology MH - DNA, Complementary/genetics MH - Fluorescence MH - Humans MH - Muscle, Skeletal/enzymology MH - N-Formylmethionine Leucyl-Phenylalanine/pharmacology MH - Neutrophils/*enzymology MH - Phosphatidylinositol Diacylglycerol-Lyase MH - Phospholipase C/metabolism MH - RNA, Messenger/metabolism MH - Recombinant Fusion Proteins/genetics MH - Research Support, Non-U.S. Gov't MH - Spodoptera/genetics EDAT- 1999/03/27 MHDA- 1999/03/27 00:01 PST - ppublish SO - Eur J Biochem 1999 Feb;259(3):866-71. DR -------------------------------------------------------------------------------- 52: Harvey TJ et al. A cellular protein which bind...[PMID: 10091999] Related Articles, Books, LinkOut PMID- 10091999 OWN - NLM STAT- MEDLINE DA - 19990415 DCOM- 19990415 LR - 20041117 PUBM- Print IS - 0022-1317 VI - 80 ( Pt 3) DP - 1999 Mar TI - A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen. PG - 607-15 AB - The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner. AD - Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Queensland, Australia. FAU - Harvey, T J AU - Harvey TJ FAU - Macnaughton, T B AU - Macnaughton TB FAU - Park, D S AU - Park DS FAU - Gowans, E J AU - Gowans EJ LA - eng PT - Journal Article PL - ENGLAND TA - J Gen Virol JID - 0077340 RN - 0 (Hepatitis B Surface Antigens) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Baculoviridae/genetics MH - Binding, Competitive MH - False Positive Reactions MH - Gene Library MH - Hepatitis B Surface Antigens/biosynthesis/genetics/isolation & purification/*metabolism MH - Hepatitis B virus/isolation & purification/*metabolism MH - Humans MH - Liver MH - Molecular Weight MH - Polymerase Chain Reaction MH - Protein Binding MH - Protein Biosynthesis MH - Proteins/genetics/isolation & purification/*metabolism MH - Recombinant Fusion Proteins/biosynthesis/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Saccharomyces cerevisiae/genetics MH - Sequence Analysis, DNA MH - Sequence Homology EDAT- 1999/03/26 MHDA- 1999/03/26 00:01 PST - ppublish SO - J Gen Virol 1999 Mar;80 ( Pt 3):607-15. DR -------------------------------------------------------------------------------- 53: McDonald OB et al. A scintillation proximity ass...[PMID: 10075822] Related Articles, Books, LinkOut PMID- 10075822 OWN - NLM STAT- MEDLINE DA - 19990503 DCOM- 19990503 LR - 20041118 PUBM- Print IS - 0003-2697 VI - 268 IP - 2 DP - 1999 Mar 15 TI - A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors. PG - 318-29 AB - We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein. CI - Copyright 1999 Academic Press. AD - Division of Biochemistry, GlaxoWellcome Inc., Research Triangle Park, North Carolina, 27709-13398, USA. FAU - McDonald, O B AU - McDonald OB FAU - Chen, W J AU - Chen WJ FAU - Ellis, B AU - Ellis B FAU - Hoffman, C AU - Hoffman C FAU - Overton, L AU - Overton L FAU - Rink, M AU - Rink M FAU - Smith, A AU - Smith A FAU - Marshall, C J AU - Marshall CJ FAU - Wood, E R AU - Wood ER LA - eng PT - Journal Article PL - UNITED STATES TA - Anal Biochem JID - 0370535 RN - 0 (DNA Primers) RN - 0 (Enzyme Inhibitors) RN - 0 (Peptides) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.7.1.- (MAP Kinase Kinase 1) RN - EC 2.7.1.- (MAP2K1 protein, human) RN - EC 2.7.1.- (Mitogen-Activated Protein Kinase Kinases) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinase 1) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (Proto-Oncogene Proteins c-raf) SB - IM MH - Amino Acid Sequence MH - Animals MH - Baculoviridae/genetics MH - Base Sequence MH - DNA Primers/genetics MH - Enzyme Inhibitors/pharmacology MH - Escherichia coli/genetics MH - Humans MH - In Vitro MH - Kinetics MH - MAP Kinase Kinase 1 MH - Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/genetics/*metabolism MH - *Mitogen-Activated Protein Kinase Kinases MH - Peptides/chemistry/metabolism MH - Protein-Serine-Threonine Kinases/antagonists & inhibitors/genetics/*metabolism MH - Protein-Tyrosine Kinase/antagonists & inhibitors/genetics/*metabolism MH - Proto-Oncogene Proteins c-raf/antagonists & inhibitors/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Scintillation Counting/*methods MH - Signal Transduction MH - Substrate Specificity EDAT- 1999/03/17 MHDA- 1999/03/17 00:01 AID - S0003269798930305 [pii] PST - ppublish SO - Anal Biochem 1999 Mar 15;268(2):318-29. NR -------------------------------------------------------------------------------- 54: Bidwai AP et al. A gene located at 56F1-2 in D...[PMID: 10329473] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Protein, GEO Profiles, Books, LinkOut PMID- 10329473 OWN - NLM STAT- MEDLINE DA - 20000921 DCOM- 20000921 LR - 20041117 PUBM- Print IS - 1522-4724 VI - 1 IP - 1 DP - 1999 Apr TI - A gene located at 56F1-2 in Drosophila melanogaster encodes a novel metazoan beta-like subunit of casein kinase II. PG - 21-8 AB - Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic alpha and regulatory beta subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a Drosophila embryo cDNA library for proteins that interact with DmCKII alpha. One of the cDNAs encodes a novel beta-like polypeptide, which we designate beta'. In situ hybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the beta gene and the Stellate family of beta-like sequences. The predicted sequence of beta' is more closely related to the beta subunit of Drosophila and other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit. In vitro reconstitution studies show that a GST-beta' fusion protein associates with the alpha subunit to generate a tetrameric complex with regulatory properties similar to those of the native alpha2beta2 holoenzyme. The data are consistent with the proposed role of the beta' subunit as an integral component of the holoenzyme. AD - Department of Biology, West Virginia University, Morgantown 26506-6057, USA. Abidwai@wvu.edu FAU - Bidwai, A P AU - Bidwai AP FAU - Zhao, W AU - Zhao W FAU - Glover, C V AU - Glover CV LA - eng GR - GM33237/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - Mol Cell Biol Res Commun JID - 100889076 RN - 0 (DNA, Complementary) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.7.1.37 (Casein Kinase II) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Animals MH - Casein Kinase II MH - DNA, Complementary/genetics/isolation & purification MH - Drosophila melanogaster/*enzymology/*genetics MH - *Genes, Insect MH - In Situ Hybridization MH - Molecular Sequence Data MH - Phylogeny MH - Protein Structure, Quaternary MH - Protein-Serine-Threonine Kinases/chemistry/*genetics/metabolism MH - Recombinant Fusion Proteins/chemistry/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Two-Hybrid System Techniques EDAT- 1999/03/08 MHDA- 2000/09/23 11:01 AID - 10.1006/mcbr.1999.0103 [doi] AID - S1522472499901034 [pii] PST - ppublish SO - Mol Cell Biol Res Commun 1999 Apr;1(1):21-8. NR -------------------------------------------------------------------------------- 55: Sheffield P et al. Overcoming expression and pur...[PMID: 10024467] Related Articles, Compound via MeSH, Substance via MeSH, Nucleotide, Protein, Cited in PMC, Books, LinkOut PMID- 10024467 OWN - NLM STAT- MEDLINE DA - 19990318 DCOM- 19990318 LR - 20041117 PUBM- Print IS - 1046-5928 VI - 15 IP - 1 DP - 1999 Feb TI - Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors. PG - 34-9 AB - We describe the construction of expression vectors based on three of the most frequently used gene fusion affinity tags [glutathione S-transferase (GST), maltose binding protein (MBP), and the His6 peptide]. The polylinkers of pGEX4T1, pMal-c2, and a pET vector were replaced with the polylinker isolated from the baculovirus expression plasmid pFastBac. Once appropriate restriction sites have been introduced into a gene, it can be fused to all three affinity tags with little effort, allowing expression-screening experiments to be performed efficiently. We discuss the development and use of these vectors with respect to overcoming purification problems encountered for the RhoA GDP/GTP nucleotide dissociation inhibitor (RhoGDI) and their advantages over commercially available expression vectors. CI - Copyright 1999 Academic Press. AD - Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, 22908, USA. pjs4n@virginia.edu FAU - Sheffield, P AU - Sheffield P FAU - Garrard, S AU - Garrard S FAU - Derewenda, Z AU - Derewenda Z LA - eng SI - GENBANK/AF097413 GR - P01 HL48807-06A1/HL/NHLBI PT - Journal Article PL - UNITED STATES TA - Protein Expr Purif JID - 9101496 RN - 0 (ATP-Binding Cassette Transporters) RN - 0 (Carrier Proteins) RN - 0 (Escherichia coli Proteins) RN - 0 (Genetic Vectors) RN - 0 (Guanine Nucleotide Dissociation Inhibitors) RN - 0 (Monosaccharide Transport Proteins) RN - 0 (Peptides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Recombinant Proteins) RN - 0 (maltose transport system, E coli) RN - 0 (maltose-binding protein) RN - 133312-85-3 (rhoB p20 GDI) RN - 71-00-1 (Histidine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.- (GTP-Binding Proteins) SB - IM MH - *ATP-Binding Cassette Transporters MH - Amino Acid Sequence MH - Baculoviridae MH - Base Sequence MH - Carrier Proteins MH - Chromatography, Affinity MH - Cloning, Molecular/methods MH - Electrophoresis, Polyacrylamide Gel MH - Escherichia coli MH - *Escherichia coli Proteins MH - GTP-Binding Proteins/biosynthesis/*genetics/*isolation & purification MH - *Genetic Vectors MH - Glutathione Transferase MH - *Guanine Nucleotide Dissociation Inhibitors MH - Histidine MH - Humans MH - Molecular Sequence Data MH - *Monosaccharide Transport Proteins MH - Mutagenesis, Site-Directed MH - Peptides MH - Point Mutation MH - Recombinant Fusion Proteins/biosynthesis/isolation & purification MH - Recombinant Proteins/biosynthesis/isolation & purification MH - Research Support, U.S. Gov't, P.H.S. MH - Restriction Mapping EDAT- 1999/02/20 MHDA- 1999/02/20 00:01 AID - S1046592898910038 [pii] PST - ppublish SO - Protein Expr Purif 1999 Feb;15(1):34-9. DR -------------------------------------------------------------------------------- 56: Adnane J et al. Cyclin D1 associates with the...[PMID: 9926939] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 9926939 OWN - NLM STAT- MEDLINE DA - 19990226 DCOM- 19990226 LR - 20050204 PUBM- Print IS - 0950-9232 VI - 18 IP - 1 DP - 1999 Jan 7 TI - Cyclin D1 associates with the TBP-associated factor TAF(II)250 to regulate Sp1-mediated transcription. PG - 239-47 AB - We have previously shown that Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. Here we investigated the mechanism(s) through which cyclin D1 represses Sp1. We examined the ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s). The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. We further demonstrate that TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and that recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduced the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) was sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250. AD - Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA. FAU - Adnane, J AU - Adnane J FAU - Shao, Z AU - Shao Z FAU - Robbins, P D AU - Robbins PD LA - eng GR - CA55227/CA/NCI PT - Journal Article PL - ENGLAND TA - Oncogene JID - 8711562 RN - 0 (DNA-Binding Proteins) RN - 0 (Nuclear Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Retinoblastoma Protein) RN - 0 (TATA-Binding Protein Associated Factors) RN - 0 (TATA-Box Binding Protein) RN - 0 (Transcription Factor TFIID) RN - 0 (Transcription Factor, Sp1) RN - 0 (Transcription Factors) RN - 136601-57-5 (Cyclin D1) RN - 138391-29-4 (TATA-binding protein associated factor 250 kDa) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (cyclin-dependent kinase 4) SB - IM MH - 3T3 Cells MH - Animals MH - Cell Line MH - Cyclin D1/genetics/*metabolism MH - Cyclin-Dependent Kinases/genetics/metabolism MH - *DNA-Binding Proteins/genetics/*metabolism MH - *Gene Expression Regulation MH - Humans MH - Mice MH - Mutagenesis MH - Nuclear Proteins/genetics/*metabolism MH - *Proto-Oncogene Proteins MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Retinoblastoma Protein/genetics/metabolism MH - Spodoptera MH - *TATA Box MH - *TATA-Binding Protein Associated Factors MH - TATA-Box Binding Protein MH - *Transcription Factor TFIID MH - Transcription Factor, Sp1/genetics/*metabolism MH - *Transcription Factors MH - Transcription, Genetic EDAT- 1999/02/02 MHDA- 1999/02/02 00:01 AID - 10.1038/sj.onc.1202297 [doi] PST - ppublish SO - Oncogene 1999 Jan 7;18(1):239-47. PR -------------------------------------------------------------------------------- 57: Shi B et al. Identification and characteri...[PMID: 9920818] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut PMID- 9920818 OWN - NLM STAT- MEDLINE DA - 19990301 DCOM- 19990301 LR - 20041117 PUBM- Print IS - 0006-291X VI - 254 IP - 3 DP - 1999 Jan 27 TI - Identification and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains. PG - 779-85 AB - The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown. CI - Copyright 1999 Academic Press. AD - OSI Pharmaceuticals, Inc., 106 Charles Lindbergh Boulevard, Uniondale, New York, 11553, USA. FAU - Shi, B AU - Shi B FAU - Triebe, D AU - Triebe D FAU - Kajiji, S AU - Kajiji S FAU - Iwata, K K AU - Iwata KK FAU - Bruskin, A AU - Bruskin A FAU - Mahajna, J AU - Mahajna J LA - eng SI - GENBANK/AF007826 PT - Journal Article PL - UNITED STATES TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (Bax epsilon protein, human) RN - 0 (DNA, Complementary) RN - 0 (Membrane Proteins) RN - 0 (Proteins) RN - 0 (Recombinant Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Apoptosis MH - Base Sequence MH - DNA, Complementary MH - Humans MH - Membrane Proteins/chemistry/*genetics MH - Mice MH - Molecular Sequence Data MH - Proteins/chemistry/*genetics MH - RNA Splicing MH - Recombinant Proteins/chemistry/genetics MH - Reverse Transcriptase Polymerase Chain Reaction EDAT- 1999/01/28 MHDA- 1999/01/28 00:01 AID - S0006291X98901309 [pii] PST - ppublish SO - Biochem Biophys Res Commun 1999 Jan 27;254(3):779-85. NR -------------------------------------------------------------------------------- 58: Ye M et al. Varicella-zoster virus Fc rec...[PMID: 9882337] Related Articles, Compound via MeSH, Substance via MeSH, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9882337 OWN - NLM STAT- MEDLINE DA - 19990218 DCOM- 19990218 LR - 20050204 PUBM- Print IS - 0022-538X VI - 73 IP - 2 DP - 1999 Feb TI - Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase. PG - 1320-30 AB - Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II. AD - Departments of Microbiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. FAU - Ye, M AU - Ye M FAU - Duus, K M AU - Duus KM FAU - Peng, J AU - Peng J FAU - Price, D H AU - Price DH FAU - Grose, C AU - Grose C LA - eng GR - AI22795/AI/NIAID GR - AI36884/AI/NIAID GR - GM35500/GM/NIGMS GR - etc. PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (Enzyme Inhibitors) RN - 0 (Purines) RN - 0 (Receptors, Fc) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein IV, varicella-zoster virus) RN - 0 (roscovitine) RN - 147-85-3 (Proline) RN - 56-45-1 (Serine) RN - 72-19-5 (Threonine) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (Positive Transcriptional Elongation Factor B) RN - EC 2.7.1.- (cyclin-dependent kinase 2) RN - EC 2.7.1.37 (CDC2 Protein Kinase) RN - EC 2.7.1.37 (CDC2-CDC28 Kinases) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Animals MH - CDC2 Protein Kinase/metabolism MH - *CDC2-CDC28 Kinases MH - Cell Line, Transformed MH - Cyclin-Dependent Kinases/metabolism MH - Drosophila/enzymology MH - Enzyme Inhibitors/pharmacology MH - Glutathione Transferase/metabolism MH - Hela Cells MH - Herpesvirus 3, Human/*metabolism MH - Humans MH - Jurkat Cells MH - Molecular Sequence Data MH - Phosphorylation MH - Positive Transcriptional Elongation Factor B MH - Proline/metabolism MH - Protein-Serine-Threonine Kinases/*metabolism MH - Purines/pharmacology MH - Receptors, Fc/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Serine/metabolism MH - Threonine MH - Transfection MH - Tumor Cells, Cultured MH - Viral Envelope Proteins/genetics/*metabolism EDAT- 1999/01/09 MHDA- 1999/01/09 00:01 PST - ppublish SO - J Virol 1999 Feb;73(2):1320-30. NR -------------------------------------------------------------------------------- 59: Uno T et al. Molecular cloning of cDNA for...[PMID: 9836423] Related Articles, Compound via MeSH, Substance via MeSH, Protein, Books, LinkOut PMID- 9836423 OWN - NLM STAT- MEDLINE DA - 19990107 DCOM- 19990107 LR - 20041117 PUBM- Print IS - 0916-8451 VI - 62 IP - 10 DP - 1998 Oct TI - Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli. PG - 1885-91 AB - From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP. AD - Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Hyogo, Japan. FAU - Uno, T AU - Uno T FAU - Ueno, M AU - Ueno M FAU - Nakajima, A AU - Nakajima A FAU - Shirai, Y AU - Shirai Y FAU - Aizono, Y AU - Aizono Y LA - eng PT - Journal Article PL - JAPAN TA - Biosci Biotechnol Biochem JID - 9205717 RN - 0 (BRab protein, Bombyx mori) RN - 0 (DNA, Complementary) RN - 0 (Insect Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 146-91-8 (Guanosine Diphosphate) RN - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate)) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.- (GTP-Binding Proteins) RN - EC 3.6.1.- (rab GTP-Binding Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Bombyx/*genetics MH - Brain Chemistry/*genetics MH - Cloning, Molecular MH - DNA, Complementary/analysis MH - Escherichia coli/metabolism MH - GTP-Binding Proteins/*genetics/metabolism MH - Glutathione Transferase/genetics MH - Guanosine 5'-O-(3-Thiotriphosphate)/metabolism MH - Guanosine Diphosphate/metabolism MH - *Insect Proteins MH - Molecular Sequence Data MH - Recombinant Fusion Proteins MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis, DNA MH - *rab GTP-Binding Proteins EDAT- 1998/12/04 MHDA- 1998/12/04 00:01 PST - ppublish SO - Biosci Biotechnol Biochem 1998 Oct;62(10):1885-91. NR -------------------------------------------------------------------------------- 60: Mouw M et al. Amino acids 16-275 of minute ...[PMID: 9813208] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9813208 OWN - NLM STAT- MEDLINE DA - 19981217 DCOM- 19981217 LR - 20041117 PUBM- Print IS - 0042-6822 VI - 251 IP - 1 DP - 1998 Nov 10 TI - Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA. PG - 123-31 AB - GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. CI - Copyright 1998 Academic Press. AD - School of Medicine, University of Missouri, Columbia, Missouri, 65212, USA. FAU - Mouw, M AU - Mouw M FAU - Pintel, D J AU - Pintel DJ LA - eng GR - RO1-AI23102/AI/NIAID PT - Journal Article PL - UNITED STATES TA - Virology JID - 0110674 RN - 0 (Amino Acids) RN - 0 (DNA, Single-Stranded) RN - 0 (DNA-Binding Proteins) RN - 0 (NS1 protein, minute virus of mice) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Nonstructural Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - 63231-63-0 (RNA) RN - 9007-49-2 (DNA) SB - IM MH - Adenosine Triphosphate/metabolism MH - Amino Acids/metabolism MH - Animals MH - Baculoviridae/genetics MH - Base Sequence MH - Binding Sites MH - DNA/*metabolism MH - DNA, Single-Stranded/metabolism MH - DNA-Binding Proteins/genetics/*metabolism MH - Escherichia coli/genetics MH - Mice MH - *Mice Minute Virus MH - Osmolar Concentration MH - Protein Conformation MH - RNA/metabolism MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Deletion MH - Thermodynamics MH - Viral Nonstructural Proteins/chemistry/genetics/*metabolism EDAT- 1998/11/14 MHDA- 1998/11/14 00:01 AID - S0042682298993758 [pii] PST - ppublish SO - Virology 1998 Nov 10;251(1):123-31. DR -------------------------------------------------------------------------------- 61: York JL et al. Reduction of MTT by glutathio...[PMID: 9793644] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 9793644 OWN - NLM STAT- MEDLINE DA - 19981230 DCOM- 19981230 LR - 20041117 PUBM- Print IS - 0736-6205 VI - 25 IP - 4 DP - 1998 Oct TI - Reduction of MTT by glutathione S-transferase. PG - 622-4, 626-8 AB - The reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a blue formazan product is widely used for assaying cell survival and proliferation. The reduction reaction is catalyzed by dehydrogenases localized in the mitochondria of viable cells. As part of an analysis of the ability of glutathione S-transferase (GST) enzymes to protect cells from electrophilic compounds, we found extremely high background levels of the formazan product produced by cells that overexpressed the mouse GST P1-1 enzyme. Further analysis with purified GST enzymes confirmed the ability of these enzymes to reduce MTT in vitro. These data suggest that cytotoxicity assays using MTT should be interpreted with caution, especially when studying the effects of compounds that can influence GST expression. AD - University of Arkansas for Medical Sciences, Little Rock 72205, USA. FAU - York, J L AU - York JL FAU - Maddox, L C AU - Maddox LC FAU - Zimniak, P AU - Zimniak P FAU - McHugh, T E AU - McHugh TE FAU - Grant, D F AU - Grant DF LA - eng GR - R01 GM 56708/GM/NIGMS PT - Technical Report PL - UNITED STATES TA - Biotechniques JID - 8306785 RN - 0 (1-chloro-2,4-dinitrobenzene-glutathione conjugate) RN - 0 (Formazans) RN - 0 (Isoenzymes) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Tetrazolium Salts) RN - 0 (Thiazoles) RN - 23305-68-2 (MTT formazan) RN - 298-93-1 (thiazolyl blue) RN - 70-18-8 (Glutathione) RN - 97-00-7 (Dinitrochlorobenzene) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Animals MH - Baculoviridae/genetics MH - Cell Count MH - Cell Line MH - Cell Survival MH - Dinitrochlorobenzene/analysis/metabolism MH - False Positive Reactions MH - Formazans/*analysis MH - Gene Transfer Techniques MH - Glutathione/analysis/metabolism MH - Glutathione Transferase/genetics/*metabolism MH - Insects MH - Isoenzymes/metabolism MH - Kinetics MH - Oxidation-Reduction MH - Recombinant Fusion Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Tetrazolium Salts/*analysis/*metabolism MH - Thiazoles/*metabolism EDAT- 1998/10/30 MHDA- 1998/10/30 00:01 PST - ppublish SO - Biotechniques 1998 Oct;25(4):622-4, 626-8. NR -------------------------------------------------------------------------------- 62: VanRenterghem B et al. Interaction of insulin recept...