Department of Biochemistry and Molecular Biology, GeorgetownUniversity Medical Center, Washington, DC 20007, USA
Received on April 12, 1999. revisedon June 3, 1999. accepted on June 4, 1999.
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Abstract |
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Introduction |
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The structure, function, and immunologic properties of MSP-1from several Plasmodium species have been extensively studied(11Holder, 1988; 6
Cooper et al., 1992; 5
Cooper,1993). MSP-1 is a polymorphic protein with a significantdegree of variable amino acid sequences and varies considerablyin Mr (185,000 to 250,000) depending on the parasite species (11
Holder, 1988; 22
McBride et al., 1985).The size variation is also present within different strains of asingle species; for example, MSP-1 from different P.falciparum strainshas an Mr that varies from 185 to 205 kDa (22
McBride et al., 1985; 11
Holder,1988). However, MSP-1 from different species and strainsdo have several common structural features: (1) an N-terminalsignal peptide sequence, (2) a C-terminal GPI anchor signal sequence, (3)1115 potential N-glycosylation sites;the positions of these sites, however, are not conserved even inproteins from different strains of a species, and none of the sitesis glycosylated to