[PMID: 9792713] Related Articles, Gene, HomoloGene, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 9792713 OWN - NLM STAT- MEDLINE DA - 19981210 DCOM- 19981210 LR - 20041118 PUBM- Print IS - 0021-9258 VI - 273 IP - 45 DP - 1998 Nov 6 TI - Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes. PG - 29942-9 AB - Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1. AD - Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA. FAU - VanRenterghem, B AU - VanRenterghem B FAU - Morin, M AU - Morin M FAU - Czech, M P AU - Czech MP FAU - Heller-Harrison, R A AU - Heller-Harrison RA LA - eng SI - GENBANK/AF084575 PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (AP3S1 protein, human) RN - 0 (Adaptor Protein Complex 3) RN - 0 (Ap3s1 protein, mouse) RN - 0 (Carrier Proteins) RN - 0 (Nerve Tissue Proteins) RN - 0 (Phosphoproteins) RN - 0 (Recombinant Proteins) RN - 0 (insulin receptor substrate-1 protein) RN - 11061-68-0 (Insulin) SB - IM MH - 3T3 Cells MH - *Adaptor Protein Complex 3 MH - Adipocytes/*metabolism MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Binding Sites MH - Carrier Proteins/genetics/*metabolism MH - Cells, Cultured MH - Humans MH - Insulin/metabolism MH - Mice MH - Molecular Sequence Data MH - *Nerve Tissue Proteins MH - Phosphoproteins/*metabolism MH - Rats MH - Rats, Inbred F344 MH - Recombinant Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Signal Transduction EDAT- 1998/10/29 MHDA- 1998/10/29 00:01 PST - ppublish SO - J Biol Chem 1998 Nov 6;273(45):29942-9. PR -------------------------------------------------------------------------------- 63: Niedziela-Majka A et al. GST-Induced dimerization of D...[PMID: 9790883] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9790883 OWN - NLM STAT- MEDLINE DA - 19981204 DCOM- 19981204 LR - 20041117 PUBM- Print IS - 1046-5928 VI - 14 IP - 2 DP - 1998 Nov TI - GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA. PG - 208-20 AB - The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the induction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfamily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp), heterodimerize to form its functional receptor. To study the receptor-DNA interaction, critical for regulating 20E-dependent gene expression, it is necessary to produce large quantities of EcR and Usp DNA-binding domains. Toward this end DNA-binding domains of EcR and Usp (EcRDBD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs were found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released from GST-chimeric proteins by thrombin cleavage and then purified by glutathione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response element from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formation of the heterodimeric EcRDBD-UspDBD complex was observed to be synergistic when equimolar mixture of both DBDs was incubated with hsp 27pal. Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than GST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the first report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, our results add to the recent findings which indicate the need for caution in interpreting the activities of GST fusion proteins. CI - Copyright 1998 Academic Press. AD - Biochemistry and Biotechnology, Division of Biochemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, Wroclaw, 50-370, Poland. FAU - Niedziela-Majka, A AU - Niedziela-Majka A FAU - Rymarczyk, G AU - Rymarczyk G FAU - Kochman, M AU - Kochman M FAU - Ozyhar, A AU - Ozyhar A LA - eng PT - Journal Article PL - UNITED STATES TA - Protein Expr Purif JID - 9101496 RN - 0 (CF1 protein, insect) RN - 0 (DNA-Binding Proteins) RN - 0 (Heat-Shock Proteins) RN - 0 (Insect Proteins) RN - 0 (Oligodeoxyribonucleotides) RN - 0 (Receptors, Cytoplasmic and Nuclear) RN - 0 (Receptors, Steroid) RN - 0 (Recombinant Proteins) RN - 0 (Transcription Factors) RN - 0 (ecdysteroid receptor) RN - 5289-74-7 (Ecdysterone) SB - IM MH - Animals MH - Binding Sites/genetics MH - DNA-Binding Proteins/*chemistry/metabolism MH - Drosophila melanogaster/physiology MH - Ecdysterone/*physiology MH - Escherichia coli MH - Genes, Insect/genetics MH - Heat-Shock Proteins/*chemistry MH - Insect Proteins/chemistry MH - Oligodeoxyribonucleotides/metabolism MH - Receptors, Cytoplasmic and Nuclear/chemistry MH - Receptors, Steroid/*chemistry MH - Recombinant Proteins/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Transcription Factors/metabolism EDAT- 1998/10/29 MHDA- 1998/10/29 00:01 AID - S1046592898909329 [pii] PST - ppublish SO - Protein Expr Purif 1998 Nov;14(2):208-20. NR -------------------------------------------------------------------------------- 64: Ciccaglione AR et al. Secretion and purification of...[PMID: 9725668] Related Articles, Books, LinkOut PMID- 9725668 OWN - NLM STAT- MEDLINE DA - 19981125 DCOM- 19981125 LR - 20041117 PUBM- Print IS - 0168-1702 VI - 55 IP - 2 DP - 1998 Jun TI - Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system. PG - 157-65 AB - We have expressed the E1 protein of Hepatitis C Virus (HCV) in a new recombinant form by using a baculovirus transfer vector directing the expression of proteins fused to the carboxy-terminus of glutathione-S-transferase (GST). The E1 domain was expressed varying at its carboxy terminus in order to retain (GST-E1) or delete (GST-E1b) the C-terminal hydrophobic region that may be involved in membrane association. Following infection with the recombinant virus, GST-E1b was efficiently secreted into the culture media and could be purified in a single step with the minimum of denaturation by glutathione affinity chromatography. The purified product was specifically immunoprecipitated by HCV positive human sera suggesting the maintenance of an immuno-relevant tertiary structure despite removal of the hydrophobic anchor. By contrast, cells infected with a recombinant baculovirus expressing GST-E1 gave a fusion protein with an appropriate molecular weight but also a series of polypeptides of lower molecular weight consistent with cleavage at the C-terminus of E1. GST-E1 was not secreted into the medium and was associated predominantly with the membrane fraction following cell disruption; the lower molecular weight forms were soluble and secreted. AD - Laboratory of Virology, Istituto Superiore di Sanita, Rome, Italy. equestre@virus1.net.iss.it FAU - Ciccaglione, A R AU - Ciccaglione AR FAU - Marcantonio, C AU - Marcantonio C FAU - Equestre, M AU - Equestre M FAU - Jones, I M AU - Jones IM FAU - Rapicetta, M AU - Rapicetta M LA - eng PT - Journal Article PL - NETHERLANDS TA - Virus Res JID - 8410979 RN - 0 (Culture Media) RN - 0 (E1 protein, hepatitis C virus) RN - 0 (Genetic Vectors) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Envelope Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.2.1.- (Hexosaminidases) SB - IM MH - Animals MH - *Baculoviridae MH - Cell Line MH - Cloning, Molecular MH - Culture Media MH - Gene Expression MH - *Genetic Vectors MH - Glutathione Transferase/genetics MH - Hepacivirus/genetics/*metabolism MH - Hexosaminidases/metabolism MH - Humans MH - Precipitin Tests MH - Recombinant Fusion Proteins/biosynthesis/genetics/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Solubility MH - Spodoptera MH - Viral Envelope Proteins/*biosynthesis/genetics/isolation & purification EDAT- 1998/09/02 MHDA- 1998/09/02 00:01 AID - S0168170298000410 [pii] PST - ppublish SO - Virus Res 1998 Jun;55(2):157-65. DR -------------------------------------------------------------------------------- 65: He X et al. Serological characterization ...[PMID: 9722879] Related Articles, Cited in PMC, Books, LinkOut PMID- 9722879 OWN - NLM STAT- MEDLINE DA - 19980914 DCOM- 19980914 LR - 20031114 PUBM- Print IS - 0304-8608 VI - 143 IP - 7 DP - 1998 TI - Serological characterization of the 3'-proximal encoded proteins of beet yellows closterovirus. PG - 1349-63 AB - The 3'-proximal open reading frames (ORFs) of beet yellows closterovirus, California isolate (BYV-CA), were sequenced and the expression of the corresponding proteins analyzed. The nucleotide sequence of ORF 5 (coding for p24) was most conserved compared with ORF 7 (coding for p20) and ORF 8 (coding for p21) among the isolates analyzed. Polyclonal antisera were produced to GST fusion proteins of p24, p20, and p21. Accumulation of p24, CP, p20 and p21 was studied in infected Tetragonia expansa plants and Chenopodium quinoa protoplasts. All four proteins were expressed in all tissues (old leaves, young leaves and stems), and most abundantly in young leaves. The subcellular localization of each protein in different tissues showed that compared with p24, CP and p21, p20 accumulated less in transfected protoplasts. Immunogold labeling in sugarbeet with p24 and CP antisera demonstrated co-localization of p24 and CP in vascular petiole tissues. In infectivity neutralization tests, antisera against p24 and CP greatly reduced transmission of BYV by viruliferous aphids compared with viruliferous aphids fed on preimmune serum or antiserum to p21. AD - Department of Plant Pathology, University of California, Riverside, USA. FAU - He, X AU - He X FAU - Harper, K AU - Harper K FAU - Grantham, G AU - Grantham G FAU - Yang, C H AU - Yang CH FAU - Creamer, R AU - Creamer R LA - eng PT - Journal Article PL - AUSTRIA TA - Arch Virol JID - 7506870 RN - 0 (Antibodies, Viral) RN - 0 (DNA Primers) RN - 0 (DNA, Viral) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Antibodies, Viral MH - Aphids/virology MH - Base Sequence MH - Closterovirus/*genetics/*immunology/pathogenicity MH - DNA Primers/genetics MH - DNA, Viral/genetics MH - Escherichia coli/genetics MH - Gene Expression MH - Immunohistochemistry MH - Microscopy, Electron MH - Molecular Sequence Data MH - Neutralization Tests MH - Open Reading Frames MH - Plants/ultrastructure/virology MH - Polymerase Chain Reaction MH - Protoplasts/virology MH - Recombinant Fusion Proteins/genetics/immunology/metabolism MH - Transfection MH - Viral Proteins/*genetics/*immunology/metabolism EDAT- 1998/09/02 MHDA- 1998/09/02 00:01 PST - ppublish SO - Arch Virol 1998;143(7):1349-63. NR -------------------------------------------------------------------------------- 66: Choice CV et al. Insulin stimulates pp120 endo...[PMID: 9712832] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9712832 OWN - NLM STAT- MEDLINE DA - 19980924 DCOM- 19980924 LR - 20050209 PUBM- Print IS - 0021-9258 VI - 273 IP - 35 DP - 1998 Aug 28 TI - Insulin stimulates pp120 endocytosis in cells co-expressing insulin receptors. PG - 22194-200 AB - pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins. AD - Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio 43614, USA. FAU - Choice, C V AU - Choice CV FAU - Howard, M J AU - Howard MJ FAU - Poy, M N AU - Poy MN FAU - Hankin, M H AU - Hankin MH FAU - Najjar, S M AU - Najjar SM LA - eng GR - HD28184/HD/NICHD PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Cell Adhesion Molecules) RN - 0 (DNA Primers) RN - 0 (Recombinant Fusion Proteins) RN - 11061-68-0 (Insulin) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (Receptor, Insulin) RN - EC 2.7.1.112 (focal adhesion kinase) SB - IM MH - 3T3 Cells MH - Animals MH - Baculoviridae/genetics MH - Base Sequence MH - Cell Adhesion Molecules/*metabolism MH - DNA Primers MH - Endocytosis/*drug effects MH - Fluorescent Antibody Technique, Indirect MH - Glutathione Transferase/metabolism MH - Insulin/*pharmacology MH - Mice MH - Phosphorylation MH - Precipitin Tests MH - Protein-Tyrosine Kinase/*metabolism MH - Rats MH - Receptor, Insulin/genetics/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1998/08/26 MHDA- 1998/08/26 00:01 PST - ppublish SO - J Biol Chem 1998 Aug 28;273(35):22194-200. PR -------------------------------------------------------------------------------- 67: Casais R et al. Hypoderma lineatum: expressio...[PMID: 9709025] Related Articles, Books, LinkOut PMID- 9709025 OWN - NLM STAT- MEDLINE DA - 19980911 DCOM- 19980911 LR - 20041117 PUBM- Print IS - 0014-4894 VI - 90 IP - 1 DP - 1998 Sep TI - Hypoderma lineatum: expression of enzymatically active hypodermin C in Escherichia coli and its use for the immunodiagnosis of hypodermosis. PG - 14-9 AB - The cDNA coding for the mature hypodermin C from first instars of Hypoderma lineatum was cloned by reverse transcription and PCR amplification of total larval RNA using specific oligonucleotide primers. This cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Mature hypodermin C was released from the GST-fusion after glutathione-Sepharose 4B affinity chromatography and proteolytic cleavage using factor Xa. The purified recombinant protein showed enzymatic activity in gelatin-polyacrylamide gels and when azocoll was used as substrate. An enzyme-linked immunosorbent assay was developed using the recombinant antigen. Positive/negative cutoff values were calculated using the mean OD percentage (1.74%) of 113 negative sera plus three standard deviations. Sensitivity and specificity according to the resulting cutoff (10.74%) were 85 and 98.2% respectively. CI - Copyright 1998 Academic Press. AD - Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, Oviedo, 33006, Spain. FAU - Casais, R AU - Casais R FAU - Martin Alonso, J M AU - Martin Alonso JM FAU - Boga, J A AU - Boga JA FAU - Parra, F AU - Parra F LA - eng PT - Journal Article PL - UNITED STATES TA - Exp Parasitol JID - 0370713 RN - 0 (DNA Primers) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.4.21 (Serine Endopeptidases) RN - EC 3.4.21.- (hypodermin C) SB - IM MH - Amino Acid Sequence MH - Animals MH - Blotting, Western MH - Cattle MH - Cattle Diseases/diagnosis MH - Chromatography, Affinity MH - Cloning, Molecular MH - DNA Primers MH - Diptera/*enzymology MH - Ectoparasitic Infestations/*diagnosis/veterinary MH - Escherichia coli MH - Glutathione Transferase MH - Humans MH - Kinetics MH - Larva MH - Polymerase Chain Reaction MH - Recombinant Fusion Proteins/biosynthesis/chemistry/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Serine Endopeptidases/*analysis/*biosynthesis/chemistry EDAT- 1998/08/26 MHDA- 1998/08/26 00:01 AID - S0014489498943028 [pii] PST - ppublish SO - Exp Parasitol 1998 Sep;90(1):14-9. NR -------------------------------------------------------------------------------- 68: Harris M. Use of GST-fusion and related...[PMID: 9664300] Related Articles, Cited in PMC, Books, LinkOut PMID- 9664300 OWN - NLM STAT- MEDLINE DA - 19981005 DCOM- 19981005 LR - 20031114 PUBM- Print IS - 1064-3745 VI - 88 DP - 1998 TI - Use of GST-fusion and related constructs for the identification of interacting proteins. PG - 87-99 AD - MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, UK. FAU - Harris, M AU - Harris M LA - eng PT - Journal Article PL - UNITED STATES TA - Methods Mol Biol JID - 9214969 RN - 0 (Genetic Vectors) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Blotting, Western MH - Cloning, Molecular MH - Escherichia coli/genetics MH - Genetic Vectors MH - Methods MH - Recombinant Fusion Proteins/*chemistry/genetics/isolation & purification MH - Spodoptera EDAT- 1998/07/17 MHDA- 1998/07/17 00:01 PST - ppublish SO - Methods Mol Biol 1998;88:87-99. DR -------------------------------------------------------------------------------- 69: Boublik Y et al. Eukaryotic virus display: eng...[PMID: 9636281] Related Articles, Cited in PMC, Books, LinkOut PMID- 9636281 OWN - NLM STAT- MEDLINE DA - 19980721 DCOM- 19980721 LR - 20041117 PUBM- Print IS - 0733-222X VI - 13 IP - 10 DP - 1995 Oct TI - Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. PG - 1079-84 AB - We describe the development of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector for the display of distinct proteins on the viral surface in a manner that is analogous to the established bacterial "phage display" systems. As a model system, the marker gene encoding the 26kDa protein glutathione-S-transferase (GST) was used to construct several fusions with the major baculovirus glycoprotein gp64 gene. Following expression in Spodoptera frugiperda (Sf9) cells, the yield and cellular distribution of each GST-gp64 protein was assessed by Western blot of both cell and supernatant fractions. One fusion, in which GST was inserted between the leader peptide and the nature protein, was found to be efficiently secreted into the cell medium. In the context of expression of the full length gp64, the hybrid GST-gp64 was shown by immunogold labelling to be incorporated onto the virion surface. In addition, the affinity purification of the soluble transmembrane gp64-GST fusion protein resulted in the co-purification of wild type gp64 suggesting that co-oligomerization of the GST-tagged fusion and the wild type molecule was the basis for virion incorporation. The HIV major surface glycoprotein, gp120 was also efficiently displayed in functional form on the viral surface following fusion to the amino terminus of gp64. A general expression vector, pAcSurf-2, was constructed in which multiple cloning sites were positioned in-phase between the gp64 signal sequence and the sequence encoding the mature protein under the control of the polyhedrin promoter. AD - NERC Institute of Virology, Oxford, UK. FAU - Boublik, Y AU - Boublik Y FAU - Di Bonito, P AU - Di Bonito P FAU - Jones, I M AU - Jones IM LA - eng PT - Journal Article PL - UNITED STATES TA - Biotechnology (N Y) JID - 8309273 RN - 0 (Antigens, CD4) RN - 0 (Genetic Vectors) RN - 0 (HIV Envelope Protein gp120) RN - 0 (Peptide Library) RN - 0 (Plasmids) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Fusion Proteins) RN - 0 (gp64 protein, baculovirus) RN - EC 2.5.1.18 (Glutathione Transferase) SB - B SB - X CIN - Biotechnology (N Y). 1995 Oct;13(10):1046. PMID: 9678911 EIN - Biotechnology 1995 Dec;13(13):1503 MH - Amino Acid Sequence MH - Animals MH - Antigens, CD4/metabolism MH - Cell Line MH - Cell Membrane/metabolism MH - Gene Deletion MH - Gene Expression MH - *Genetic Vectors MH - Glutathione Transferase/genetics MH - HIV Envelope Protein gp120/analysis/chemistry/genetics MH - Molecular Sequence Data MH - Nucleopolyhedrovirus/*genetics/ultrastructure MH - *Peptide Library MH - Plasmids/genetics MH - Recombinant Fusion Proteins/*analysis/chemistry/genetics MH - Research Support, Non-U.S. Gov't MH - Spodoptera/metabolism MH - Viral Fusion Proteins/*genetics EDAT- 1998/06/24 MHDA- 1998/06/24 00:01 PST - ppublish SO - Biotechnology (N Y) 1995 Oct;13(10):1079-84. DR -------------------------------------------------------------------------------- 70: Wu C et al. Development and characterizat...[PMID: 9632740] Related Articles, Books, LinkOut PMID- 9632740 OWN - NLM STAT- MEDLINE DA - 19980716 DCOM- 19980716 LR - 20041117 PUBM- Print IS - 0022-1554 VI - 46 IP - 7 DP - 1998 Jul TI - Development and characterization of monoclonal antibodies specific to the serotonin 5-HT2A receptor. PG - 811-24 AB - Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies. AD - Molecular and Cellular Biology, PharMingen, San Diego, California, USA. FAU - Wu, C AU - Wu C FAU - Yoder, E J AU - Yoder EJ FAU - Shih, J AU - Shih J FAU - Chen, K AU - Chen K FAU - Dias, P AU - Dias P FAU - Shi, L AU - Shi L FAU - Ji, X D AU - Ji XD FAU - Wei, J AU - Wei J FAU - Conner, J M AU - Conner JM FAU - Kumar, S AU - Kumar S FAU - Ellisman, M H AU - Ellisman MH FAU - Singh, S K AU - Singh SK LA - eng GR - 1R43MH54437-1/MH/NIMH GR - NS14718/NS/NINDS GR - NS24635/NS/NINDS GR - etc. PT - Journal Article PL - UNITED STATES TA - J Histochem Cytochem JID - 9815334 RN - 0 (Antibodies, Monoclonal) RN - 0 (Receptor, Serotonin, 5-HT2A) RN - 0 (Receptors, Serotonin) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Aged MH - Animals MH - Animals, Newborn MH - Antibodies, Monoclonal/*biosynthesis MH - Blotting, Western MH - Brain/metabolism MH - Cells, Cultured MH - Dendrites/metabolism MH - Humans MH - Immunohistochemistry MH - Male MH - Mice MH - Mice, Inbred BALB C MH - Microscopy, Confocal MH - Microscopy, Fluorescence MH - Pyramidal Cells/metabolism MH - Rats MH - Rats, Sprague-Dawley MH - Receptor, Serotonin, 5-HT2A MH - Receptors, Serotonin/*immunology/metabolism MH - Recombinant Fusion Proteins/immunology MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Schwann Cells/metabolism EDAT- 1998/06/20 MHDA- 1998/06/20 00:01 PST - ppublish SO - J Histochem Cytochem 1998 Jul;46(7):811-24. DR -------------------------------------------------------------------------------- 71: Matsumoto I et al. Functional expression and enz...[PMID: 9538263] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 9538263 OWN - NLM STAT- MEDLINE DA - 19980713 DCOM- 19980713 LR - 20041117 PUBM- Print IS - 0021-924X VI - 123 IP - 4 DP - 1998 Apr TI - Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro. PG - 693-700 AB - To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study [Matsumoto et al. (1997) J. Biochem. 121, 464-476], we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli. The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column. GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA. The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower. The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1. AD - Department of Applied Biological Chemistry, Faculty of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657. FAU - Matsumoto, I AU - Matsumoto I FAU - Abe, K AU - Abe K FAU - Arai, S AU - Arai S FAU - Emori, Y AU - Emori Y LA - eng PT - Journal Article PL - JAPAN TA - J Biochem (Tokyo) JID - 0376600 RN - 0 (DNA, Complementary) RN - 0 (Enzyme Precursors) RN - 0 (Insect Proteins) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.4.22 (Cysteine Endopeptidases) RN - EC 3.4.22.- (SCPc1 protein, Sitophilus zeamais) RN - EC 3.4.22.- (SCPg3 protein, Sitophilus zeamais) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Beetles/*enzymology MH - Cloning, Molecular MH - Cysteine Endopeptidases/genetics/isolation & purification/*metabolism MH - DNA, Complementary MH - Enzyme Precursors/metabolism MH - Escherichia coli/genetics MH - Glutathione Transferase/genetics MH - Hydrogen-Ion Concentration MH - Hydrolysis MH - *Insect Proteins MH - Molecular Sequence Data MH - *Protein Processing, Post-Translational MH - Recombinant Fusion Proteins/genetics MH - Substrate Specificity EDAT- 1998/05/21 MHDA- 1998/05/21 00:01 PST - ppublish SO - J Biochem (Tokyo) 1998 Apr;123(4):693-700. NR -------------------------------------------------------------------------------- 72: Patel IR et al. TNF-alpha convertase enzyme f...[PMID: 9574564] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, OMIM, GEO Profiles, Books, LinkOut PMID- 9574564 OWN - NLM STAT- MEDLINE DA - 19980521 DCOM- 19980521 LR - 20041117 PUBM- Print IS - 0022-1767 VI - 160 IP - 9 DP - 1998 May 1 TI - TNF-alpha convertase enzyme from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-alpha. PG - 4570-9 AB - A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-alpha convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCdeltaDETCy), expressed in baculovirus could cleave preferentially (approximately 12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-alpha to TNF-alpha (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-alpha mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alphaR fused to IgG Fc fragment (TNF-alphaR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-alpha in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE. AD - Department of Rheumatology and Medicine, Hospital for Joint Diseases, New York, NY 10003, USA. FAU - Patel, I R AU - Patel IR FAU - Attur, M G AU - Attur MG FAU - Patel, R N AU - Patel RN FAU - Stuchin, S A AU - Stuchin SA FAU - Abagyan, R A AU - Abagyan RA FAU - Abramson, S B AU - Abramson SB FAU - Amin, A R AU - Amin AR LA - eng SI - GENBANK/U92649 PT - Journal Article PL - UNITED STATES TA - J Immunol JID - 2985117R RN - 0 (DNA, Complementary) RN - 0 (Tumor Necrosis Factor-alpha) RN - EC 3.4.24 (Metalloendopeptidases) RN - EC 3.4.24.- (TNF-alpha converting enzyme) SB - AIM SB - IM MH - Arthritis, Rheumatoid/enzymology/*genetics MH - Cartilage/*enzymology MH - Cloning, Molecular MH - DNA, Complementary/*genetics/isolation & purification MH - Gene Expression Regulation, Enzymologic/*drug effects MH - Humans MH - Metalloendopeptidases/biosynthesis/*genetics MH - Molecular Sequence Data MH - Organ Culture Techniques MH - Research Support, Non-U.S. Gov't MH - Sequence Analysis MH - Tumor Necrosis Factor-alpha/*pharmacology EDAT- 1998/05/09 MHDA- 1998/05/09 00:01 PST - ppublish SO - J Immunol 1998 May 1;160(9):4570-9. NR -------------------------------------------------------------------------------- 73: Clayton JD et al. Interaction of troponin-H and...[PMID: 9536439] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9536439 OWN - NLM STAT- MEDLINE DA - 19980514 DCOM- 19980514 LR - 20041203 PUBM- Print IS - 0142-4319 VI - 19 IP - 2 DP - 1998 Feb TI - Interaction of troponin-H and glutathione S-transferase-2 in the indirect flight muscles of Drosophila melanogaster. PG - 117-27 AB - Drosophila indirect flight muscles (IFMs) contain a 35 kDa protein which cross-reacts with antibodies to the IFM specific protein troponin-H isoform 34 (TnH-34). Peptide fingerprinting and peptide sequencing showed that this 35 kDa protein is glutathione S-transferase-2 (GST-2). GST-2 is present in the asynchronous indirect flight muscles but not in the synchronous tergal depressor of the trochanter (jump muscle). Genetic dissection of the sarcomere showed that GST-2 is stably associated with the thin filaments but the presence of myosin is required to achieve the correct stoichiometry, suggesting that there is also an interaction with the thick filament. The two Drosophila TnHs (isoforms 33 and 34) are naturally occurring fusion proteins in which a proline-rich extension of approximately 250 amino acids replaces the 27 C-terminal residues of the muscle-specific tropomyosin II isoform. The proteolytic enzyme, Igase, cleaves the hydrophobic C-terminal sequence of TnH-34 at three sites and TnH-33 at one site. This results in the release of GST-2 from the myofibril. The amount of GST-2 stably bound to the myofibril is directly proportional to the total amount of undigested TnH. It is concluded that GST-2 in the thin filament is stabilized there by interaction with TnH. We speculate that the hydrophobic N-terminal region of GST-2 interacts with the hydrophobic C-terminal extension of TnH, and that both are close to a myosin cross-bridge. AD - European Molecular Biology Laboratory, Heidelberg, Germany. FAU - Clayton, J D AU - Clayton JD FAU - Cripps, R M AU - Cripps RM FAU - Sparrow, J C AU - Sparrow JC FAU - Bullard, B AU - Bullard B LA - eng PT - Journal Article PL - ENGLAND TA - J Muscle Res Cell Motil JID - 8006298 RN - 0 (Drosophila Proteins) RN - 0 (Insect Proteins) RN - 0 (Tm1 protein, Drosophila) RN - 0 (Tropomyosin) RN - 0 (Troponin) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Animals MH - *Drosophila Proteins MH - Drosophila melanogaster MH - Glutathione Transferase/chemistry/isolation & purification/*physiology MH - Insect Proteins/*physiology MH - Muscle, Skeletal/*metabolism MH - Peptide Mapping MH - Research Support, Non-U.S. Gov't MH - *Tropomyosin MH - Troponin/*physiology MH - Wing/*metabolism EDAT- 1998/04/16 MHDA- 1998/04/16 00:01 PST - ppublish SO - J Muscle Res Cell Motil 1998 Feb;19(2):117-27. NR -------------------------------------------------------------------------------- 74: Yu J et al. Regulation of the p85/p110 ph...[PMID: 9488453] Related Articles, Compound, Compound via MeSH, Substance, Substance via MeSH, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9488453 OWN - NLM STAT- MEDLINE DA - 19980319 DCOM- 19980319 LR - 20041117 PUBM- Print IS - 0270-7306 VI - 18 IP - 3 DP - 1998 Mar TI - Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. PG - 1379-87 AB - We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85. AD - Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. FAU - Yu, J AU - Yu J FAU - Zhang, Y AU - Zhang Y FAU - McIlroy, J AU - McIlroy J FAU - Rordorf-Nikolic, T AU - Rordorf-Nikolic T FAU - Orr, G A AU - Orr GA FAU - Backer, J M AU - Backer JM LA - eng GR - GM55692/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - Mol Cell Biol JID - 8109087 RN - 0 (Enzyme Inhibitors) RN - 0 (Recombinant Fusion Proteins) RN - 56-45-1 (Serine) RN - 72-19-5 (Threonine) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase/biosynthesis/genetics/*metabolism MH - Amino Acid Sequence MH - Animals MH - Catalysis MH - Cell Line MH - Enzyme Inhibitors/*metabolism MH - Enzyme Stability MH - Humans MH - Molecular Sequence Data MH - Phosphorylation MH - Rabbits MH - Recombinant Fusion Proteins/biosynthesis/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Serine/metabolism MH - Spodoptera/cytology MH - Temperature MH - Threonine/metabolism EDAT- 1998/03/06 MHDA- 1998/03/06 00:01 PST - ppublish SO - Mol Cell Biol 1998 Mar;18(3):1379-87. PR -------------------------------------------------------------------------------- 75: Monahan SJ et al. Interaction between the herpe...[PMID: 9454723] Related Articles, Cited in PMC, Books, LinkOut PMID- 9454723 OWN - NLM STAT- MEDLINE DA - 19980226 DCOM- 19980226 LR - 20041117 PUBM- Print IS - 0042-6822 VI - 241 IP - 1 DP - 1998 Feb 1 TI - Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins. PG - 122-30 AB - Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9-UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 microM bound increasing amounts of UL9 expressed by in vitro transcription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis. CI - Copyright 1998 Academic Press. AD - Department of Medical Microbiology and Immunology, Ohio State University, 333 West Tenth Avenue, Columbus, Ohio, 43210, USA. FAU - Monahan, S J AU - Monahan SJ FAU - Grinstead, L A AU - Grinstead LA FAU - Olivieri, W AU - Olivieri W FAU - Parris, D S AU - Parris DS LA - eng GR - F32 AI08566/AI/NIAID GR - GM34930/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - Virology JID - 0110674 RN - 0 (DNA-Binding Proteins) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) RN - 115004-77-8 (UL9 protein, Human herpesvirus 1) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.7.7 (DNA-Directed DNA Polymerase) RN - EC 3.1.- (Exodeoxyribonucleases) RN - EC 3.1.11.- (DNA polymerase, Simplexvirus) SB - IM MH - Animals MH - Cell Line MH - Chromatography, Affinity MH - DNA-Binding Proteins/immunology/*metabolism MH - *DNA-Directed DNA Polymerase MH - *Exodeoxyribonucleases MH - Glutathione Transferase/genetics/metabolism MH - Herpesvirus 1, Human/*metabolism MH - Humans MH - Peptide Fragments/metabolism MH - Precipitin Tests MH - Rabbits MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera/cytology MH - Viral Proteins/immunology/*metabolism EDAT- 1998/02/28 MHDA- 1998/02/28 00:01 AID - S0042682297989534 [pii] PST - ppublish SO - Virology 1998 Feb 1;241(1):122-30. PR -------------------------------------------------------------------------------- 76: Zhou ZH et al. A complex glutathione transfe...[PMID: 9349710] Related Articles, Nucleotide, Protein, Cited in PMC, Books, LinkOut PMID- 9349710 OWN - NLM STAT- MEDLINE DA - 19971124 DCOM- 19971124 LR - 20041118 PUBM- Print IS - 0026-8925 VI - 256 IP - 2 DP - 1997 Sep TI - A complex glutathione transferase gene family in the housefly Musca domestica. PG - 187-94 AB - In most metazoans, the glutathione S-transferases (GST) are encoded by gene families, and are used to detoxify xenobiotics. We describe the structure of genomic loci coding for the GSTs in the housefly that have been implicated, by both genetic and biochemical means, in mediating insecticide resistance. In earlier work, we showed that one of the theta-class enzymes, MdGST-3, is overproduced in resistant flies and degrades certain insecticides. We used a fragment from a cDNA clone of MdGST-3 as a probe to screen a housefly genomic DNA bank in phage lambda. This probe detected multiple gst loci. Genes for GSTs were found in five different, nonoverlapping lambda clones, three of which carry multiple, closely linked gsts. Multiple genes for both MdGST-3 and MdGST-4 were found; some of which have introns in their 5' untranslated regions. In adults, the only MdGST-3 enzymes that are expressed are encoded by the intron-free genes. A new theta-class GST (called MdGST-5) was also discovered. Fusion genes comprising 5' MdGST-3 sequences and either MdGST-4 or MdGST-5 sequences in their 3' halves were encountered at three separate loci. The genes described here are found in both the ancestral sensitive strain and the insecticide-resistant strains. AD - Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616, USA. FAU - Zhou, Z H AU - Zhou ZH FAU - Syvanen, M AU - Syvanen M LA - eng SI - GENBANK/X73575 SI - GENBANK/X94279 SI - GENBANK/X94280 SI - GENBANK/X94281 SI - GENBANK/X94282 SI - GENBANK/X94283 SI - GENBANK/X94284 SI - GENBANK/X94285 SI - GENBANK/X94286 SI - GENBANK/X94287 SI - GENBANK/X94288 PT - Journal Article PL - GERMANY TA - Mol Gen Genet JID - 0125036 RN - 0 (DNA Probes) RN - 0 (Insecticides) RN - 0 (Organophosphorus Compounds) RN - 0 (Plasmids) RN - EC 2.5.1.- (glutathione S-transferase T1) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Animals MH - Base Sequence MH - Cloning, Molecular MH - DNA Probes/genetics MH - Gene Fusion MH - Gene Library MH - Glutathione Transferase/*genetics MH - Houseflies/enzymology/*genetics MH - Insecticide Resistance/*genetics MH - Insecticides/metabolism/toxicity MH - Introns MH - Molecular Sequence Data MH - Multigene Family MH - Organophosphorus Compounds MH - Plasmids MH - Polymerase Chain Reaction MH - Restriction Mapping MH - Sequence Analysis, DNA EDAT- 1998/02/12 MHDA- 1998/02/12 00:01 PST - ppublish SO - Mol Gen Genet 1997 Sep;256(2):187-94. NR -------------------------------------------------------------------------------- 77: Angeles TS et al. Kinetics of trkA tyrosine kin...[PMID: 9448714] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9448714 OWN - NLM STAT- MEDLINE DA - 19980218 DCOM- 19980218 LR - 20041117 PUBM- Print IS - 0003-9861 VI - 349 IP - 2 DP - 1998 Jan 15 TI - Kinetics of trkA tyrosine kinase activity and inhibition by K-252a. PG - 267-74 AB - The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases. AD - Cephalon, Inc., West Chester, Pennsylvania 19380, USA. TAngeles@cephalon.com FAU - Angeles, T S AU - Angeles TS FAU - Yang, S X AU - Yang SX FAU - Steffler, C AU - Steffler C FAU - Dionne, C A AU - Dionne CA LA - eng PT - Journal Article PL - UNITED STATES TA - Arch Biochem Biophys JID - 0372430 RN - 0 (Carbazoles) RN - 0 (Isoenzymes) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Receptors, Nerve Growth Factor) RN - 0 (Recombinant Fusion Proteins) RN - 3469-78-1 (5'-adenylyl (beta,gamma-methylene)diphosphonate) RN - 56-65-5 (Adenosine Triphosphate) RN - 58-64-0 (Adenosine Diphosphate) RN - 7292-42-4 (alpha,beta-methyleneadenosine 5'-triphosphate) RN - 97161-97-2 (K 252) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) RN - EC 2.7.1.112 (Receptor, trkA) RN - EC 3.1.4.- (phospholipase C gamma) RN - EC 3.1.4.3 (Phospholipase C) SB - IM MH - Adenosine Diphosphate/pharmacology MH - Adenosine Triphosphate/analogs & derivatives/pharmacology MH - Animals MH - Carbazoles/*pharmacology MH - Cell Line MH - Glutathione Transferase/*metabolism MH - Humans MH - Isoenzymes/*metabolism MH - Kinetics MH - Phospholipase C/*metabolism MH - Phosphorylation MH - Proto-Oncogene Proteins/antagonists & inhibitors/*metabolism MH - Receptor Protein-Tyrosine Kinases/antagonists & inhibitors/*metabolism MH - Receptor, trkA MH - Receptors, Nerve Growth Factor/antagonists & inhibitors/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Spodoptera MH - Substrate Specificity MH - Transfection EDAT- 1998/02/04 MHDA- 1998/02/04 00:01 AID - S0003986197904902 [pii] PST - ppublish SO - Arch Biochem Biophys 1998 Jan 15;349(2):267-74. PR -------------------------------------------------------------------------------- 78: Coss MC et al. The immunophilin FKBP65 forms...[PMID: 9438387] Related Articles, Cited in PMC, Books, LinkOut PMID- 9438387 OWN - NLM STAT- MEDLINE DA - 19980206 DCOM- 19980206 LR - 20041203 PUBM- Print IS - 1044-9523 VI - 9 IP - 1 DP - 1998 Jan TI - The immunophilin FKBP65 forms an association with the serine/threonine kinase c-Raf-1. PG - 41-8 AB - FKBP65 is a member of the FK506-binding protein class of immunophilins and is the only member reported to contain four peptidylprolyl cis-trans isomerase domains and an unrelated COOH-terminal domain. In this report, we show that the heat shock protein hsp90 and the serine/threonine protein kinase c-Raf-1 are components of FKBP65 immune complexes. The NH2-terminal regulatory domain of c-Raf-1 appears to be required for its interaction with FKBP65. Using GST-FKBP65 fusion protein and purified Raf proteins, we show that full-length FKBP65 can interact with c-Raf-1 but not B-Raf. The activation kinetics of c-Raf-1 after v-H-RasV12 injection of Xenopus oocytes appear to correlate with FKBP65/c-Raf-1 interaction, suggesting that FKBP65 may preferentially associate with forms of c-Raf-1 that are more posttranslationally modified. The interaction of FKBP65 with the c-Raf-heat shock protein 90 heterocomplex implicates this immunophilin in signal-transduction processes. AD - Division of Basic Science, National Cancer Institute-Frederick Cancer Research and Development Center, NIH, Frederick, Maryland 21702-1201, USA. FAU - Coss, M C AU - Coss MC FAU - Stephens, R M AU - Stephens RM FAU - Morrison, D K AU - Morrison DK FAU - Winterstein, D AU - Winterstein D FAU - Smith, L M AU - Smith LM FAU - Simek, S L AU - Simek SL LA - eng PT - Journal Article PL - UNITED STATES TA - Cell Growth Differ JID - 9100024 RN - 0 (Carrier Proteins) RN - 0 (DNA-Binding Proteins) RN - 0 (FK506-binding protein, Xenopus) RN - 0 (Heat-Shock Proteins 90) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Xenopus Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.37 (Proto-Oncogene Proteins c-raf) RN - EC 5.2.1.- (Tacrolimus Binding Proteins) SB - IM MH - Animals MH - Carrier Proteins/*metabolism MH - DNA-Binding Proteins/*metabolism MH - Glutathione Transferase/metabolism MH - Heat-Shock Proteins 90/metabolism MH - Mutagenesis MH - Protein Binding MH - Proto-Oncogene Proteins c-raf/genetics/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Spodoptera MH - *Tacrolimus Binding Proteins MH - *Xenopus Proteins MH - Xenopus laevis EDAT- 1998/01/23 MHDA- 1998/01/23 00:01 PST - ppublish SO - Cell Growth Differ 1998 Jan;9(1):41-8. PR -------------------------------------------------------------------------------- 79: Amano S et al. Identification of endogenous ...[PMID: 9399593] Related Articles, Books, LinkOut PMID- 9399593 OWN - NLM STAT- MEDLINE DA - 19980217 DCOM- 19980217 LR - 20041117 PUBM- Print IS - 0021-924X VI - 122 IP - 4 DP - 1997 Oct TI - Identification of endogenous substrates for Drosophila calpain from a salt-extracted fraction of Drosophila ovaries. PG - 865-71 AB - Drosophila calpain (Dm-calpain) produced in Escherichia coli has a distinct Ca2+-dependent activity. By using a recombinant Dm-calpain, we searched for its substrates occurring in Drosophila ovaries, where Dm-calpain is expressed. Among a number of major proteins, several proteins in a salt-extracted fraction were selectively degraded by Dm-calpain in a Ca2+-dependent manner. The major substrates were identified by microsequencing the lysylendopeptidase-digested proteins. Three ribosomal proteins, the L5, L7, and L8 subunits of the 60S ribosome, were found to be potential Dm-calpain substrates. In addition, the alpha subunit of elongation factor-1 (EF-1alpha), a multi-functional protein involved in both protein synthesis and cytoskeletal regulation, was shown to be cleaved by Dm-calpain into several distinct fragments when expressed as a GST-fusion protein. Endogenous EF-1alpha in ovary extracts was also shown by western blot analysis to be similarly degraded. These observations suggest that Dm-calpain may regulate protein synthesis and cytoskeletal structure through its degradative or processing activity. AD - Department of Molecular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo. FAU - Amano, S AU - Amano S FAU - Kawasaki, H AU - Kawasaki H FAU - Ishiura, S AU - Ishiura S FAU - Kawashima, S AU - Kawashima S FAU - Suzuki, K AU - Suzuki K FAU - Emori, Y AU - Emori Y LA - eng PT - Journal Article PL - JAPAN TA - J Biochem (Tokyo) JID - 0376600 RN - 0 (Peptide Elongation Factor 1) RN - 0 (Peptide Elongation Factors) RN - 0 (Recombinant Proteins) RN - EC 3.4.22.17 (Calpain) SB - IM MH - Amino Acid Sequence MH - Animals MH - Calpain/genetics/*metabolism MH - Chromatography, High Pressure Liquid MH - Cloning, Molecular MH - Drosophila melanogaster/*enzymology MH - Escherichia coli/genetics MH - Female MH - Hydrolysis MH - Ovary/*metabolism MH - Peptide Elongation Factor 1 MH - Peptide Elongation Factors/isolation & purification/*metabolism MH - Recombinant Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Substrate Specificity EDAT- 1997/12/17 MHDA- 1997/12/17 00:01 PST - ppublish SO - J Biochem (Tokyo) 1997 Oct;122(4):865-71. NR -------------------------------------------------------------------------------- 80: Schwartz JL et al. Production of recombinant her...[PMID: 9366090] Related Articles, Books, LinkOut PMID- 9366090 OWN - NLM STAT- MEDLINE DA - 19971204 DCOM- 19971204 LR - 20031114 PUBM- Print IS - 1367-5435 VI - 19 IP - 2 DP - 1997 Aug TI - Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus-insect cell expression system. PG - 87-91 AB - A gene expression system using recombinant Autographa california nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6-10 x 10(6) cells ml-1) in shake flasks in serum-free media. With these procedures, cell densities of 5 x 10(6) cells ml-1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell-1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11-38 mg L-1 of recombinant protein and > or = 90% purity. Maximum production of this protein was observed at a cell density of 5.0 x 10(6) cells ml-1. AD - Department of Microbial Products-Fermentation, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539, USA. FAU - Schwartz, J L AU - Schwartz JL FAU - Ferrari, E B AU - Ferrari EB FAU - Terracciano, J AU - Terracciano J FAU - Troyanovich, J AU - Troyanovich J FAU - Gunnarsson, I AU - Gunnarsson I FAU - Wright-Minogue, J AU - Wright-Minogue J FAU - Chen, J W AU - Chen JW FAU - Kwong, A D AU - Kwong AD LA - eng PT - Journal Article PL - ENGLAND TA - J Ind Microbiol Biotechnol JID - 9705544 RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Proteins) RN - EC 3.4.- (Endopeptidases) SB - B MH - Animals MH - Baculoviridae/genetics MH - Endopeptidases/*biosynthesis MH - Recombinant Fusion Proteins/*biosynthesis MH - Simplexvirus/*enzymology MH - Spodoptera MH - Viral Proteins/*biosynthesis EDAT- 1997/11/20 MHDA- 1997/11/20 00:01 PST - ppublish SO - J Ind Microbiol Biotechnol 1997 Aug;19(2):87-91. DR -------------------------------------------------------------------------------- 81: Chen H et al. Protein-protein interactions ...[PMID: 9325319] Related Articles, Books, LinkOut PMID- 9325319 OWN - NLM STAT- MEDLINE DA - 19971113 DCOM- 19971113 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 272 IP - 41 DP - 1997 Oct 10 TI - Protein-protein interactions are implied in glucocorticoid receptor mutant 465*-mediated cell death. PG - 25873-80 AB - Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid "tail," beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* "trapped" several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function. AD - Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0645, USA. FAU - Chen, H AU - Chen H FAU - Srinivasan, G AU - Srinivasan G FAU - Thompson, E B AU - Thompson EB LA - eng GR - CA 41407/CA/NCI PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Proto-Oncogene Proteins c-jun) RN - 0 (Receptors, Glucocorticoid) RN - 0 (Recombinant Proteins) RN - 0 (Transcription Factor AP-1) SB - IM MH - Animals MH - *Apoptosis MH - Humans MH - Mutagenesis MH - Protein Binding MH - Proto-Oncogene Proteins c-jun/metabolism MH - Receptors, Glucocorticoid/*genetics/physiology MH - Recombinant Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Transcription Factor AP-1/metabolism MH - Transcription, Genetic MH - Transfection MH - Tumor Cells, Cultured EDAT- 1997/11/05 MHDA- 1997/11/05 00:01 PST - ppublish SO - J Biol Chem 1997 Oct 10;272(41):25873-80. PR -------------------------------------------------------------------------------- 82: Yamaguchi M et al. Distinct roles of E2F recogni...[PMID: 9380507] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9380507 OWN - NLM STAT- MEDLINE DA - 19971112 DCOM- 19971112 LR - 20041208 PUBM- Print IS - 0305-1048 VI - 25 IP - 19 DP - 1997 Oct 1 TI - Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase alpha 180 kDa catalytic subunit gene promoter during Drosophila development. PG - 3847-54 AB - The transcription factor E2F plays a key role in transcriptional control during the growth cycle of higher eukaryotic cells. The promoter region of the DrosophilaDNA polymerase alpha 180 kDa catalytic subunit gene contains three E2F recognition sequences located at positions -353 to -342 (E2F site 1), -21 to -14 (E2F site 2) and -12 to -5 (E2F site 3) with respect to the transcription initiation site. Various base substitutions were generated in each or all of the three E2F sites in vitro to allow examination of their effects on E2F binding and promoter function in cultured Kc cells as well as in living flies. Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins were found to cooperate in binding to the three E2F sites in the DNA polymerase alpha gene promoter in vitro. In contrast, an E2F-specific activity detected in nuclear extracts of Kc cells showed little affinity for E2F site 1 but strong binding to sites 2 and 3. Transient expression of Drosophila E2F in Kc cells activated the DNA polymerase alpha gene promoter and the target sites for activation coincided with E2F sites 2 and 3. However, analyses with transgenic flies indicate that E2F site 3 functions positively in terms of DNA polymerase alpha gene promoter activity, while E2F sites 1 and 2 rather have a negative control function. Thus E2F sites play distinct roles as positive or negative elements in regulation of the DNA polymerase alpha gene promoter during Drosophila development. AD - Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan. myamaguc@aichi-cc.pref.aichi.jp FAU - Yamaguchi, M AU - Yamaguchi M FAU - Hayashi, Y AU - Hayashi Y FAU - Hirose, F AU - Hirose F FAU - Nishimoto, Y AU - Nishimoto Y FAU - Matsukage, A AU - Matsukage A LA - eng PT - Journal Article PL - ENGLAND TA - Nucleic Acids Res JID - 0411011 RN - 0 (Carrier Proteins) RN - 0 (Cell Cycle Proteins) RN - 0 (DNA-Binding Proteins) RN - 0 (Dp protein, Drosophila) RN - 0 (Drosophila Proteins) RN - 0 (E2F transcription factors) RN - 0 (Oligodeoxyribonucleotides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Trans-Activators) RN - 0 (Transcription Factors) RN - 0 (retinoblastoma binding protein 1) RN - EC 2.7.7.- (DNA Polymerase I) SB - IM MH - Animals MH - Animals, Genetically Modified MH - Base Sequence MH - Binding Sites/genetics MH - *Carrier Proteins MH - *Cell Cycle Proteins MH - Cell Line MH - Chromosome Mapping MH - DNA Polymerase I/chemistry/*genetics MH - *DNA-Binding Proteins MH - Drosophila/*genetics/growth & development/*metabolism MH - *Drosophila Proteins MH - Gene Expression Regulation, Developmental MH - *Genes, Insect MH - Molecular Weight MH - Mutation MH - Oligodeoxyribonucleotides/genetics/metabolism MH - *Promoter Regions (Genetics) MH - Protein Conformation MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - *Trans-Activators MH - Transcription Factors/genetics/*metabolism EDAT- 1997/10/10 MHDA- 1997/10/10 00:01 AID - gka616 [pii] PST - ppublish SO - Nucleic Acids Res 1997 Oct 1;25(19):3847-54. NR -------------------------------------------------------------------------------- 83: Kawaguchi Y et al. Herpes simplex virus 1 alpha ...[PMID: 9311810] Related Articles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9311810 OWN - NLM STAT- MEDLINE DA - 19971020 DCOM- 19971020 LR - 20050204 PUBM- Print IS - 0022-538X VI - 71 IP - 10 DP - 1997 Oct TI - Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. PG - 7328-36 AB - The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0. (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase. AD - The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Illinois 60637, USA. FAU - Kawaguchi, Y AU - Kawaguchi Y FAU - Van Sant, C AU - Van Sant C FAU - Roizman, B AU - Roizman B LA - eng GR - AI124009/AI/NIAID GR - CA47451/CA/NCI GR - GM07183-22/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (Chimeric Proteins) RN - 0 (Cyclins) RN - 0 (Immediate-Early Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Trans-Activators) RN - 0 (herpes simplex virus type 1 alpha ICP0 protein) RN - 147954-86-7 (cyclin D3) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (cyclin-dependent kinase 4) SB - IM MH - Animals MH - Binding Sites MH - Cercopithecus aethiops MH - Chimeric Proteins/biosynthesis MH - Cloning, Molecular MH - Cyclin-Dependent Kinases/metabolism MH - Cyclins/chemistry/isolation & purification/*metabolism MH - Genome, Viral MH - Glutathione Transferase MH - Herpesvirus 1, Human/genetics/*physiology MH - Humans MH - Immediate-Early Proteins/chemistry/isolation & purification/*metabolism MH - Protein Binding MH - *Proto-Oncogene Proteins MH - Recombinant Fusion Proteins/chemistry/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae MH - Spodoptera MH - Trans-Activators MH - Transfection MH - Tumor Cells, Cultured MH - Vero Cells EDAT- 1997/10/06 MHDA- 1997/10/06 00:01 PST - ppublish SO - J Virol 1997 Oct;71(10):7328-36. NR -------------------------------------------------------------------------------- 84: Georgel PT et al. Role of histone tails in nucl...[PMID: 9303316] Related Articles, HomoloGene, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9303316 OWN - NLM STAT- MEDLINE DA - 19971015 DCOM- 19971015 LR - 20041117 PUBM- Print IS - 0261-4189 VI - 16 IP - 15 DP - 1997 Aug 1 TI - Role of histone tails in nucleosome remodeling by Drosophila NURF. PG - 4717-26 AB - The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. Here, we show that the interaction between NURF and nucleosomes is impaired by proteolytic removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation. AD - Laboratory of Molecular Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA. FAU - Georgel, P T AU - Georgel PT FAU - Tsukiyama, T AU - Tsukiyama T FAU - Wu, C AU - Wu C LA - eng PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (Cross-Linking Reagents) RN - 0 (Histones) RN - 0 (Nucleosomes) RN - 0 (Oligodeoxyribonucleotides) RN - 0 (Recombinant Fusion Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - 9007-49-2 (DNA) RN - EC 3.6.1.3 (Adenosinetriphosphatase) SB - IM MH - Acetylation MH - Adenosine Triphosphate/metabolism MH - Adenosinetriphosphatase/antagonists & inhibitors MH - Animals MH - Base Sequence MH - Cross-Linking Reagents MH - DNA/metabolism MH - Drosophila/genetics/*metabolism MH - Histones/chemistry/genetics/*metabolism MH - Nucleosomes/*metabolism MH - Oligodeoxyribonucleotides/genetics MH - Recombinant Fusion Proteins/chemistry/genetics/metabolism MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1997/09/26 MHDA- 1997/09/26 00:01 AID - 10.1093/emboj/16.15.4717 [doi] PST - ppublish SO - EMBO J 1997 Aug 1;16(15):4717-26. NR -------------------------------------------------------------------------------- 85: Ju H et al. Direct interaction of endothe...[PMID: 9228013] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9228013 OWN - NLM STAT- MEDLINE DA - 19970909 DCOM- 19970909 LR - 20050127 PUBM- Print IS - 0021-9258 VI - 272 IP - 30 DP - 1997 Jul 25 TI - Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. PG - 18522-5 AB - Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1. AD - Vascular Biology Center, Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA. FAU - Ju, H AU - Ju H FAU - Zou, R AU - Zou R FAU - Venema, V J AU - Venema VJ FAU - Venema, R C AU - Venema RC LA - eng SI - GENBANK/U07645 SI - GENBANK/U86639 SI - GENBANK/Z12161 SI - GENBANK/Z18951 SI - GENBANK/Z46424 GR - HL57201/HL/NHLBI PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Calmodulin) RN - 0 (Caveolins) RN - 0 (Enzyme Inhibitors) RN - 0 (Membrane Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 149720-12-7 (caveolin 1) RN - 7440-70-2 (Calcium) RN - EC 1.14.13.39 (Nitric-Oxide Synthase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Calcium/metabolism MH - Calmodulin/metabolism MH - Catalysis MH - Cattle MH - *Caveolins MH - Cells, Cultured MH - Chickens MH - Dogs MH - Endothelium, Vascular/*enzymology MH - Enzyme Inhibitors MH - Humans MH - Membrane Proteins/chemistry/*metabolism MH - Mice MH - Molecular Sequence Data MH - Nitric-Oxide Synthase/antagonists & inhibitors/*metabolism MH - Protein Binding MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1997/07/25 MHDA- 1997/07/25 00:01 PST - ppublish SO - J Biol Chem 1997 Jul 25;272(30):18522-5. NR -------------------------------------------------------------------------------- 86: Cai XY et al. Expression, purification, and...[PMID: 9226723] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 9226723 OWN - NLM STAT- MEDLINE DA - 19970818 DCOM- 19970818 LR - 20041118 PUBM- Print IS - 1046-5928 VI - 10 IP - 2 DP - 1997 Jul TI - Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells. PG - 263-74 AB - An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal conditions, 2-3 mg of GST-CSBP2 could be obtained per liter of infected cell culture. The fusion protein was easily purified from the soluble fraction of the total cell lysate under nondenaturing conditions by using a glutathione-Sepharose 4B affinity resin. As expected, the purified GST-CSBP2 fusion protein was approximately 68 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and reacted with antibodies directed toward either the GST or the CSBP amino terminus. To obtain activated CSBP2, SF9 cells were coinfected with two recombinant baculovirus vectors: one that directed the synthesis of the GST-CSBP2 fusion protein and a second vector that directed the synthesis of a constitutively active form of the CSBP activating kinase, MKK3. Coexpression of GST-CSBP2 kinase with the MKK3 activator increased GST-CSBP2 activity 8- to 10-fold based on the ability of GST-CSBP2 to phosphorylate the substrate, myelin basic protein (MBP), and the ATF2 transcription factor, in vitro. Moreover, activated GST-CSBP2 was capable of activating a bacterially derived mitogen-activated protein kinase-activating protein kinase 2 in vitro. The activity of insect-derived GST-CSBP2 was also inhibited by the CSBP inhibitor, SB202190. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of this kinase. AD - Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. FAU - Cai, X Y AU - Cai XY FAU - Shanahan, M AU - Shanahan M FAU - Miller, K AU - Miller K FAU - Gommoll, C AU - Gommoll C FAU - Lundell, D AU - Lundell D FAU - Zavodny, P AU - Zavodny P FAU - Dalie, B AU - Dalie B LA - eng PT - Journal Article PL - UNITED STATES TA - Protein Expr Purif JID - 9101496 RN - 0 (Anti-Inflammatory Agents, Non-Steroidal) RN - 0 (Cytokines) RN - 0 (DNA, Complementary) RN - 0 (Genetic Vectors) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (MAP Kinase Kinase 3) RN - EC 2.7.1.- (MAP2K3 protein, human) RN - EC 2.7.1.- (Mitogen-Activated Protein Kinase Kinases) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.123 (Ca(2+)-Calmodulin Dependent Protein Kinase) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.1.37 (p38 Mitogen-Activated Protein Kinases) SB - IM MH - Animals MH - Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis/isolation & purification MH - Ca(2+)-Calmodulin Dependent Protein Kinase/*biosynthesis/genetics/*isolation & purification MH - Cell Line MH - Cloning, Molecular MH - Cytokines/antagonists & inhibitors MH - DNA, Complementary/genetics MH - Enzyme Activation MH - Genetic Vectors/chemical synthesis MH - Glutathione Transferase/genetics MH - Humans MH - MAP Kinase Kinase 3 MH - *Mitogen-Activated Protein Kinase Kinases MH - *Mitogen-Activated Protein Kinases MH - Protein-Serine-Threonine Kinases/genetics MH - Protein-Tyrosine Kinase/genetics MH - Recombinant Fusion Proteins/biosynthesis/genetics/isolation & purification MH - Spodoptera/cytology/enzymology/genetics MH - p38 Mitogen-Activated Protein Kinases EDAT- 1997/07/01 MHDA- 2000/05/20 09:00 AID - S1046592897907440 [pii] PST - ppublish SO - Protein Expr Purif 1997 Jul;10(2):263-74. DR -------------------------------------------------------------------------------- 87: Screen S et al. Carbon regulation of the cuti...[PMID: 9211795] Related Articles, Compound via MeSH, Substance via MeSH, Nucleotide, Protein, Cited in PMC, Books, LinkOut PMID- 9211795 OWN - NLM STAT- MEDLINE DA - 19970903 DCOM- 19970903 LR - 20041117 PUBM- Print IS - 0172-8083 VI - 31 IP - 6 DP - 1997 Jun TI - Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CREA protein. PG - 511-8 AB - The pr1 gene of the entomopathogenic fungus Metarhizium anisopliae encodes a serine protease that is highly active towards the insect cuticle and whose synthesis is subject to both carbon and nitrogen repression. The pr1 promoter region was sequenced revealing the presence of putative CREA- and AREA-binding sites. In vitro bandshift experiments demonstrated that an Aspergillus nidulans GST-CREA fusion protein was capable of binding to two of the three putative CREA sites. Using a PCR-based strategy the M. anisopliae crr1 gene was identified; it encodes a putative C2H2-type DNA-binding protein with significant sequence similarity to A. nidulans CREA. Complementation experiments with an A. nidulans strain carrying creA204 demonstrated that CRR1 can partially substitute for CREA function. AD - Microbial Pathogenicity Group, School of Biology and Biochemistry, University of Bath, Bath, UK. FAU - Screen, S AU - Screen S FAU - Bailey, A AU - Bailey A FAU - Charnley, K AU - Charnley K FAU - Cooper, R AU - Cooper R FAU - Clarkson, J AU - Clarkson J LA - eng SI - GENBANK/Y10265 PT - Journal Article PL - UNITED STATES TA - Curr Genet JID - 8004904 RN - 0 (CRR1 protein, Metarhizium anisopliae) RN - 0 (Fungal Proteins) RN - 0 (Repressor Proteins) RN - 0 (Trans-Activators) RN - 144516-87-0 (CreA protein, Aspergillus nidulans) RN - 7440-44-0 (Carbon) RN - EC 3.4.21 (Serine Endopeptidases) RN - EC 3.4.21.- (Pr1 serine protease, fungal) SB - IM MH - Amino Acid Sequence MH - Aspergillus nidulans/chemistry/genetics/metabolism MH - Base Sequence MH - Binding Sites MH - Carbon/*metabolism MH - Cloning, Molecular MH - Fungal Proteins/*genetics/*metabolism MH - Fungi/*enzymology/genetics MH - Gene Expression Regulation, Fungal MH - Genes, Fungal MH - Molecular Sequence Data MH - Open Reading Frames MH - Promoter Regions (Genetics) MH - Regulatory Sequences, Nucleic Acid MH - Repressor Proteins/*metabolism MH - Sequence Analysis MH - Sequence Analysis, DNA MH - Sequence Homology, Amino Acid MH - Serine Endopeptidases/*genetics/*metabolism MH - Trans-Activators/*genetics/metabolism MH - Transcription, Genetic EDAT- 1997/06/01 MHDA- 1997/06/01 00:01 PST - ppublish SO - Curr Genet 1997 Jun;31(6):511-8. NR -------------------------------------------------------------------------------- 88: Lacy S et al. Identification of a p130 doma...[PMID: 9188854] Related Articles, Gene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 9188854 OWN - NLM STAT- MEDLINE DA - 19970627 DCOM- 19970627 LR - 20050204 PUBM- Print IS - 0950-9232 VI - 14 IP - 20 DP - 1997 May 22 TI - Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes. PG - 2395-406 AB - P130 shares structural and functional homology with pRb and p107. One property common to p107 and p130, but not to pRb, is the ability to stably interact with cyclin A/cdk2 and cyclin E/cdk2 complexes in vitro and in vivo. Using GST-p130 fusion proteins representing various regions of p130, baculovirus-produced cyclin A/cdk2 and cyclin E/cdk2 complexes were found to interact with residues within a part of p130 known as the spacer region. Cyclin E was able to bind the p130 spacer region in the presence or absence of cdk2 whereas cyclin A binding was dependent upon the presence of cdk2. The smallest p130 fusion protein sufficient to interact with cyclin A/cdk2 or cyclin E/cdk2 complexes contained p130 amino acids 652-698 and deletion of p130 amino acids 680-682 abolished binding to both of the cyclin/cdk2 complexes. When overexpressed in C33A cells, a p130 mutant containing a deletion of amino acids 620-697 was unable to form complexes with either cyclin A or cyclin E. This p130 mutant was at least as active as wild type p130 in suppressing the growth of G418 resistant colonies when overexpressed in C33A or SAOS-2 cells. AD - Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada. FAU - Lacy, S AU - Lacy S FAU - Whyte, P AU - Whyte P LA - eng PT - Journal Article PL - ENGLAND TA - Oncogene JID - 8711562 RN - 0 (Cyclins) RN - 0 (Nuclear Proteins) RN - 0 (Peptide Fragments) RN - 0 (Phosphoproteins) RN - 0 (Proteins) RN - 0 (Recombinant Proteins) RN - 0 (Retinoblastoma like protein p130) RN - 0 (retinoblastoma like protein p107) RN - EC 2.7.1.- (cyclin-dependent kinase 2) RN - EC 2.7.1.37 (CDC2-CDC28 Kinases) RN - EC 2.7.1.37 (Cyclin-Dependent Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - *CDC2-CDC28 Kinases MH - Cyclin-Dependent Kinases/*metabolism MH - Cyclins/*metabolism MH - Humans MH - Molecular Sequence Data MH - Nuclear Proteins/genetics/metabolism MH - Peptide Fragments/metabolism MH - Peptide Mapping MH - Phosphoproteins/*genetics/*metabolism MH - Protein-Serine-Threonine Kinases/*metabolism MH - *Proteins MH - Recombinant Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - *Sequence Homology, Amino Acid MH - Tumor Cells, Cultured EDAT- 1997/05/22 MHDA- 1997/05/22 00:01 AID - 10.1038/sj.onc.1201085 [doi] PST - ppublish SO - Oncogene 1997 May 22;14(20):2395-406. NR -------------------------------------------------------------------------------- 89: Meisner H et al. Interactions of Drosophila Cb...[PMID: 9121472] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9121472 OWN - NLM STAT- MEDLINE DA - 19970424 DCOM- 19970424 LR - 20041118 PUBM- Print IS - 0270-7306 VI - 17 IP - 4 DP - 1997 Apr TI - Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development. PG - 2217-25 AB - The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases. AD - Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA. Herman.Meisner@ummed.edu FAU - Meisner, H AU - Meisner H FAU - Daga, A AU - Daga A FAU - Buxton, J AU - Buxton J FAU - Fernandez, B AU - Fernandez B FAU - Chawla, A AU - Chawla A FAU - Banerjee, U AU - Banerjee U FAU - Czech, M P AU - Czech MP LA - eng SI - GENBANK/U87925 GR - RO1EY08152-06/EY/NEI PT - Journal Article PL - UNITED STATES TA - Mol Cell Biol JID - 8109087 RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (CBL protein, human) RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (growth factor receptor-bound protein-2) RN - EC 2.7.1.112 (Receptor, Epidermal Growth Factor) RN - EC 6.3.2.- (proto-oncogene protein c-cbl) RN - EC 6.3.2.19 (Ubiquitin-Protein Ligases) SB - IM MH - *Adaptor Proteins, Signal Transducing MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Binding Sites MH - Caenorhabditis elegans/genetics MH - DNA Primers/genetics MH - DNA, Complementary/genetics MH - Drosophila melanogaster/genetics/growth & development/*metabolism MH - Humans MH - Molecular Sequence Data MH - Photoreceptors, Invertebrate/cytology/growth & development/*metabolism MH - Protein Binding MH - Proteins/metabolism MH - Proto-Oncogene Proteins/genetics/*metabolism MH - Proto-Oncogenes MH - Receptor, Epidermal Growth Factor/*metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - *Ubiquitin-Protein Ligases MH - src Homology Domains EDAT- 1997/04/01 MHDA- 1997/04/01 00:01 PST - ppublish SO - Mol Cell Biol 1997 Apr;17(4):2217-25. NR -------------------------------------------------------------------------------- 90: Ceci JD et al. Tpl-2 is an oncogenic kinase ...[PMID: 9087424] Related Articles, Cited in PMC, Books, LinkOut PMID- 9087424 OWN - NLM STAT- MEDLINE DA - 19970502 DCOM- 19970502 LR - 20041118 PUBM- Print IS - 0890-9369 VI - 11 IP - 6 DP - 1997 Mar 15 TI - Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. PG - 688-700 AB - Provirus insertion in the last intron of the Tpl-2 gene in retrovirus-induced rat T-cell lymphomas results in the enhanced expression of a carboxy-terminally truncated Tpl-2 kinase. Here we show that the truncated protein exhibits an approximately sevenfold higher catalytic activity and is two- to threefold more efficient in activating the MAPK and SAPK pathways relative to the wild-type protein. The truncated Tpl-2 protein and a GST fusion of the Tpl-2 carboxy-terminal tail interact when coexpressed in Sf9 cells. Their interaction down-regulates the kinase activity of the truncated protein suggesting that tail-directed intramolecular interactions regulate the Tpl-2 kinase. Tpl-2 transgenic mice expressing the wild-type protein from the proximal Lck promoter fail to show a biological phenotype, whereas mice expressing the truncated protein develop large-cell lymphoblastic lymphomas of T-cell origin. These results show that Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. AD - National Cancer Institute-Frederick Cancer Research Facility and Development Center, Maryland 21702, USA. FAU - Ceci, J D AU - Ceci JD FAU - Patriotis, C P AU - Patriotis CP FAU - Tsatsanis, C AU - Tsatsanis C FAU - Makris, A M AU - Makris AM FAU - Kovatch, R AU - Kovatch R FAU - Swing, D A AU - Swing DA FAU - Jenkins, N A AU - Jenkins NA FAU - Tsichlis, P N AU - Tsichlis PN FAU - Copeland, N G AU - Copeland NG LA - eng GR - CA06927/CA/NCI GR - CA38047/CA/NCI PT - Journal Article PL - UNITED STATES TA - Genes Dev JID - 8711660 RN - 0 (Peptide Fragments) RN - 0 (Proto-Oncogene Proteins) RN - EC 2.7.1.123 (Ca(2+)-Calmodulin Dependent Protein Kinase) RN - EC 2.7.1.37 (MAP Kinase Kinase Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 2.7.10.- (Map3k8 protein, mouse) RN - EC 2.7.10.- (Map3k8 protein, rat) SB - IM MH - Amino Acid Sequence MH - Animals MH - Ca(2+)-Calmodulin Dependent Protein Kinase/metabolism MH - Cell Line MH - Enzyme Activation MH - In Vitro MH - Introns MH - Lymphoma, T-Cell/enzymology/genetics/virology MH - *MAP Kinase Kinase Kinases MH - Mice MH - Mice, Transgenic MH - Molecular Sequence Data MH - Moloney murine leukemia virus/genetics MH - Peptide Fragments/genetics MH - Protein-Serine-Threonine Kinases/*chemistry/genetics/*metabolism MH - Proto-Oncogene Proteins/*chemistry/genetics/*metabolism MH - Proto-Oncogenes MH - Proviruses/genetics MH - Rats MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Retroviridae Infections/enzymology/genetics/virology MH - Tumor Virus Infections/enzymology/genetics/virology EDAT- 1997/03/15 MHDA- 1997/03/15 00:01 PST - ppublish SO - Genes Dev 1997 Mar 15;11(6):688-700. DR -------------------------------------------------------------------------------- 91: Adler V et al. Conformation-dependent phosph...[PMID: 9050839] Related Articles, Compound via MeSH, Substance via MeSH, Free in PMC, Cited in PMC, Books, LinkOut PMID- 9050839 OWN - NLM STAT- MEDLINE DA - 19970407 DCOM- 19970407 LR - 20041117 PUBM- Print IS - 0027-8424 VI - 94 IP - 5 DP - 1997 Mar 4 TI - Conformation-dependent phosphorylation of p53. PG - 1686-91 AB - Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase. AD - Molecular Carcinogenesis Program, American Health Foundation, Valhalla, NY 10595, USA. FAU - Adler, V AU - Adler V FAU - Pincus, M R AU - Pincus MR FAU - Minamoto, T AU - Minamoto T FAU - Fuchs, S Y AU - Fuchs SY FAU - Bluth, M J AU - Bluth MJ FAU - Brandt-Rauf, P W AU - Brandt-Rauf PW FAU - Friedman, F K AU - Friedman FK FAU - Robinson, R C AU - Robinson RC FAU - Chen, J M AU - Chen JM FAU - Wang, X W AU - Wang XW FAU - Harris, C C AU - Harris CC FAU - Ronai, Z AU - Ronai Z LA - eng PT - Journal Article PL - UNITED STATES TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Peptide Fragments) RN - 0 (Protein p53) RN - 0 (Recombinant Fusion Proteins) RN - 56-65-5 (Adenosine Triphosphate) RN - 9007-49-2 (DNA) RN - EC 2.7.1.123 (Ca(2+)-Calmodulin Dependent Protein Kinase) RN - EC 2.7.1.37 (Casein Kinase II) RN - EC 2.7.1.37 (Cyclic AMP-Dependent Protein Kinases) RN - EC 2.7.1.37 (JNK Mitogen-Activated Protein Kinases) RN - EC 2.7.1.37 (Mitogen-Activated Protein Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Adenosine Triphosphate/metabolism MH - Amino Acid Sequence MH - Ca(2+)-Calmodulin Dependent Protein Kinase/metabolism MH - Casein Kinase II MH - Cyclic AMP-Dependent Protein Kinases/metabolism MH - DNA/metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Humans MH - JNK Mitogen-Activated Protein Kinases MH - *Mitogen-Activated Protein Kinases MH - Models, Molecular MH - Molecular Sequence Data MH - Peptide Fragments/chemical synthesis/metabolism/pharmacology MH - Peptide Mapping MH - Phosphorylation MH - Point Mutation MH - *Protein Conformation MH - Protein p53/*chemistry/genetics/*metabolism MH - Protein-Serine-Threonine Kinases/metabolism MH - Recombinant Fusion Proteins/chemistry/metabolism MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1997/03/04 MHDA- 1997/03/04 00:01 PST - ppublish SO - Proc Natl Acad Sci U S A 1997 Mar 4;94(5):1686-91. NR -------------------------------------------------------------------------------- 92: Furlong MT et al. Identification of the major s...[PMID: 9042338] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 9042338 OWN - NLM STAT- MEDLINE DA - 19970325 DCOM- 19970325 LR - 20041117 PUBM- Print IS - 0006-3002 VI - 1355 IP - 2 DP - 1997 Feb 4 TI - Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk. PG - 177-90 AB - The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1. AD - Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA. FAU - Furlong, M T AU - Furlong MT FAU - Mahrenholz, A M AU - Mahrenholz AM FAU - Kim, K H AU - Kim KH FAU - Ashendel, C L AU - Ashendel CL FAU - Harrison, M L AU - Harrison ML FAU - Geahlen, R L AU - Geahlen RL LA - eng GR - CA37372/CA/NCI GR - CA46882/CA/NCI PT - Journal Article PL - NETHERLANDS TA - Biochim Biophys Acta JID - 0217513 RN - 0 (Enzyme Precursors) RN - 0 (Isoenzymes) RN - 0 (Recombinant Proteins) RN - 21820-51-9 (Phosphotyrosine) RN - 55520-40-6 (Tyrosine) RN - EC 2.7.1.- (autophosphorylation-dependent multifunctional protein kinase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (SYK protein, human) RN - EC 2.7.1.37 (Protein Kinases) RN - EC 3.1.4.- (phospholipase C gamma) RN - EC 3.1.4.3 (Phospholipase C) SB - IM MH - Amino Acid Sequence MH - Animals MH - Binding Sites/physiology MH - Cell Line MH - Chromatography, High Pressure Liquid MH - Cloning, Molecular MH - Enzyme Precursors/*chemistry MH - Isoenzymes/chemistry MH - Mice MH - Molecular Sequence Data MH - Phospholipase C/chemistry MH - Phosphotyrosine/*chemistry MH - Protein Kinases/chemistry MH - Protein-Tyrosine Kinase/*chemistry MH - Recombinant Proteins/chemistry MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Tyrosine/*chemistry MH - src Homology Domains EDAT- 1997/02/04 MHDA- 1997/02/04 00:01 PST - ppublish SO - Biochim Biophys Acta 1997 Feb 4;1355(2):177-90. PR -------------------------------------------------------------------------------- 93: Rubio I et al. Real-time assay of the intera...[PMID: 9043696] Related Articles, Books, LinkOut PMID- 9043696 OWN - NLM STAT- MEDLINE DA - 19970502 DCOM- 19970502 LR - 20041117 PUBM- Print IS - 0736-6205 VI - 22 IP - 2 DP - 1997 Feb TI - Real-time assay of the interaction of a GST fusion protein with a protein ligate using resonant mirror technique. PG - 269-71 AD - University of Jena, Germany. FAU - Rubio, I AU - Rubio I FAU - Buckle, P AU - Buckle P FAU - Trutnau, H AU - Trutnau H FAU - Wetzker, R AU - Wetzker R LA - eng PT - Journal Article PL - UNITED STATES TA - Biotechniques JID - 8306785 RN - 0 (Enzymes, Immobilized) RN - 0 (Ligands) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1 (Phosphotransferases (Alcohol Group Acceptor)) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase MH - Animals MH - Baculoviridae/genetics MH - Enzymes, Immobilized MH - Glutathione Transferase/*genetics MH - *Ligands MH - Phosphotransferases (Alcohol Group Acceptor)/genetics MH - Proteins/*metabolism MH - Recombinant Fusion Proteins/*metabolism MH - Research Support, Non-U.S. Gov't MH - Spodoptera/metabolism EDAT- 1997/02/01 MHDA- 1997/02/01 00:01 PST - ppublish SO - Biotechniques 1997 Feb;22(2):269-71. PR -------------------------------------------------------------------------------- 94: Jousset C et al. A domain of TEL conserved in ...[PMID: 9009269] Related Articles, Cited in PMC, Books, LinkOut PMID- 9009269 OWN - NLM STAT- MEDLINE DA - 19970213 DCOM- 19970213 LR - 20050610 PUBM- Print IS - 0261-4189 VI - 16 IP - 1 DP - 1997 Jan 2 TI - A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein. PG - 69-82 AB - TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators. AD - CNRS UMR 146, Institut Curie-Section de Recherche, Centre Universitaire, Orsay, France. FAU - Jousset, C AU - Jousset C FAU - Carron, C AU - Carron C FAU - Boureux, A AU - Boureux A FAU - Quang, C T AU - Quang CT FAU - Oury, C AU - Oury C FAU - Dusanter-Fourt, I AU - Dusanter-Fourt I FAU - Charon, M AU - Charon M FAU - Levin, J AU - Levin J FAU - Bernard, O AU - Bernard O FAU - Ghysdael, J AU - Ghysdael J LA - eng SI - GENBANK/L19541 PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (DNA-Binding Proteins) RN - 0 (ETS translocation variant 6 protein) RN - 0 (Oncogene Proteins) RN - 0 (Repressor Proteins) RN - 0 (Transcription Factors) RN - EC 2.7.1.112 (Receptor, Platelet-Derived Growth Factor beta) RN - EC 2.7.1.112 (Receptors, Platelet-Derived Growth Factor) SB - IM MH - Amino Acid Sequence MH - Animals MH - Binding Sites MH - Cell Division MH - Conserved Sequence MH - DNA-Binding Proteins/chemistry/*physiology MH - Drosophila MH - Hela Cells MH - Humans MH - Molecular Sequence Data MH - Oncogene Proteins/chemistry/*physiology MH - Receptor, Platelet-Derived Growth Factor beta MH - Receptors, Platelet-Derived Growth Factor/chemistry/*physiology MH - *Repressor Proteins MH - Research Support, Non-U.S. Gov't MH - Sequence Deletion MH - Sequence Homology, Amino Acid MH - Transcription Factors/chemistry/*physiology EDAT- 1997/01/02 MHDA- 1997/01/02 00:01 AID - 10.1093/emboj/16.1.69 [doi] PST - ppublish SO - EMBO J 1997 Jan 2;16(1):69-82. NR -------------------------------------------------------------------------------- 95: Elke C et al. Expression of EcR and USP in ...[PMID: 9131781] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 9131781 OWN - NLM STAT- MEDLINE DA - 19970604 DCOM- 19970604 LR - 20041117 PUBM- Print IS - 0739-4462 VI - 35 IP - 1-2 DP - 1997 TI - Expression of EcR and USP in Escherichia coli: purification and functional studies. PG - 59-69 AB - The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B. AD - Institut Fur Zellbiologie, ETH-Honggerberg, Zurich, Switzerland. FAU - Elke, C AU - Elke C FAU - Vogtli, M AU - Vogtli M FAU - Rauch, P AU - Rauch P FAU - Spindler-Barth, M AU - Spindler-Barth M FAU - Lezzi, M AU - Lezzi M LA - eng PT - Journal Article PL - UNITED STATES TA - Arch Insect Biochem Physiol JID - 8501752 RN - 0 (CF1 protein, insect) RN - 0 (DNA-Binding Proteins) RN - 0 (Invertebrate Hormones) RN - 0 (Ligands) RN - 0 (Receptors, Steroid) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - 0 (ecdysteroid receptor) RN - 5289-74-7 (Ecdysterone) RN - 9007-49-2 (DNA) SB - IM MH - Animals MH - Chironomidae/genetics/metabolism MH - Cloning, Molecular MH - DNA/metabolism MH - DNA-Binding Proteins/*genetics/metabolism MH - Drosophila melanogaster/genetics/metabolism MH - *Ecdysterone MH - Escherichia coli MH - Invertebrate Hormones/*genetics/metabolism MH - Ligands MH - Receptors, Steroid/*genetics/metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Transcription Factors/*genetics/metabolism EDAT- 1997/01/01 MHDA- 2000/06/20 09:00 AID - 10.1002/(SICI)1520-6327(1997)35:1/2<59::AID-ARCH6>3.0.CO;2-S [pii] PST - ppublish SO - Arch Insect Biochem Physiol 1997;35(1-2):59-69. NR -------------------------------------------------------------------------------- 96: Yacoub A et al. Drosophila ribosomal protein ...[PMID: 8932386] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 8932386 OWN - NLM STAT- MEDLINE DA - 19961220 DCOM- 19961220 LR - 20041117 PUBM- Print IS - 0305-1048 VI - 24 IP - 21 DP - 1996 Nov 1 TI - Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity. PG - 4298-303 AB - Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA. AD - Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA. FAU - Yacoub, A AU - Yacoub A FAU - Kelley, M R AU - Kelley MR FAU - Deutsch, W A AU - Deutsch WA LA - eng GR - RR 09884/RR/NCRR PT - Journal Article PL - ENGLAND TA - Nucleic Acids Res JID - 0411011 RN - 0 (Antibodies) RN - 0 (Drosophila Proteins) RN - 0 (Escherichia coli Proteins) RN - 0 (Insect Hormones) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Ribosomal Proteins) RN - 0 (Ribosomal protein P0, Drosophila) RN - 9007-49-2 (DNA) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.1.21.2 (Deoxyribonuclease IV (Phage T4-Induced)) RN - EC 3.1.21.2 (endonuclease IV, E coli) RN - EC 4. (Lyases) RN - EC 4.2.99.18 (DNA-(Apurinic or Apyrimidinic Site) Lyase) SB - IM MH - Animals MH - Antibodies/immunology MH - Blotting, Western MH - DNA/metabolism MH - DNA-(Apurinic or Apyrimidinic Site) Lyase MH - Deoxyribonuclease IV (Phage T4-Induced) MH - *Drosophila Proteins MH - Drosophila melanogaster MH - Escherichia coli MH - *Escherichia coli Proteins MH - Glutathione Transferase/genetics/metabolism MH - Insect Hormones/genetics/immunology/*metabolism MH - Lyases/genetics/*metabolism MH - Mutation MH - Nuclear Matrix/metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Ribosomal Proteins/genetics/*metabolism EDAT- 1996/11/01 MHDA- 1996/11/01 00:01 AID - 6s0322 [pii] PST - ppublish SO - Nucleic Acids Res 1996 Nov 1;24(21):4298-303. NR -------------------------------------------------------------------------------- 97: Hirao M et al. Regulation mechanism of ERM (...[PMID: 8858161] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Domains, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8858161 OWN - NLM STAT- MEDLINE DA - 19961125 DCOM- 19961125 LR - 20041117 PUBM- Print IS - 0021-9525 VI - 135 IP - 1 DP - 1996 Oct TI - Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. PG - 37-51 AB - The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism. AD - Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan. FAU - Hirao, M AU - Hirao M FAU - Sato, N AU - Sato N FAU - Kondo, T AU - Kondo T FAU - Yonemura, S AU - Yonemura S FAU - Monden, M AU - Monden M FAU - Sasaki, T AU - Sasaki T FAU - Takai, Y AU - Takai Y FAU - Tsukita, S AU - Tsukita S FAU - Tsukita, S AU - Tsukita S LA - eng PT - Journal Article PL - UNITED STATES TA - J Cell Biol JID - 0375356 RN - 0 (Antibodies, Monoclonal) RN - 0 (Antigens, CD44) RN - 0 (Blood Proteins) RN - 0 (Botulinum Toxins) RN - 0 (Cytoskeletal Proteins) RN - 0 (Guanine Nucleotide Dissociation Inhibitors) RN - 0 (Membrane Proteins) RN - 0 (Microfilament Proteins) RN - 0 (Phosphatidylinositol 4,5-Diphosphate) RN - 0 (Phospholipids) RN - 0 (Phosphoproteins) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (ezrin) RN - 0 (rhoB GTP-Binding Protein) RN - 133312-85-3 (rhoB p20 GDI) RN - 144131-77-1 (moesin) RN - 144517-21-5 (radixin) RN - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate)) RN - EC 2.4.2.- (ADP Ribose Transferases) RN - EC 2.4.2.- (exoenzyme C3, Clostridium botulinum) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.- (GTP-Binding Proteins) SB - IM MH - ADP Ribose Transferases/pharmacology MH - Animals MH - Antibodies, Monoclonal MH - Antigens, CD44/genetics/metabolism MH - Binding, Competitive/drug effects MH - Blood Proteins/genetics/*metabolism MH - *Botulinum Toxins MH - Cell Line MH - Cell Membrane/*metabolism MH - Cytoplasm MH - *Cytoskeletal Proteins MH - GTP-Binding Proteins/genetics/*physiology MH - Glutathione Transferase/genetics/metabolism MH - *Guanine Nucleotide Dissociation Inhibitors MH - Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology MH - Hamsters MH - Kidney MH - Membrane Proteins/genetics/*metabolism/*physiology MH - Mice MH - *Microfilament Proteins MH - Phosphatidylinositol 4,5-Diphosphate/*metabolism MH - Phospholipids/pharmacology MH - Phosphoproteins/genetics/*metabolism MH - Protein Binding/drug effects MH - Proteins/genetics/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Signal Transduction MH - rhoB GTP-Binding Protein EDAT- 1996/10/01 MHDA- 1996/10/01 00:01 PST - ppublish SO - J Cell Biol 1996 Oct;135(1):37-51. DR -------------------------------------------------------------------------------- 98: Settlage SB et al. Interactions between geminivi...[PMID: 8794317] Related Articles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 8794317 OWN - NLM STAT- MEDLINE DA - 19961122 DCOM- 19961122 LR - 20041117 PUBM- Print IS - 0022-538X VI - 70 IP - 10 DP - 1996 Oct TI - Interactions between geminivirus replication proteins. PG - 6790-5 AB - Geminiviruses are small DNA viruses that replicate in the nuclei of infected plant cells. The closely related geminiviruses tomato golden mosaic virus and bean golden mosaic virus each encode a protein, AL1, that catalyzes the initiation of rolling-circle replication. Both viruses also specify a second replication protein, AL3, that greatly enhances the level of viral DNA accumulation. Using recombinant proteins produced in a baculovirus expression system, we showed that AL1 copurifies with a protein fusion of glutathione S-transferase (GST) and AL1, independent of the GST domain. Similarly, authentic AL3 cofractionates with a GST-AL3 fusion protein. These results demonstrated that both AL1 and AL3 form oligomers. Immunoprecipitation of protein extracts from insect cells expressing both AL1 and AL3 showed that the two proteins also complex with each other. None of the protein interactions displayed virus specificity; the tomato and bean golden mosaic virus proteins complexed with each other. The addition of heterologous replication proteins had no effect on the efficiency of geminivirus replication in transient-replication assays, suggesting that heteroprotein complexes might be functional. The significance of these protein interactions is discussed with respect to geminivirus replication in plant cells. AD - Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA. FAU - Settlage, S B AU - Settlage SB FAU - Miller, A B AU - Miller AB FAU - Hanley-Bowdoin, L AU - Hanley-Bowdoin L LA - eng GR - GM-16681-02/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (Viral Proteins) RN - 0 (replication protein AL1, Begomovirus) SB - IM MH - Geminiviridae/*physiology MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Research Support, U.S. Gov't, P.H.S. MH - Viral Proteins/*metabolism MH - *Virus Replication EDAT- 1996/10/01 MHDA- 1996/10/01 00:01 PST - ppublish SO - J Virol 1996 Oct;70(10):6790-5. NR -------------------------------------------------------------------------------- 99: Stein E et al. Ligand activation of ELK rece...[PMID: 8798570] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8798570 OWN - NLM STAT- MEDLINE DA - 19961119 DCOM- 19961119 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 271 IP - 38 DP - 1996 Sep 20 TI - Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells. PG - 23588-93 AB - ELK is a member of the Eph-related tyrosine kinase family that includes receptors signaling axonal guidance, neuronal bundling, and angiogenesis. We recently identified ELK expression in human renal microvascular endothelial cells and sought to identify intracellular proteins through which it signals responses. The cytoplasmic domain of ELK was used as "bait" in a yeast two-hybrid screen to identify interactive proteins expressed from a randomly primed embryonic murine library (E9.5-10.5). Among interactive products of 76 cDNAs characterized, 10 nonidentical, overlapping clones encoded the SH2 domain of the recently reported Grb10 adapter protein, and an additional 3 encoded Grb2. A self-phosphorylated recombinant, baculovirus-expressed GST-ELKcy fusion protein bound Grb10 and Grb2 from human renal microvascular endothelial cell extracts, while the unphosphorylated fusion form did not. Site-directed mutation identified Tyr-929 as a putative phosphorylation site required for Grb10, but not Grb2, interaction in yeast and recombinant protein assays. The ELK ligand, LERK-2/Fc, stimulated tyrosine phosphorylation of ELK, and recruitment of Grb10 and Grb2 to endothelial ELK receptors recovered by wheat germ agglutinin lectin and immunoprecipitation. These findings define ligand-activated interaction between ELK and the SH2 domains of Grb2 and the newly identified Grb10 protein that shares homology with a Caenorhabditis elegans gene product implicated in neural cell migration. AD - Departments of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. FAU - Stein, E AU - Stein E FAU - Cerretti, D P AU - Cerretti DP FAU - Daniel, T O AU - Daniel TO LA - eng GR - DK38517/DK/NIDDK GR - DK47078/DK/NIDDK PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (DNA-Binding Proteins) RN - 0 (Ephrin-B1) RN - 0 (Ligands) RN - 0 (Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - 0 (ets-domain protein elk-1) RN - 0 (growth factor receptor-bound protein-2) RN - 151441-47-3 (growth factor receptor-bound protein 10) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) SB - IM MH - *Adaptor Proteins, Signal Transducing MH - Animals MH - *DNA-Binding Proteins MH - Endothelium, Vascular/*metabolism MH - Ephrin-B1 MH - Humans MH - Kidney/blood supply/cytology MH - Ligands MH - Mice MH - Phosphorylation MH - Protein Binding/genetics MH - Proteins/*metabolism MH - Proto-Oncogene Proteins/*metabolism MH - Receptor Protein-Tyrosine Kinases/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Saccharomyces cerevisiae/genetics MH - Signal Transduction MH - *Transcription Factors EDAT- 1996/09/20 MHDA- 1996/09/20 00:01 PST - ppublish SO - J Biol Chem 1996 Sep 20;271(38):23588-93. DR -------------------------------------------------------------------------------- 100: Khromykh AA et al. Expression and purification o...[PMID: 8882936] Related Articles, Cited in PMC, Books, LinkOut PMID- 8882936 OWN - NLM STAT- MEDLINE DA - 19970114 DCOM- 19970114 LR - 20041203 PUBM- Print IS - 0166-0934 VI - 61 IP - 1-2 DP - 1996 Sep TI - Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E. coli and the baculovirus expression systems. PG - 47-58 AB - All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST). High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E. coli. The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg). NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E. coli cultures transformed with the expression plasmid and induced with IPTG. Only delta NS3 protein was recovered intact by removing GST from the fusion protein by digestion with Factor Xa protease. Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient cleavage or in degradation of the proteins. No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-delta NS3, GST-NS4A, and GST-NS4B was eluted off the GS beads by adding glutathione. Thus, KUN purified recombinant proteins, either in eluted form or while immobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions. AD - Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, QLD, Australia. A.Khromykh@mailbox.uq.oz.au FAU - Khromykh, A A AU - Khromykh AA FAU - Harvey, T J AU - Harvey TJ FAU - Abedinia, M AU - Abedinia M FAU - Westaway, E G AU - Westaway EG LA - eng PT - Journal Article PL - NETHERLANDS TA - J Virol Methods JID - 8005839 RN - 0 (NS1 protein, Flavivirus) RN - 0 (NS2B protein, flavivirus) RN - 0 (NS3 protein, flavivirus) RN - 0 (NS4B protein, flavivirus) RN - 0 (NS5 protein, flavivirus) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Nonstructural Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.7.- (RNA Helicases) RN - EC 3.4.21 (Serine Endopeptidases) SB - IM MH - Animals MH - Baculoviridae/*metabolism MH - Cell Line MH - Escherichia coli/*metabolism MH - Flavivirus/*genetics/metabolism MH - Gene Expression MH - Glutathione Transferase/genetics MH - RNA Helicases MH - Recombinant Fusion Proteins/genetics/isolation & purification MH - Research Support, Non-U.S. Gov't MH - Serine Endopeptidases MH - Spodoptera/cytology MH - Viral Nonstructural Proteins/*genetics/isolation & purification EDAT- 1996/09/01 MHDA- 1996/09/01 00:01 PST - ppublish SO - J Virol Methods 1996 Sep;61(1-2):47-58. DR -------------------------------------------------------------------------------- 101: Banin S et al. Wiskott-Aldrich syndrome prot...[PMID: 8805332] Related Articles, Gene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8805332 OWN - NLM STAT- MEDLINE DA - 19961107 DCOM- 19961107 LR - 20050201 PUBM- Print IS - 0960-9822 VI - 6 IP - 8 DP - 1996 Aug 1 TI - Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases. PG - 981-8 AB - BACKGROUND. Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS. We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS. Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells. AD - Leukaemia Research Fund Centre for Childhood Leukaemia, Molecular Haematology Unit, Institute of Child Health, 30 Guilford Street, LondonWC1N 1EH, UK. FAU - Banin, S AU - Banin S FAU - Truong, O AU - Truong O FAU - Katz, D R AU - Katz DR FAU - Waterfield, M D AU - Waterfield MD FAU - Brickell, P M AU - Brickell PM FAU - Gout, I AU - Gout I LA - eng PT - Journal Article PL - ENGLAND TA - Curr Biol JID - 9107782 RN - 0 (Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Wiskott-Aldrich syndrome protein) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (proto-oncogene protein c-fyn) RN - EC 2.7.1.112 (src-Family Kinases) SB - IM MH - Blotting, Western MH - Cell Line MH - Chromatography, Affinity MH - Glutathione Transferase/metabolism MH - Humans MH - Protein Binding MH - Proteins/isolation & purification/*metabolism MH - Proto-Oncogene Proteins/metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Wiskott-Aldrich Syndrome/*metabolism MH - src Homology Domains MH - src-Family Kinases/*metabolism EDAT- 1996/08/01 MHDA- 1996/08/01 00:01 PST - ppublish SO - Curr Biol 1996 Aug 1;6(8):981-8. NR -------------------------------------------------------------------------------- 102: Sugimoto K et al. Human homolog of Drosophila h...[PMID: 8864858] Related Articles, Gene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8864858 OWN - NLM STAT- MEDLINE DA - 19970114 DCOM- 19970114 LR - 20041117 PUBM- Print IS - 0021-924X VI - 120 IP - 1 DP - 1996 Jul TI - Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C). PG - 153-9 AB - Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal component tightly associated with the pericentromeric heterochromatic region of fruit fly, mouse, and human throughout the cell cycle. Drosophila HP1 has been shown to be involved in position effect variegation and to be required for the correct chromosome segregation in vivo, while the biological activity of human homolog (HP1Hsa) has not yet been characterized. We previously reported that human CENP-B and CENP-C, two major centromere heterochromatin autoantigens often recognized by autosera in scleroderma patients, possess DNA-binding activity in vitro. Here, we show that human HP1, which is also an autoantigen targeted by some types of anticentromere autosera, is a DNA-binding protein. Human HP1 was expressed as a GST-fusion in Escherichia coli and purified with glutathione-Sepharose. The DNA-binding activity of the recombinant HP1 was demonstrated by gel mobility shift assay and South-Western-type blotting. The minimum DNA-binding region was further limited to the internal 64-amino acid stretch that is less-conserved between human and fruit fly but retains a helix-enriched motif with weak similarity to CENP-C. This suggests that HP1 is involved in the pericentromeric heterochromatin formation by directly associating with genomic DNA. AD - Department of Applied Biochemistry, University of Osaka Prefecture. FAU - Sugimoto, K AU - Sugimoto K FAU - Yamada, T AU - Yamada T FAU - Muro, Y AU - Muro Y FAU - Himeno, M AU - Himeno M LA - eng PT - Journal Article PL - JAPAN TA - J Biochem (Tokyo) JID - 0376600 RN - 0 (Autoantigens) RN - 0 (Chromosomal Proteins, Non-Histone) RN - 0 (DNA-Binding Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (centromere autoantigen 140K) RN - 107283-02-3 (heterochromatin-specific nonhistone chromosomal protein HP-1) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Autoantigens/chemistry MH - Chromosomal Proteins, Non-Histone/*chemistry/genetics/isolation & purification MH - Cloning, Molecular MH - DNA-Binding Proteins/*chemistry/genetics/isolation & purification MH - Drosophila MH - Epitope Mapping MH - Escherichia coli/genetics MH - Glutathione Transferase/genetics MH - Hela Cells MH - Humans MH - Molecular Sequence Data MH - Protein Structure, Secondary MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - *Sequence Homology, Amino Acid EDAT- 1996/07/01 MHDA- 1996/07/01 00:01 PST - ppublish SO - J Biochem (Tokyo) 1996 Jul;120(1):153-9. NR -------------------------------------------------------------------------------- 103: Seif SA et al. A 27 amino acid coding region...[PMID: 8822638] Related Articles, Cited in PMC, Books, LinkOut PMID- 8822638 OWN - NLM STAT- MEDLINE DA - 19961204 DCOM- 19961204 LR - 20041117 PUBM- Print IS - 0168-1702 VI - 43 IP - 1 DP - 1996 Jul TI - A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. PG - 91-6 AB - We have recently shown that neutralizing epitope(s) exist near the C-terminal of JE virus E-protein by expressing the coding gene cDNA fragments as fusion proteins with protein A. Among four cDNA fragments, the fragment (B3) carrying the coding sequence of amino acid number 373-399 of E protein elicited the highest neutralizing (N) antibody titer (1:75). To exclude the possible influence of protein A contained in the expressed gene products on the mouse immune response, we expressed (B3) using pGEX-3X expression vector as fusion with glutathione-S transferase (GST). The mice immunized with recombinant GST-B3 fusion protein induced an immune response (mean average ELISA: 3364; N: 1:75) almost similar to that by recombinant protein A-B3 fusion protein (mean average ELISA: 3476; N: 1:75). While hemagglutination-inhibition (HI) antibodies were not induced by this fusion protein. These results indicate that 27 amino acid sequence on the E protein (373-399) was sufficient to induce neutralizing antibodies without association with protein A moiety. AD - Department of Virology, Nagasaki University, Japan. FAU - Seif, S A AU - Seif SA FAU - Morita, K AU - Morita K FAU - Igarashi, A AU - Igarashi A LA - eng PT - Journal Article PL - NETHERLANDS TA - Virus Res JID - 8410979 RN - 0 (Antibodies, Viral) RN - 0 (Membrane Glycoproteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Viral Envelope Proteins) RN - 0 (glycoprotein E, Japanese encephalitis virus) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Aedes/cytology MH - Animals MH - Antibodies, Viral/*immunology MH - Cell Line MH - Encephalitis Virus, Japanese/*immunology MH - Escherichia coli MH - Glutathione Transferase/immunology MH - Hamsters MH - Membrane Glycoproteins/genetics/*immunology MH - Mice MH - Neutralization Tests MH - Recombinant Fusion Proteins/genetics/immunology MH - Research Support, Non-U.S. Gov't MH - Viral Envelope Proteins/genetics/*immunology EDAT- 1996/07/01 MHDA- 1996/07/01 00:01 AID - 0168170296013238 [pii] PST - ppublish SO - Virus Res 1996 Jul;43(1):91-6. NR -------------------------------------------------------------------------------- 104: Moore JD et al. A point mutation in the micro...[PMID: 8670831] Related Articles, Gene, HomoloGene, UniGene, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8670831 OWN - NLM STAT- MEDLINE DA - 19960830 DCOM- 19960830 LR - 20041117 PUBM- Print IS - 0261-4189 VI - 15 IP - 13 DP - 1996 Jul 1 TI - A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity. PG - 3306-14 AB - Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role. AD - Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA. FAU - Moore, J D AU - Moore JD FAU - Song, H AU - Song H FAU - Endow, S A AU - Endow SA LA - eng PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (Drosophila Proteins) RN - 0 (Microtubule Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (ncd protein, Drosophila) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.6.1.- (Kinesin) RN - EC 3.6.1.3 (Adenosinetriphosphatase) SB - IM MH - Adenosinetriphosphatase/antagonists & inhibitors/metabolism MH - Animals MH - Binding Sites MH - Biological Transport MH - Drosophila/genetics MH - *Drosophila Proteins MH - Enzyme Activation MH - Glutathione Transferase/genetics MH - *Kinesin MH - Microtubule Proteins/genetics/*metabolism MH - Microtubules/*metabolism MH - *Point Mutation MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1996/07/01 MHDA- 1996/07/01 00:01 PST - ppublish SO - EMBO J 1996 Jul 1;15(13):3306-14. NR -------------------------------------------------------------------------------- 105: Matsuzaki K et al. Reconstitution of a pentameri...[PMID: 8842749] Related Articles, Substance via MeSH, Books, LinkOut PMID- 8842749 OWN - NLM STAT- MEDLINE DA - 19961219 DCOM- 19961219 LR - 20041117 PUBM- Print IS - 1071-2690 VI - 32 IP - 6 DP - 1996 Jun TI - Reconstitution of a pentameric complex of dimeric transforming growth factor beta ligand and a type I, II, III receptor in baculoviral-infected insect cells. PG - 345-60 AB - Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric ligand participate in the transforming growth factor beta type one (TGF beta 1) signal transduction complex. The expression of recombinant receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among the ectodomains of the three types of receptors and the TGF beta 1 ligand in absence of uncontrollable extrinsic factors in mammalian cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed among receptor subunits as follows: type III-III, type I-I, type III-I, and type II-I. The homeotypic complex of type II-II receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor rather than specificity for ligand. A monomeric subunit of the TGF beta 1 ligand bound concurrently to type III and type II or type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGF beta 1 to the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit. The combined results suggest a pentameric TGF beta signal transduction complex in which one unit each of the type III, type II, and type I components is assembled around the two subunits of the dimeric TGF beta ligand. An immobilized GST-tagged subunit of the receptor complex was utilized to assemble multi-subunit complexes in vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation occurs in the type II kinase; (c) both the type III and type I subunits are trans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit and dimeric TGF beta 1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits. AD - Albert B. Alkek Institute of Biosciences and Technology, Department of Biochemistry and Biophysics, Texas A&M University, Houston 77030-3303, USA. FAU - Matsuzaki, K AU - Matsuzaki K FAU - Kan, M AU - Kan M FAU - McKeehan, W L AU - McKeehan WL LA - eng GR - CA59971/CA/NCI GR - DK35310/DK/NIDDK GR - DK38639/DK/NIDDK PT - Journal Article PL - UNITED STATES TA - In Vitro Cell Dev Biol Anim JID - 9418515 RN - 0 (Macromolecular Substances) RN - 0 (Receptors, Transforming Growth Factor beta) RN - 0 (Recombinant Proteins) RN - 0 (Transforming Growth Factor beta) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Animals MH - Baculoviridae/*genetics MH - Cell Line MH - Gene Expression MH - Macromolecular Substances MH - Mutagenesis, Site-Directed MH - Phosphorylation MH - Protein-Serine-Threonine Kinases/metabolism MH - Receptors, Transforming Growth Factor beta/chemistry/genetics/*metabolism MH - Recombinant Proteins MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera/*metabolism MH - Transfection MH - Transforming Growth Factor beta/chemistry/*metabolism EDAT- 1996/06/01 MHDA- 1996/06/01 00:01 PST - ppublish SO - In Vitro Cell Dev Biol Anim 1996 Jun;32(6):345-60. DR -------------------------------------------------------------------------------- 106: Lehr RV et al. Production, purification and ...[PMID: 8647461] Related Articles, Books, LinkOut PMID- 8647461 OWN - NLM STAT- MEDLINE DA - 19960722 DCOM- 19960722 LR - 20031114 PUBM- Print IS - 0378-1119 VI - 169 IP - 2 DP - 1996 Mar 9 TI - Production, purification and characterization of non-myristylated human T-cell protein tyrosine kinase in a baculovirus expression system. PG - 275-9 AB - A non-myristylated form (LCK M) of the human T-lymphocyte-specific protein tyrosine kinase (LCK) was produced at high levels in a baculovirus expression system (BVES) using two strategies. First, LCK M was produced by direct expression of a Gly2 --> Ala mutant of LCK. Second, LCK was produced as a glutathione S-transferase (GST) fusion, and LCK M was derived from the fusion protein by cleavage with thrombin. Both recombinant proteins (re-proteins) were produced at 5% of the total protein of infected Spodoptera frugiperda (Sf9) cells and were purified to >95% homogeneity. The enzymatic properties of the re-proteins and their inhibition by protein kinase inhibitors were comparable to the native enzyme (LCK N) derived from Jurkat cells and wild-type LCK derived from the BVES. The high production levels will facilitate the recovery of large quantities of re-protein for use in biochemical and biophysical studies. AD - Department of Molecular and Cellular Biology, Sterling Winthrop Inc., Collegeville, PA 19426-0900, USA. FAU - Lehr, R V AU - Lehr RV FAU - Ma, Y G AU - Ma YG FAU - Kratz, D AU - Kratz D FAU - Brake, P G AU - Brake PG FAU - Wang, S AU - Wang S FAU - Faltynek, C R AU - Faltynek CR FAU - Wang, X M AU - Wang XM FAU - Stevis, P E AU - Stevis PE LA - eng PT - Journal Article PL - NETHERLANDS TA - Gene JID - 7706761 RN - 0 (Genetic Vectors) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (src-Family Kinases) RN - EC 2.7.1.37 (Lymphocyte Specific Protein Tyrosine Kinase p56(lck)) SB - IM MH - Amino Acid Sequence MH - Animals MH - Baculoviridae/*genetics MH - Base Sequence MH - Cell Line MH - Gene Expression/*genetics MH - Genetic Vectors/*genetics MH - Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MH - Molecular Sequence Data MH - Protein-Tyrosine Kinase/*genetics MH - Recombinant Fusion Proteins/chemistry/genetics/metabolism MH - Spodoptera/cytology MH - T-Lymphocytes/*enzymology MH - src-Family Kinases/chemistry/*genetics/metabolism EDAT- 1996/03/09 MHDA- 1996/03/09 00:01 AID - 0378111995008179 [pii] PST - ppublish SO - Gene 1996 Mar 9;169(2):275-9. DR -------------------------------------------------------------------------------- 107: Soker S et al. Characterization of novel vas...[PMID: 8621443] Related Articles, Cited in PMC, Cited in Books, Books, LinkOut PMID- 8621443 OWN - NLM STAT- MEDLINE DA - 19960620 DCOM- 19960620 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 271 IP - 10 DP - 1996 Mar 8 TI - Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. PG - 5761-7 AB - Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked 125I-VEGF165 to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF165 binding site, with a Kd of 2.8 x 10(-10) M and 0.95 1.1 x 10(5) binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a Kd of 7.5 x 10(-12) M, presumably FLK/KDR, and the other with a Kd of 2 x 10(-10) M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked 125I-VEGF165 but not 125I-VEGF121. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF165 that are absent in VEGF121, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF165 and inhibited the binding of 125I-VEGF165 to 231 cells. Cross-linking of 125I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF165 receptors. It was concluded that certain tumor-derived cell lines express novel surface-associated receptors that selectively bind VEGF165 via the exon 7-encoded domain, which is absent in VEGF121. AD - Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. FAU - Soker, S AU - Soker S FAU - Fidder, H AU - Fidder H FAU - Neufeld, G AU - Neufeld G FAU - Klagsbrun, M AU - Klagsbrun M LA - eng GR - CA37392/CA/NCI GR - GM47397/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Cross-Linking Reagents) RN - 0 (DNA Primers) RN - 0 (Endothelial Growth Factors) RN - 0 (Lymphokines) RN - 0 (Receptors, Growth Factor) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Vascular Endothelial Growth Factor A) RN - 0 (Vascular Endothelial Growth Factors) RN - 0 (vascular endothelial growth factor A, human) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) RN - EC 2.7.1.112 (Receptors, Vascular Endothelial Growth Factor) SB - IM MH - Animals MH - Base Sequence MH - Binding Sites MH - Cell Line MH - Cells, Cultured MH - Chromatography, Affinity MH - Cross-Linking Reagents MH - DNA Primers MH - Endothelial Growth Factors/*genetics/isolation & purification/*metabolism MH - Endothelium, Vascular/*metabolism MH - *Exons MH - Glutathione Transferase/metabolism MH - Humans MH - Kinetics MH - Lymphokines/*genetics/isolation & purification/*metabolism MH - Molecular Sequence Data MH - Polymerase Chain Reaction MH - Receptor Protein-Tyrosine Kinases/isolation & purification/*metabolism MH - Receptors, Growth Factor/isolation & purification/*metabolism MH - Receptors, Vascular Endothelial Growth Factor MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Transfection MH - Tumor Cells, Cultured MH - Umbilical Veins MH - Vascular Endothelial Growth Factor A MH - Vascular Endothelial Growth Factors EDAT- 1996/03/08 MHDA- 1996/03/08 00:01 PST - ppublish SO - J Biol Chem 1996 Mar 8;271(10):5761-7. PR -------------------------------------------------------------------------------- 108: Otto JC et al. The hepatitis delta virus lar...[PMID: 8617711] Related Articles, Compound via MeSH, Substance via MeSH, Protein, Cited in PMC, Books, LinkOut PMID- 8617711 OWN - NLM STAT- MEDLINE DA - 19960611 DCOM- 19960611 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 271 IP - 9 DP - 1996 Mar 1 TI - The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells. PG - 4569-72 AB - The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent Km = 0.8 microM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection. AD - Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686, USA. FAU - Otto, J C AU - Otto JC FAU - Casey, P J AU - Casey PJ LA - eng GR - GM46372/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Hepatitis Antigens) RN - 0 (Hepatitis Delta Virus) RN - 0 (Hepatitis delta Antigens) RN - 0 (Polyisoprenyl Phosphates) RN - 0 (Recombinant Fusion Proteins) RN - 0 (hepatitis delta virus large antigen) RN - 13058-04-3 (farnesyl pyrophosphate) RN - 6699-20-3 (geranylgeranyl pyrophosphate) RN - EC 2.5.1.1 (Dimethylallyltranstransferase) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Brain/enzymology MH - Cattle MH - Cell Line MH - Cercopithecus aethiops MH - Chromatography, High Pressure Liquid MH - Cytosol/enzymology MH - Dimethylallyltranstransferase/*metabolism MH - Glutathione Transferase/metabolism MH - Hepatitis Antigens/chemistry/isolation & purification/*metabolism MH - Hepatitis Delta Virus/*metabolism MH - Hepatitis delta Antigens MH - Kinetics MH - Molecular Sequence Data MH - Polyisoprenyl Phosphates/metabolism MH - *Protein Isoprenylation MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Transfection EDAT- 1996/03/01 MHDA- 1996/03/01 00:01 PST - ppublish SO - J Biol Chem 1996 Mar 1;271(9):4569-72. PR -------------------------------------------------------------------------------- 109: Hirose F et al. Isolation and characterizatio...[PMID: 8632015] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8632015 OWN - NLM STAT- MEDLINE DA - 19960702 DCOM- 19960702 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 271 IP - 7 DP - 1996 Feb 16 TI - Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. PG - 3930-7 AB - DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknullt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences. AD - Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan. FAU - Hirose, F AU - Hirose F FAU - Yamaguchi, M AU - Yamaguchi M FAU - Kuroda, K AU - Kuroda K FAU - Omori, A AU - Omori A FAU - Hachiya, T AU - Hachiya T FAU - Ikeda, M AU - Ikeda M FAU - Nishimoto, Y AU - Nishimoto Y FAU - Matsukage, A AU - Matsukage A LA - eng SI - GENBANK/AB010823 PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (Dref protein, Drosophila) RN - 0 (Drosophila Proteins) RN - 0 (RNA, Messenger) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - EC 2.3.1.28 (Chloramphenicol O-Acetyltransferase) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Cell Line MH - Chloramphenicol O-Acetyltransferase/biosynthesis MH - DNA Footprinting MH - DNA Primers MH - DNA Replication/*genetics MH - DNA, Complementary/isolation & purification/metabolism MH - *Drosophila Proteins MH - Drosophila melanogaster/*genetics MH - Embryo, Nonmammalian/metabolism MH - Gene Expression MH - *Genes, Insect MH - Glutathione Transferase/biosynthesis MH - Immunohistochemistry MH - Molecular Sequence Data MH - Mutagenesis MH - RNA, Messenger/analysis/biosynthesis MH - Recombinant Fusion Proteins/biosynthesis/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Restriction Mapping MH - Sequence Deletion MH - Templates, Genetic MH - Transcription Factors/biosynthesis/isolation & purification/*metabolism MH - *Transcription, Genetic MH - Transfection EDAT- 1996/02/16 MHDA- 1996/02/16 00:01 PST - ppublish SO - J Biol Chem 1996 Feb 16;271(7):3930-7. NR -------------------------------------------------------------------------------- 110: Weiss S et al. Recombinant prion protein rPr...[PMID: 8619803] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 8619803 OWN - NLM STAT- MEDLINE DA - 19960613 DCOM- 19960613 LR - 20041117 PUBM- Print IS - 0006-291X VI - 219 IP - 1 DP - 1996 Feb 6 TI - Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity. PG - 173-9 AB - PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity. AD - Laboratorium fur Molekulare Biologie-Genzentrum-Institut fur Biochemie der LMU Munchen, Germany. FAU - Weiss, S AU - Weiss S FAU - Rieger, R AU - Rieger R FAU - Edenhofer, F AU - Edenhofer F FAU - Fisch, E AU - Fisch E FAU - Winnacker, E L AU - Winnacker EL LA - eng PT - Journal Article PL - UNITED STATES TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (Prions) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Recombinant Proteins) RN - 105268-27-7 (PrP 27-30 Protein) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 3.4.21 (Serine Endopeptidases) RN - EC 3.4.21.5 (Thrombin) RN - EC 3.4.21.64 (Endopeptidase K) SB - IM MH - Animals MH - Cell Line MH - Cloning, Molecular MH - Endopeptidase K MH - Escherichia coli MH - Glutathione Transferase/biosynthesis MH - Hamsters MH - Mesocricetus MH - PrP 27-30 Protein/chemistry/*metabolism MH - Prions/chemistry/isolation & purification/*metabolism MH - Recombinant Fusion Proteins/biosynthesis/isolation & purification MH - Recombinant Proteins/chemistry/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Serine Endopeptidases/*metabolism MH - Spodoptera MH - Substrate Specificity MH - Thrombin/metabolism MH - Transfection EDAT- 1996/02/06 MHDA- 1996/02/06 00:01 AID - S0006291X96902016 [pii] PST - ppublish SO - Biochem Biophys Res Commun 1996 Feb 6;219(1):173-9. DR -------------------------------------------------------------------------------- 111: Ema M et al. cDNA cloning of a murine homo...[PMID: 8561800] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8561800 OWN - NLM STAT- MEDLINE DA - 19960223 DCOM- 19960223 LR - 20050430 PUBM- Print IS - 0006-291X VI - 218 IP - 2 DP - 1996 Jan 17 TI - cDNA cloning of a murine homologue of Drosophila single-minded, its mRNA expression in mouse development, and chromosome localization. PG - 588-94 AB - A combination of the RT-PCR method and subsequent screening of the cDNA library of mouse skeletal muscle with the cDNA isolated by RT-PCR used as a probe led to isolation of cDNAs encoding a polypeptide (mSim) with bHLH and PAS domains which show high similarity to the corresponding regions of Drosophila Sim, a master regulator in neurogenesis. Experiments using a GST-fusion protein demonstrated that mSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. RNA blot analysis using RNAs from various tissues of mice indicated that mSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of mSim mRNA was always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. Chromosomal location of the mSim gene was determined by fluorescent in situ hybridization to be localized on the C3.3-C4 band of mouse chromosome 16 which is syntenic with the human chromosome 21q22 carrying the Down syndrome critical region, where a gene highly homologous to the Drosophila sim was localized. Whole mount in situ hybridization using a unique part of mSim cDNA sequence showed that mSim mRNA was expressed in the ventral diencephalon, branchial arches and limbs. These findings will provide an approach to the cause of the Down syndrome as well as the elucidation of the functional roles of mSim in animal development. AD - Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan. FAU - Ema, M AU - Ema M FAU - Suzuki, M AU - Suzuki M FAU - Morita, M AU - Morita M FAU - Hirose, K AU - Hirose K FAU - Sogawa, K AU - Sogawa K FAU - Matsuda, Y AU - Matsuda Y FAU - Gotoh, O AU - Gotoh O FAU - Saijoh, Y AU - Saijoh Y FAU - Fujii, H AU - Fujii H FAU - Hamada, H AU - Hamada H FAU - Fujii-Kuriyama, Y AU - Fujii-Kuriyama Y LA - eng SI - GENBANK/D63383 PT - Journal Article PL - UNITED STATES TA - Biochem Biophys Res Commun JID - 0372516 RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (DNA-Binding Proteins) RN - 0 (Nuclear Proteins) RN - 0 (RNA, Messenger) RN - 0 (Receptors, Aryl Hydrocarbon) RN - 0 (Recombinant Proteins) RN - 0 (Transcription Factors) RN - 0 (sim protein, Drosophila) RN - 138391-32-9 (aryl hydrocarbon receptor nuclear translocator) SB - IM MH - Age Factors MH - Animals MH - Base Sequence MH - Chromosome Mapping MH - Cloning, Molecular MH - Comparative Study MH - DNA Primers/chemistry MH - DNA, Complementary/*genetics MH - DNA-Binding Proteins/*genetics MH - Drosophila melanogaster/genetics MH - Gene Expression Regulation, Developmental MH - Helix-Loop-Helix Motifs MH - In Situ Hybridization MH - Mice MH - Molecular Sequence Data MH - Nuclear Proteins/*genetics MH - Protein Binding MH - RNA, Messenger/genetics MH - *Receptors, Aryl Hydrocarbon MH - Recombinant Proteins MH - Research Support, Non-U.S. Gov't MH - Sequence Alignment MH - Sequence Homology, Amino Acid MH - Tissue Distribution MH - Transcription Factors/metabolism EDAT- 1996/01/17 MHDA- 1996/01/17 00:01 AID - S0006291X96901047 [pii] PST - ppublish SO - Biochem Biophys Res Commun 1996 Jan 17;218(2):588-94. NR -------------------------------------------------------------------------------- 112: Yamaguchi M et al. Essential role of E2F recogni...[PMID: 7559650] Related Articles, HomoloGene, Cited in PMC, Books, LinkOut PMID- 7559650 OWN - NLM STAT- MEDLINE DA - 19951121 DCOM- 19951121 LR - 20041208 PUBM- Print IS - 0021-9258 VI - 270 IP - 42 DP - 1995 Oct 20 TI - Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development. PG - 25159-65 AB - We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism. AD - Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Nagoya, Japan. FAU - Yamaguchi, M AU - Yamaguchi M FAU - Hayashi, Y AU - Hayashi Y FAU - Matsukage, A AU - Matsukage A LA - eng PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Carrier Proteins) RN - 0 (Cell Cycle Proteins) RN - 0 (DNA-Binding Proteins) RN - 0 (Dp protein, Drosophila) RN - 0 (Drosophila Proteins) RN - 0 (E2F transcription factors) RN - 0 (Proliferating Cell Nuclear Antigen) RN - 0 (Trans-Activators) RN - 0 (Transcription Factors) RN - 0 (retinoblastoma binding protein 1) SB - IM MH - Animals MH - Base Sequence MH - Binding Sites MH - *Carrier Proteins MH - *Cell Cycle Proteins MH - *DNA-Binding Proteins MH - Drosophila/genetics/*physiology MH - *Drosophila Proteins MH - Molecular Sequence Data MH - Proliferating Cell Nuclear Antigen/*genetics MH - *Promoter Regions (Genetics) MH - Research Support, Non-U.S. Gov't MH - *Trans-Activators MH - Transcription Factors/*physiology EDAT- 1995/10/20 MHDA- 1995/10/20 00:01 PST - ppublish SO - J Biol Chem 1995 Oct 20;270(42):25159-65. NR -------------------------------------------------------------------------------- 113: Michaud D et al. Carboxy-terminal truncation o...[PMID: 7574723] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 7574723 OWN - NLM STAT- MEDLINE DA - 19951106 DCOM- 19951106 LR - 20041117 PUBM- Print IS - 0003-9861 VI - 322 IP - 2 DP - 1995 Oct 1 TI - Carboxy-terminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. PG - 469-74 AB - The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a approximately 10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearance of OCIIi resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control. AD - Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, British Columbia. FAU - Michaud, D AU - Michaud D FAU - Cantin, L AU - Cantin L FAU - Vrain, T C AU - Vrain TC LA - eng PT - Journal Article PL - UNITED STATES TA - Arch Biochem Biophys JID - 0372430 RN - 0 (Cystatins) RN - 0 (Cysteine Proteinase Inhibitors) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Fusion Proteins) RN - 9000-70-8 (Gelatin) RN - EC 3.4.- (Endopeptidases) SB - IM MH - Animals MH - Beetles/*enzymology MH - Binding Sites MH - Cystatins/genetics/*metabolism/pharmacology MH - Cysteine Proteinase Inhibitors/genetics/*metabolism/pharmacology MH - Digestive System/*enzymology MH - Dose-Response Relationship, Drug MH - Electrophoresis, Polyacrylamide Gel MH - Endopeptidases/drug effects/*metabolism MH - Gelatin/metabolism MH - Host-Parasite Relations MH - Peptide Fragments/metabolism MH - Protein Conformation MH - Recombinant Fusion Proteins/metabolism/pharmacology MH - Research Support, Non-U.S. Gov't EDAT- 1995/10/01 MHDA- 1995/10/01 00:01 AID - S0003986185714907 [pii] PST - ppublish SO - Arch Biochem Biophys 1995 Oct 1;322(2):469-74. NR -------------------------------------------------------------------------------- 114: Jung JU et al. Identification of Lck-binding...[PMID: 7544793] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 7544793 OWN - NLM STAT- MEDLINE DA - 19951004 DCOM- 19951004 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 270 IP - 35 DP - 1995 Sep 1 TI - Identification of Lck-binding elements in tip of herpesvirus saimiri. PG - 20660-7 AB - A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri. AD - Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102, USA. FAU - Jung, J U AU - Jung JU FAU - Lang, S M AU - Lang SM FAU - Friedrich, U AU - Friedrich U FAU - Jun, T AU - Jun T FAU - Roberts, T M AU - Roberts TM FAU - Desrosiers, R C AU - Desrosiers RC FAU - Biesinger, B AU - Biesinger B LA - eng GR - CA31363/CA/NCI GR - RR00168/RR/NCRR PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (DNA Primers) RN - 0 (Peptide Fragments) RN - 0 (Phosphoproteins) RN - 0 (Recombinant Proteins) RN - 0 (Viral Proteins) RN - 0 (tyrosine kinase interacting protein, Saimiriine herpesvirus 2) RN - 1114-81-4 (Phosphothreonine) RN - 17885-08-4 (Phosphoserine) RN - 21820-51-9 (Phosphotyrosine) RN - 55520-40-6 (Tyrosine) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.37 (Lymphocyte Specific Protein Tyrosine Kinase p56(lck)) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Binding Sites MH - Cell Line MH - Cercopithecus aethiops MH - Comparative Study MH - DNA Primers MH - Herpesvirus 2, Saimiriine/*metabolism MH - Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Peptide Fragments/chemistry/isolation & purification MH - Phosphoproteins/isolation & purification/*metabolism MH - Phosphoserine/analysis MH - Phosphothreonine/analysis MH - Phosphotyrosine MH - Point Mutation MH - Polymerase Chain Reaction MH - Protein-Tyrosine Kinase/isolation & purification/*metabolism MH - Rats MH - Recombinant Proteins/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Spodoptera MH - Structure-Activity Relationship MH - Transfection MH - Tyrosine/analogs & derivatives/analysis MH - Viral Proteins/isolation & purification/*metabolism EDAT- 1995/09/01 MHDA- 1995/09/01 00:01 PST - ppublish SO - J Biol Chem 1995 Sep 1;270(35):20660-7. PR -------------------------------------------------------------------------------- 115: Weiss S et al. Overexpression of active Syri...[PMID: 7609044] Related Articles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 7609044 OWN - NLM STAT- MEDLINE DA - 19950811 DCOM- 19950811 LR - 20041117 PUBM- Print IS - 0022-538X VI - 69 IP - 8 DP - 1995 Aug TI - Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems. PG - 4776-83 AB - This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced. AD - Laboratorium fur Molekulare Biologie-Genzentrum-Institut fur Biochemie der LMU Munchen, Germany. FAU - Weiss, S AU - Weiss S FAU - Famulok, M AU - Famulok M FAU - Edenhofer, F AU - Edenhofer F FAU - Wang, Y H AU - Wang YH FAU - Jones, I M AU - Jones IM FAU - Groschup, M AU - Groschup M FAU - Winnacker, E L AU - Winnacker EL LA - eng PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (Prions) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM GS - GST GS - PrPc MH - Animals MH - Baculoviridae/genetics MH - Cell Line MH - Cloning, Molecular MH - Escherichia coli/genetics MH - Glutathione Transferase/*genetics MH - Hamsters MH - Mesocricetus MH - Pichia/genetics MH - Prions/*genetics MH - Recombinant Fusion Proteins/genetics MH - Research Support, Non-U.S. Gov't MH - Spodoptera EDAT- 1995/08/01 MHDA- 1995/08/01 00:01 PST - ppublish SO - J Virol 1995 Aug;69(8):4776-83. DR -------------------------------------------------------------------------------- 116: Briggs SD et al. The Ras GTPase-activating pro...[PMID: 7782336] Related Articles, Gene, HomoloGene, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 7782336 OWN - NLM STAT- MEDLINE DA - 19950717 DCOM- 19950717 LR - 20050224 PUBM- Print IS - 0021-9258 VI - 270 IP - 24 DP - 1995 Jun 16 TI - The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck. PG - 14718-24 AB - The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of Hck.GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (delta CAT) or the Src homology region (delta SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, delta CAT, and delta SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain. AD - Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198, USA. FAU - Briggs, S D AU - Briggs SD FAU - Bryant, S S AU - Bryant SS FAU - Jove, R AU - Jove R FAU - Sanderson, S D AU - Sanderson SD FAU - Smithgall, T E AU - Smithgall TE LA - eng GR - CA55652/CA/NCI GR - CA58667/CA/NCI GR - P30 CA36727/CA/NCI PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (GTPase-Activating Proteins) RN - 0 (Proteins) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (ras GTPase-Activating Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (proto-oncogene protein c-hck) SB - IM MH - Amino Acid Sequence MH - Animals MH - Baculoviridae/genetics MH - Catalysis MH - Cattle MH - Cell Line MH - Cloning, Molecular MH - GTPase-Activating Proteins MH - Glutathione Transferase/genetics/metabolism MH - Humans MH - Molecular Sequence Data MH - Phosphorylation MH - Protein Binding MH - Protein-Tyrosine Kinase/genetics/*metabolism MH - Proteins/*metabolism MH - Proto-Oncogene Proteins/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Spodoptera MH - Substrate Specificity MH - ras GTPase-Activating Proteins EDAT- 1995/06/16 MHDA- 1995/06/16 00:01 PST - ppublish SO - J Biol Chem 1995 Jun 16;270(24):14718-24. PR -------------------------------------------------------------------------------- 117: Nolan LA et al. The Epstein-Barr virus open r...[PMID: 7782767] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 7782767 OWN - NLM STAT- MEDLINE DA - 19950720 DCOM- 19950720 LR - 20041117 PUBM- Print IS - 0022-1317 VI - 76 ( Pt 6) DP - 1995 Jun TI - The Epstein-Barr virus open reading frame BDLF3 codes for a 100-150 kDa glycoprotein. PG - 1381-92 AB - The Epstein-Barr virus (EBV) open reading frame BDLF3 is predicted to code for a glycoprotein on the basis that it contains sequences with signal peptide and transdomain characteristics and nine potential N-linked glycosylation sites. No sequential or positional homologues of BDLF3 have been located in other herpesviruses. A bacterial glutathione S-transferase (GST)-BDLF3 fusion protein was used to demonstrate that over one-third of EBV-immune human sera tested recognized the fusion protein but not GST alone on Western blots. The fusion protein was used to raise polyclonal sera in rabbits. A BDLF3 recombinant baculovirus was constructed using the full-length BDLF3 sequence (AcBDLF3). Rabbit anti-fusion protein sera and some human EBV-immune sera recognized products of approximately 30 and 55 kDa from AcBDLF3-infected insect cells by Western blotting. A peptide representing the carboxy-terminal amino acids 215-234 of the BDLF3 sequence was used to raise anti-peptide sera in rabbits. Anti-peptide serum detected a product by indirect immunofluorescence in acetone-fixed EBV-infected B cells from all cell lines tested. A diffuse band with a molecular mass of 100-150 kDa was detected by Western blot in B95-8 cell lysates, partially purified B95-8 virus and B95-8-infected cell membranes after probing with anti-BDLF3 peptide serum. This product was shown to be glycosylated after enzymatic deglycosylation of a B95-8 virus preparation using neuraminidase, O-glycosidase or N-glycosidase F. The BDLF3 protein products have no known function. AD - Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, UK. FAU - Nolan, L A AU - Nolan LA FAU - Morgan, A J AU - Morgan AJ LA - eng PT - Journal Article PL - ENGLAND TA - J Gen Virol JID - 0077340 RN - 0 (Antibodies, Viral) RN - 0 (DNA Primers) RN - 0 (Glycoproteins) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Animals MH - Antibodies, Viral/blood MH - Base Sequence MH - Blotting, Western MH - Cell Line MH - DNA Primers MH - Electrophoresis, Polyacrylamide Gel MH - Glycoproteins/biosynthesis/*genetics/immunology MH - Herpesvirus 4, Human/*genetics/*metabolism MH - Humans MH - Molecular Sequence Data MH - Molecular Weight MH - *Open Reading Frames MH - Polymerase Chain Reaction MH - Protein Biosynthesis MH - Rabbits/immunology MH - Recombinant Fusion Proteins/biosynthesis/immunology MH - Research Support, Non-U.S. Gov't MH - Spodoptera MH - Transcription, Genetic MH - Transfection EDAT- 1995/06/01 MHDA- 1995/06/01 00:01 PST - ppublish SO - J Gen Virol 1995 Jun;76 ( Pt 6):1381-92. NR -------------------------------------------------------------------------------- 118: Millward T et al. Molecular cloning and charact...[PMID: 7761441] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, OMIM, GEO Profiles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 7761441 OWN - NLM STAT- MEDLINE DA - 19950629 DCOM- 19950629 LR - 20041117 PUBM- Print IS - 0027-8424 VI - 92 IP - 11 DP - 1995 May 23 TI - Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase. PG - 5022-6 AB - Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division. AD - Friedrich Miescher-Institut, Basel, Switzerland. FAU - Millward, T AU - Millward T FAU - Cron, P AU - Cron P FAU - Hemmings, B A AU - Hemmings BA LA - eng SI - GENBANK/Z34989 SI - GENBANK/Z35102 SI - GENBANK/Z35103 PT - Journal Article PL - UNITED STATES TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (Nuclear Proteins) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Caenorhabditis elegans/enzymology MH - Cell Line MH - Cell Nucleus/*enzymology MH - Cercopithecus aethiops MH - Cloning, Molecular MH - Comparative Study MH - Conserved Sequence MH - DNA Primers MH - DNA, Complementary MH - Drosophila/embryology/enzymology MH - Glutathione Transferase/biosynthesis MH - Hela Cells MH - Humans MH - Kinetics MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Nuclear Proteins/analysis/*biosynthesis/*chemistry MH - Polymerase Chain Reaction MH - Protein-Serine-Threonine Kinases/analysis/*biosynthesis/*chemistry MH - Recombinant Fusion Proteins/analysis/biosynthesis/chemistry MH - Sequence Deletion MH - Sequence Homology, Amino Acid MH - Transfection EDAT- 1995/05/23 MHDA- 1995/05/23 00:01 PST - ppublish SO - Proc Natl Acad Sci U S A 1995 May 23;92(11):5022-6. NR -------------------------------------------------------------------------------- 119: Coates K et al. The human immunodeficiency vi...[PMID: 9049329] Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut PMID- 9049329 OWN - NLM STAT- MEDLINE DA - 19970331 DCOM- 19970331 LR - 20041117 PUBM- Print IS - 0022-1317 VI - 76 ( Pt 4) DP - 1995 Apr TI - The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro. PG - 837-44 AB - The human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [gamma32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components. AD - MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, UK. FAU - Coates, K AU - Coates K FAU - Harris, M AU - Harris M LA - eng PT - Journal Article PL - ENGLAND TA - J Gen Virol JID - 0077340 RN - 0 (Gene Products, nef) RN - 0 (Recombinant Fusion Proteins) RN - 56-45-1 (Serine) RN - EC 2.7.1.37 (Protein Kinase C) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) SB - IM SB - X MH - Animals MH - Cell Line MH - Gene Products, nef/genetics/*metabolism MH - HIV-1/*metabolism MH - Hela Cells MH - Humans MH - Jurkat Cells MH - Phosphorylation MH - Protein Kinase C/*metabolism MH - Protein-Serine-Threonine Kinases/metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Serine/metabolism MH - Spodoptera/cytology MH - Substrate Specificity MH - Tumor Cells, Cultured EDAT- 1995/04/01 MHDA- 1995/04/01 00:01 PST - ppublish SO - J Gen Virol 1995 Apr;76 ( Pt 4):837-44. DR -------------------------------------------------------------------------------- 120: DebBurman SK et al. Lipid-mediated regulation of ...[PMID: 7890702] Related Articles, Cited in PMC, Books, LinkOut PMID- 7890702 OWN - NLM STAT- MEDLINE DA - 19950418 DCOM- 19950418 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 270 IP - 11 DP - 1995 Mar 17 TI - Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3. PG - 5742-7 AB - G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs. AD - Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611. FAU - DebBurman, S K AU - DebBurman SK FAU - Ptasienski, J AU - Ptasienski J FAU - Boetticher, E AU - Boetticher E FAU - Lomasney, J W AU - Lomasney JW FAU - Benovic, J L AU - Benovic JL FAU - Hosey, M M AU - Hosey MM LA - eng GR - GM 44944/GM/NIGMS GR - HL 50121/HL/NHLBI PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Macromolecular Substances) RN - 0 (Phospholipids) RN - 0 (Receptors, Muscarinic) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (G-protein-coupled receptor kinase 3) RN - EC 2.7.1.- (beta-adrenergic receptor kinase) RN - EC 2.7.1.112 (Receptor Protein-Tyrosine Kinases) RN - EC 2.7.1.37 (Cyclic AMP-Dependent Protein Kinases) RN - EC 2.7.1.37 (Protein-Serine-Threonine Kinases) RN - EC 3.6.1.- (GTP-Binding Proteins) SB - IM MH - Animals MH - Binding, Competitive MH - Cell Membrane/metabolism MH - Comparative Study MH - Cyclic AMP-Dependent Protein Kinases/biosynthesis/*metabolism MH - Enzyme Activation MH - GTP-Binding Proteins/*metabolism MH - Glutathione Transferase/metabolism MH - Humans MH - Kinetics MH - Macromolecular Substances MH - Phospholipids/pharmacology MH - *Protein-Serine-Threonine Kinases MH - Receptor Protein-Tyrosine Kinases/*metabolism MH - Receptors, Muscarinic/biosynthesis/isolation & purification/*metabolism MH - Recombinant Fusion Proteins/biosynthesis/isolation & purification/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Transfection EDAT- 1995/03/17 MHDA- 1995/03/17 00:01 PST - ppublish SO - J Biol Chem 1995 Mar 17;270(11):5742-7. PR -------------------------------------------------------------------------------- 121: Matsuguchi T et al. Tyrosine phosphorylation of p...[PMID: 7530656] Related Articles, Gene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 7530656 OWN - NLM STAT- MEDLINE DA - 19950227 DCOM- 19950227 LR - 20050223 PUBM- Print IS - 0261-4189 VI - 14 IP - 2 DP - 1995 Jan 16 TI - Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL. PG - 257-65 AB - Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS) AD - Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA 02115. FAU - Matsuguchi, T AU - Matsuguchi T FAU - Inhorn, R C AU - Inhorn RC FAU - Carlesso, N AU - Carlesso N FAU - Xu, G AU - Xu G FAU - Druker, B AU - Druker B FAU - Griffin, J D AU - Griffin JD LA - eng GR - CA36167/CA/NCI PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (Cell Cycle Proteins) RN - 0 (Fusion Proteins, bcr-abl) RN - 0 (Hematopoietic Cell Growth Factors) RN - 0 (Interleukin-3) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Stem Cell Factor) RN - 0 (proto-oncogene protein c-vav) RN - 55520-40-6 (Tyrosine) RN - 83869-56-1 (Granulocyte-Macrophage Colony-Stimulating Factor) RN - EC 2.7.1.112 (Janus kinase 2) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 3.1.3.48 (Protein-Tyrosine-Phosphatase) SB - IM CIN - NIH Guide Grants Contracts. 1995 Dec 8;24(42):1-2. PMID: 7495607 GS - ABL GS - BCR GS - JAK2 MH - Animals MH - Baculoviridae/genetics MH - Bone Marrow/metabolism MH - Bone Marrow Cells MH - *Cell Cycle Proteins MH - Cell Line MH - Fusion Proteins, bcr-abl/*physiology MH - Granulocyte-Macrophage Colony-Stimulating Factor/*pharmacology MH - Hematopoietic Cell Growth Factors/*pharmacology MH - Humans MH - Interleukin-3/*pharmacology MH - Phosphorylation MH - Protein-Tyrosine Kinase/metabolism MH - Protein-Tyrosine-Phosphatase/antagonists & inhibitors MH - Proto-Oncogene Proteins/*metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Spodoptera MH - Stem Cell Factor MH - Tyrosine/*metabolism EDAT- 1995/01/16 MHDA- 1995/01/16 00:01 PST - ppublish SO - EMBO J 1995 Jan 16;14(2):257-65. PR -------------------------------------------------------------------------------- 122: Skantar AM et al. Identifying a transcription f...[PMID: 7488860] Related Articles, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 7488860 OWN - NLM STAT- MEDLINE DA - 19960104 DCOM- 19960104 LR - 20041117 PUBM- Print IS - 1052-2166 VI - 5 IP - 1 DP - 1995 TI - Identifying a transcription factor interaction site on RNA polymerase II. PG - 49-69 AB - We have generated a series of fusion proteins carrying portions of subunit IIc, the second largest subunit of Drosophila RNA polymerase I, and have used them in a domain interference assay to identify a fragment of the IIc subunit that carries the binding site for a basal transcription factor. Fusion proteins carrying a subunit IIc fragment spanning residues Ala519-Gly992 strongly inhibit promoter-driven transcription in both unfractionated nuclear extracts and in reconstituted systems. The same fusion proteins similarly inhibit dTFIIF stimulation of Pol II elongation on dC-tailed templates, suggesting that the IIc(A519-G992) fragment, which carries conserved regions D-H, interferes with transcription by binding to dTFIIF. Finally, dTFIIF can be specifically cross-linked to a GST-IIc(A519-G992) fusion protein or to subunit IIc in intact Pol II. AD - Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA. FAU - Skantar, A M AU - Skantar AM FAU - Greenleaf, A L AU - Greenleaf AL LA - eng GR - GM 28078/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - Gene Expr JID - 9200651 RN - 0 (Peptide Fragments) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Transcription Factors) RN - EC 2.7.7.- (RNA Polymerase II) RN - EC 3.2.1.23 (beta-Galactosidase) SB - IM MH - Animals MH - Base Sequence MH - Binding Sites MH - Drosophila/genetics MH - Genes, Structural, Insect MH - Molecular Sequence Data MH - Peptide Fragments/metabolism MH - Protein Binding MH - Protein Structure, Tertiary MH - RNA Polymerase II/chemistry/genetics/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. MH - Transcription Factors/*metabolism MH - beta-Galactosidase/genetics EDAT- 1995/01/01 MHDA- 1995/01/01 00:01 PST - ppublish SO - Gene Expr 1995;5(1):49-69. NR -------------------------------------------------------------------------------- 123: Colamonici O et al. Direct binding to and tyrosin...[PMID: 7526154] Related Articles, Compound via MeSH, Substance via MeSH, Protein, Free in PMC, Cited in PMC, Books, LinkOut PMID- 7526154 OWN - NLM STAT- MEDLINE DA - 19941220 DCOM- 19941220 LR - 20041117 PUBM- Print IS - 0270-7306 VI - 14 IP - 12 DP - 1994 Dec TI - Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase. PG - 8133-42 AB - Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the alpha and beta subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3 alpha (Stat113, Stat84, and Stat91), and the p135tyk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the alpha subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the p135tyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the alpha subunit can bind the p135tyk2 contained in human cell lysates. The association between the alpha subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, p135tyk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFN alpha-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the alpha subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs. AD - Department of Pathology, University of Tennessee, Memphis 38163. FAU - Colamonici, O AU - Colamonici O FAU - Yan, H AU - Yan H FAU - Domanski, P AU - Domanski P FAU - Handa, R AU - Handa R FAU - Smalley, D AU - Smalley D FAU - Mullersman, J AU - Mullersman J FAU - Witte, M AU - Witte M FAU - Krishnan, K AU - Krishnan K FAU - Krolewski, J AU - Krolewski J LA - eng GR - CA55079/CA/NCI GR - CA56862/CA/NCI PT - Journal Article PL - UNITED STATES TA - Mol Cell Biol JID - 8109087 RN - 0 (DNA-Binding Proteins) RN - 0 (Interferon-alpha) RN - 0 (Proteins) RN - 0 (Receptors, Interferon) RN - 0 (Recombinant Proteins) RN - 0 (Stat2 protein) RN - 0 (Trans-Activators) RN - 0 (interferon alpha receptor) RN - 21820-51-9 (Phosphotyrosine) RN - 55520-40-6 (Tyrosine) RN - EC 2.7.1.112 (Janus kinase 1) RN - EC 2.7.1.112 (Protein-Tyrosine Kinase) RN - EC 2.7.1.112 (TYK2 protein, human) SB - IM MH - DNA-Binding Proteins/metabolism MH - Humans MH - In Vitro MH - Interferon-alpha/pharmacology MH - Phosphotyrosine MH - Protein Binding MH - Protein-Tyrosine Kinase/*metabolism MH - Proteins/*metabolism MH - Receptors, Interferon/*metabolism MH - Recombinant Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Signal Transduction MH - Trans-Activators/metabolism MH - Tumor Cells, Cultured MH - Tyrosine/analogs & derivatives/metabolism EDAT- 1994/12/01 MHDA- 1994/12/01 00:01 PST - ppublish SO - Mol Cell Biol 1994 Dec;14(12):8133-42. NR -------------------------------------------------------------------------------- 124: Beekman JM et al. A rapid one-step method to pu...[PMID: 8076833] Related Articles, Books, LinkOut PMID- 8076833 OWN - NLM STAT- MEDLINE DA - 19941004 DCOM- 19941004 LR - 20041117 PUBM- Print IS - 0378-1119 VI - 146 IP - 2 DP - 1994 Sep 2 TI - A rapid one-step method to purify baculovirus-expressed human estrogen receptor to be used in the analysis of the oxytocin promoter. PG - 285-9 AB - We have produced a truncated form of the human estrogen receptor (hER) as a fusion protein with glutathione S-transferase (GST) in Spodoptera frugiperda (Sf) cells using the baculovirus expression vector (BEV) system. The protein is correctly produced and can be purified from crude whole-cell extracts by a single-step, batch-wise affinity-purification procedure. We show that this GST-hER fusion protein binds at its DNA-binding site specifically and in a hormone-inducible manner. Furthermore, we used the purified hER to analyze the complex estrogen response element (ERE) in the promoter of the oxytocin-encoding gene. AD - Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030. FAU - Beekman, J M AU - Beekman JM FAU - Cooney, A J AU - Cooney AJ FAU - Elliston, J F AU - Elliston JF FAU - Tsai, S Y AU - Tsai SY FAU - Tsai, M J AU - Tsai MJ LA - eng PT - Journal Article PL - NETHERLANDS TA - Gene JID - 7706761 RN - 0 (Genetic Vectors) RN - 0 (Receptors, Estrogen) RN - 0 (Recombinant Fusion Proteins) RN - 9007-49-2 (DNA) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Baculoviridae/genetics MH - Base Sequence MH - DNA/*metabolism MH - Genetic Vectors/chemistry/*genetics MH - Glutathione Transferase/chemistry/*genetics/metabolism MH - Humans MH - Molecular Sequence Data MH - Receptors, Estrogen/chemistry/*isolation & purification/metabolism MH - Recombinant Fusion Proteins/chemistry/*genetics/metabolism EDAT- 1994/09/02 MHDA- 1994/09/02 00:01 PST - ppublish SO - Gene 1994 Sep 2;146(2):285-9. DR -------------------------------------------------------------------------------- 125: Guy PM et al. Insect cell-expressed p180erb...[PMID: 8058768] Related Articles, Substance via MeSH, Free in PMC, Cited in PMC, Books, LinkOut PMID- 8058768 OWN - NLM STAT- MEDLINE DA - 19940914 DCOM- 19940914 LR - 20041117 PUBM- Print IS - 0027-8424 VI - 91 IP - 17 DP - 1994 Aug 16 TI - Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. PG - 8132-6 AB - Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity. AD - Department of Pharmacology, Cornell University, Ithaca, NY 14853. FAU - Guy, P M AU - Guy PM FAU - Platko, J V AU - Platko JV FAU - Cantley, L C AU - Cantley LC FAU - Cerione, R A AU - Cerione RA FAU - Carraway, K L 3rd AU - Carraway KL 3rd LA - eng GR - GM40654/GM/NIGMS GR - GM41890/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 62229-50-9 (Epidermal Growth Factor) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1 (Phosphotransferases (Alcohol Group Acceptor)) RN - EC 2.7.1.112 (Receptor, Epidermal Growth Factor) RN - EC 2.7.1.112 (Receptor, erbB-3) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase MH - Amino Acid Sequence MH - Animals MH - Cattle MH - Comparative Study MH - Epidermal Growth Factor/pharmacology MH - Glutathione Transferase/metabolism MH - Humans MH - Insects MH - Kinetics MH - Molecular Sequence Data MH - Phosphotransferases (Alcohol Group Acceptor)/metabolism MH - Proto-Oncogene Proteins/biosynthesis/isolation & purification/*metabolism MH - Receptor, Epidermal Growth Factor/biosynthesis/isolation & purification/*metabolism MH - Receptor, erbB-3 MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - Research Support, U.S. Gov't, P.H.S. MH - Sequence Homology, Amino Acid MH - Transfection EDAT- 1994/08/16 MHDA- 2001/03/28 10:01 PST - ppublish SO - Proc Natl Acad Sci U S A 1994 Aug 16;91(17):8132-6. DR -------------------------------------------------------------------------------- 126: Harris MP et al. Myristoylation-dependent bind...[PMID: 8057354] Related Articles, Cited in PMC, Books, LinkOut PMID- 8057354 OWN - NLM STAT- MEDLINE DA - 19940909 DCOM- 19940909 LR - 20041117 PUBM- Print IS - 0022-2836 VI - 241 IP - 2 DP - 1994 Aug 12 TI - Myristoylation-dependent binding of HIV-1 Nef to CD4. PG - 136-42 AB - The nef gene is conserved throughout the primate lentivirus family. Although dispensable in vitro, an important role for nef in vivo is suggested by the failure of SIV nef mutants to establish persistent viraemia. Although the biochemical function of the Nef protein remains equivocal, a consistent theme has emerged with the reproducible observation that Nef expression results in the down-modulation of the cell surface marker CD4. Down-modulation requires amino acid sequences within the cytoplasmic domain of CD4 but occurs by a mechanism distinct from the normal serine phosphorylation-dependent pathway. As CD4 is a transmembrane glycoprotein and Nef a myristoylated protein targeted to the cytoplasmic face of the plasma membrane we considered that a direct interaction between Nef and CD4 might play a role in down-modulation. Here we demonstrate that a baculovirus-expressed Nef-GST fusion protein interacts specifically with CD4. This interaction requires co-expression in the same cell and is dependent on Nef myristoylation. The site of Nef interaction maps to the cytoplasmic domain of CD4, as a deletion mutant lacking this domain fails to interact with Nef. This observation sheds new light on the biochemical function of Nef and offers new opportunities for the future development of HIV chemotherapy. AD - Department of Veterinary Pathology, University of Glasgow, Scotland, U.K. FAU - Harris, M P AU - Harris MP FAU - Neil, J C AU - Neil JC LA - eng PT - Journal Article PL - ENGLAND TA - J Mol Biol JID - 2985088R RN - 0 (Antigens, CD4) RN - 0 (Gene Products, nef) RN - 0 (Myristates) RN - 0 (Recombinant Fusion Proteins) SB - IM SB - X GS - nef MH - Animals MH - Antigens, CD4/genetics/*metabolism MH - Base Sequence MH - Binding Sites MH - Cell Line MH - Down-Regulation MH - Flow Cytometry MH - Gene Products, nef/*metabolism MH - Genes, nef MH - *HIV-1 MH - Humans MH - Immunoblotting MH - Mice MH - Molecular Sequence Data MH - Myristates/metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, Non-U.S. Gov't MH - SIV/genetics/metabolism EDAT- 1994/08/12 MHDA- 1994/08/12 00:01 AID - S0022283684714835 [pii] PST - ppublish SO - J Mol Biol 1994 Aug 12;241(2):136-42. DR -------------------------------------------------------------------------------- 127: Quest AF et al. The regulatory region of prot...[PMID: 8051084] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Books, LinkOut PMID- 8051084 OWN - NLM STAT- MEDLINE DA - 19940908 DCOM- 19940908 LR - 20041117 PUBM- Print IS - 0021-9258 VI - 269 IP - 31 DP - 1994 Aug 5 TI - The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations. PG - 20000-12 AB - The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function. AD - Department of Molecular Cancer Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710. FAU - Quest, A F AU - Quest AF FAU - Bell, R M AU - Bell RM LA - eng GR - GM38737/GM/NIGMS PT - Journal Article PL - UNITED STATES TA - J Biol Chem JID - 2985121R RN - 0 (Cations, Divalent) RN - 0 (DNA Primers) RN - 0 (Isoenzymes) RN - 0 (Phosphatidylserines) RN - 0 (Recombinant Fusion Proteins) RN - 37558-16-0 (Phorbol 12,13-Dibutyrate) RN - 52-90-4 (Cysteine) RN - 7440-70-2 (Calcium) RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1.- (protein kinase C gamma) RN - EC 2.7.1.37 (Protein Kinase C) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Calcium/metabolism MH - Cations, Divalent MH - Cells, Cultured MH - Cysteine/metabolism MH - DNA Primers MH - Glutathione Transferase/*metabolism MH - Isoenzymes/genetics/*metabolism MH - Molecular Sequence Data MH - Moths MH - Phorbol 12,13-Dibutyrate/*metabolism MH - Phosphatidylserines/*metabolism MH - Protein Kinase C/genetics/*metabolism MH - Recombinant Fusion Proteins/isolation & purification/metabolism MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1994/08/05 MHDA- 1994/08/05 00:01 PST - ppublish SO - J Biol Chem 1994 Aug 5;269(31):20000-12. NR -------------------------------------------------------------------------------- 128: Lechner MS et al. Inhibition of p53 DNA binding...[PMID: 8207801] Related Articles, Free in PMC, Cited in PMC, Books, LinkOut PMID- 8207801 OWN - NLM STAT- MEDLINE DA - 19940713 DCOM- 19940713 LR - 20041117 PUBM- Print IS - 0022-538X VI - 68 IP - 7 DP - 1994 Jul TI - Inhibition of p53 DNA binding by human papillomavirus E6 proteins. PG - 4262-73 AB - Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types. AD - Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637. FAU - Lechner, M S AU - Lechner MS FAU - Laimins, L A AU - Laimins LA LA - eng GR - CA49670/CA/NCI GR - CA60607/CA/NCI PT - Journal Article PL - UNITED STATES TA - J Virol JID - 0113724 RN - 0 (DNA-Binding Proteins) RN - 0 (E6 protein, Human papillomavirus type 18) RN - 0 (Oncogene Proteins, Viral) RN - 0 (Protein p53) RN - 0 (Recombinant Fusion Proteins) RN - 9007-49-2 (DNA) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Baculoviridae/genetics MH - Base Sequence MH - Cell Line MH - DNA/*metabolism MH - DNA-Binding Proteins/antagonists & inhibitors/*metabolism MH - Glutathione Transferase/metabolism MH - Humans MH - Molecular Sequence Data MH - Moths MH - Oncogene Proteins, Viral/*metabolism MH - Papillomavirus, Human/*metabolism MH - Protein p53/antagonists & inhibitors/*metabolism MH - Recombinant Fusion Proteins/metabolism MH - Research Support, U.S. Gov't, P.H.S. EDAT- 1994/07/01 MHDA- 1994/07/01 00:01 PST - ppublish SO - J Virol 1994 Jul;68(7):4262-73. NR -------------------------------------------------------------------------------- 129: Ando A et al. A complex of GRB2-dynamin bin...[PMID: 8039498] Related Articles, Compound via MeSH, Substance via MeSH, Cited in PMC, Cited in Books, Books, LinkOut PMID- 8039498 OWN - NLM STAT- MEDLINE DA - 19940819 DCOM- 19940819 LR - 20041117 PUBM- Print IS - 0261-4189 VI - 13 IP - 13 DP - 1994 Jul 1 TI - A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment. PG - 3033-8 AB - Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) AD - Second Department of Internal Medicine, Kobe University School of Medicine, Japan. FAU - Ando, A AU - Ando A FAU - Yonezawa, K AU - Yonezawa K FAU - Gout, I AU - Gout I FAU - Nakata, T AU - Nakata T FAU - Ueda, H AU - Ueda H FAU - Hara, K AU - Hara K FAU - Kitamura, Y AU - Kitamura Y FAU - Noda, Y AU - Noda Y FAU - Takenawa, T AU - Takenawa T FAU - Hirokawa, N AU - Hirokawa N AU - et al. LA - eng PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (Membrane Proteins) RN - 0 (Phosphoproteins) RN - 0 (Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Son of Sevenless Proteins) RN - 0 (growth factor receptor-bound protein-2) RN - 0 (insulin receptor substrate-1 protein) RN - 11061-68-0 (Insulin) RN - 55520-40-6 (Tyrosine) RN - EC 2.7.1 (Phosphotransferases (Alcohol Group Acceptor)) RN - EC 2.7.1.112 (Receptor, Insulin) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) RN - EC 3.6.1.- (GTP Phosphohydrolases) RN - EC 3.6.1.50 (Dynamins) SB - IM MH - 1-Phosphatidylinositol 3-Kinase MH - *Adaptor Proteins, Signal Transducing MH - Amino Acid Sequence MH - Animals MH - Brain/metabolism MH - CHO Cells MH - Dynamins MH - GTP Phosphohydrolases/*metabolism MH - Hamsters MH - Insulin/*pharmacology MH - Membrane Proteins/metabolism MH - Molecular Sequence Data MH - Phosphoproteins/*metabolism MH - Phosphorylation/drug effects MH - Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism MH - Protein Binding/drug effects MH - Proteins/genetics/*metabolism MH - Rats MH - Receptor, Insulin/genetics/*metabolism MH - Recombinant Fusion Proteins/genetics/metabolism MH - Research Support, Non-U.S. Gov't MH - Son of Sevenless Proteins MH - Tyrosine/*metabolism EDAT- 1994/07/01 MHDA- 1994/07/01 00:01 PST - ppublish SO - EMBO J 1994 Jul 1;13(13):3033-8. NR -------------------------------------------------------------------------------- 130: Stroumbakis ND et al. RNA- and single-stranded DNA-...[PMID: 8206370] Related Articles, Gene, HomoloGene, Compound via MeSH, Substance via MeSH, UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut PMID- 8206370 OWN - NLM STAT- MEDLINE DA - 19940711 DCOM- 19940711 LR - 20041117 PUBM- Print IS - 0378-1119 VI - 143 IP - 2 DP - 1994 Jun 10 TI - RNA- and single-stranded DNA-binding (SSB) proteins expressed during Drosophila melanogaster oogenesis: a homolog of bacterial and eukaryotic mitochondrial SSBs. PG - 171-7 AB - Little is known about the identity and involvement of single-stranded (ss) DNA-binding (SSB) and RNA-binding proteins in developmental processes that occur during oogenesis in Drosophila melanogaster (Dm). Here, we describe a molecular approach designed to identify such proteins by virtue of their ssDNA-binding activity. We have constructed a directional ovarian cDNA library and conducted expression cloning screens which identified five unique cDNAs that encode proteins capable of binding ssDNA. All five represent previously unreported sequences. The remainder of this paper focuses on one of these cDNAs which encodes a Dm protein displaying significant sequence homology to Escherichia coli ssDNA-binding protein (SSB, involved in DNA replication, repair and recombination), as well as eukaryotic SSBs isolated from the mitochondria (mt) of rats, frogs, humans and yeast. The deduced amino acid (aa) sequence of this 15.6-kDa protein, which we will refer to as Dm mtSSB, displays average identities of 38.3% with eukaryotic mtSSBs and 23.4% with bacterial SSBs. Gel retardation analysis with an affinity-purified GST fusion protein confirms that Dm mtSSB specifically binds ss, but not double stranded DNA. Dm mtSSB is encoded by a nuclear gene whose expression appears to be developmentally regulated. It is expressed as a single 600-nucleotide (nt) transcript during oogenesis and embryogenesis. A larger transcript of 1500 nt is prevalent in some later stages of Dm development. AD - Public Health Research Institute, New York, NY 10016. FAU - Stroumbakis, N D AU - Stroumbakis ND FAU - Li, Z AU - Li Z FAU - Tolias, P P AU - Tolias PP LA - eng SI - GENBANK/U00669 SI - GENBANK/U26549 PT - Journal Article PL - NETHERLANDS TA - Gene JID - 7706761 RN - 0 (Bacterial Proteins) RN - 0 (DNA, Complementary) RN - 0 (DNA, Single-Stranded) RN - 0 (DNA-Binding Proteins) RN - 0 (RNA-Binding Proteins) SB - IM MH - Amino Acid Sequence MH - Animals MH - Bacterial Proteins/chemistry MH - Base Sequence MH - DNA, Complementary/chemistry MH - DNA, Single-Stranded/metabolism MH - DNA-Binding Proteins/*biosynthesis/chemistry MH - Drosophila melanogaster/cytology/*metabolism MH - Female MH - Mitochondria/chemistry MH - Molecular Sequence Data MH - Oogenesis/physiology MH - Ovary/chemistry MH - RNA-Binding Proteins/*biosynthesis/chemistry MH - Research Support, U.S. Gov't, Non-P.H.S. MH - Sequence Homology, Amino Acid EDAT- 1994/06/10 MHDA- 1994/06/10 00:01 PST - ppublish SO - Gene 1994 Jun 10;143(2):171-7. NR -------------------------------------------------------------------------------- 131: Dhand R et al. PI 3-kinase: structural and f...[PMID: 8313896] Related Articles, Domains, Cited in PMC, Cited in Books, Books, LinkOut PMID- 8313896 OWN - NLM STAT- MEDLINE DA - 19940321 DCOM- 19940321 LR - 20031114 PUBM- Print IS - 0261-4189 VI - 13 IP - 3 DP - 1994 Feb 1 TI - PI 3-kinase: structural and functional analysis of intersubunit interactions. PG - 511-21 AB - Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85 alpha) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85 alpha and a related protein p85 beta are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the p110 protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defined 88 amino acids in the N-terminal region of p110 which mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel alpha-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed. AD - Ludwig Institute for Cancer Research, London, UK. FAU - Dhand, R AU - Dhand R FAU - Hara, K AU - Hara K FAU - Hiles, I AU - Hiles I FAU - Bax, B AU - Bax B FAU - Gout, I AU - Gout I FAU - Panayotou, G AU - Panayotou G FAU - Fry, M J AU - Fry MJ FAU - Yonezawa, K AU - Yonezawa K FAU - Kasuga, M AU - Kasuga M FAU - Waterfield, M D AU - Waterfield MD LA - eng PT - Journal Article PL - ENGLAND TA - EMBO J JID - 8208664 RN - EC 2.5.1.18 (Glutathione Transferase) RN - EC 2.7.1 (Phosphotransferases (Alcohol Group Acceptor)) RN - EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase) SB - IM MH - 1-Phosphatidylinositol 3-Kinase MH - Amino Acid Sequence MH - Animals MH - Binding Sites MH - Cattle MH - Cell Line MH - Electrophoresis, Polyacrylamide Gel MH - Glutathione Transferase/metabolism MH - L Cells (Cell Line) MH - Mice MH - Molecular Sequence Data MH - Moths MH - Mutation MH - Phosphotransferases (Alcohol Group Acceptor)/chemistry/*metabolism MH - Restriction Mapping MH - Structure-Activity Relationship MH - Transfection EDAT- 1994/02/01 MHDA- 1994/02/01 00:01 PST - ppublish SO - EMBO J 1994 Feb 1;13(3):511-21. NR -------------------------------------------------------------------------------- 132: Schoneck R et al. Trypanosoma cruzi cDNA encode...[PMID: 8054880] Related Articles, Compound via MeSH, Substance via MeSH, Nucleotide, Protein, Cited in PMC, Books, LinkOut PMID- 8054880 OWN - NLM STAT- MEDLINE DA - 19940912 DCOM- 19940912 LR - 20041117 PUBM- Print IS - 0248-4900 VI - 80 IP - 1 DP - 1994 TI - Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferases. PG - 1-10 AB - The isolation, characterization, and expression of a novel cDNA encoding a Trypanosoma cruzi polypeptide (TcAc2), homologous to various small stress proteins and glutathione S-transferases, are described. The deduced amino-acid sequence revealed two domains sharing 27% identity and an additional 27% similarity to each other suggesting that the molecule may have evolved from a single domain by a process of gene duplication and fusion. The TcAc2 cDNA was subcloned into the pGEX-2T vector for expression in E coli. In vitro translation products of epimastigote mRNA, immunoprecipitated with anti-TXepi serum, showed a major radioactive band of 52 kDa. Immunoprecipitation of [35S]methionine labelled epimastigote and trypomastigote antigens after pulse chase experiments, using anti-TcAc2 fusion protein antibodies, showed that the protein is released into the culture medium. Moreover, Western blot analysis revealed a single band of 52 kDa with epimastigote, trypomastigote and amastigote antigens. Primary structure homology searches revealed that each TcAc2 domain contained within its N-terminus significant homology to Solanum tuberosum pathogenesis-related protein PR1, soybean heat shock protein 26-A, auxin regulated clone pCNT103 from Nicotiana tabacum and Drosophila melanogaster glutathione S-transferase 27 (GST27). This finding was supported by a comparison of hydrophobicity profiles of TcAc2 and these proteins. Most of them play a central role in protection mechanisms against stress. Based on the homology between TcAc2, glutathione S-transferases (GST) and small stress proteins, it is likely that the TcAc2 gene product may play a crucial role in parasite's adaptation to its microenvironment. These molecules could be considered as members of the GST superfamily, where the T cruzi protein may take a particular place because of its internal gene duplication. AD - Research Laboratory on Trypanosomatids, INSERM U415, Lille, France. FAU - Schoneck, R AU - Schoneck R FAU - Plumas-Marty, B AU - Plumas-Marty B FAU - Taibi, A AU - Taibi A FAU - Billaut-Mulot, O AU - Billaut-Mulot O FAU - Loyens, M AU - Loyens M FAU - Gras-Masse, H AU - Gras-Masse H FAU - Capron, A AU - Capron A FAU - Ouaissi, A AU - Ouaissi A LA - eng SI - GENBANK/L07519 PT - Journal Article PL - FRANCE TA - Biol Cell JID - 8108529 RN - 0 (DNA Primers) RN - 0 (DNA Probes) RN - 0 (DNA, Complementary) RN - 0 (DNA, Protozoan) RN - 0 (Heat-Shock Proteins) RN - 0 (RNA, Messenger) RN - 0 (Sulfur Radioisotopes) RN - 63-68-3 (Methionine) RN - EC 2.5.1.18 (Glutathione Transferase) SB - IM MH - Amino Acid Sequence MH - Animals MH - Base Sequence MH - Blotting, Western MH - Cloning, Molecular MH - Comparative Study MH - DNA Primers MH - DNA Probes MH - DNA, Complementary MH - DNA, Protozoan/*genetics MH - Drosophila melanogaster/genetics MH - Escherichia coli MH - Gene Library MH - Glutathione Transferase/*biosynthesis/genetics/isolation & purification MH - Heat-Shock Proteins/*biosynthesis/genetics/isolation & purification MH - Methionine/metabolism MH - Molecular Sequence Data MH - Plants/genetics MH - Polymerase Chain Reaction MH - RNA, Messenger/biosynthesis MH - *Repetitive Sequences, Nucleic Acid MH - Research Support, Non-U.S. Gov't MH - Restriction Mapping MH - Sequence Homology, Amino Acid MH - Sulfur Radioisotopes MH - Trypanosoma cruzi/*genetics EDAT- 1994/01/01 MHDA- 1994/01/01 00:01 PST - ppublish SO - Biol Cell 1994;80(1):1-10. 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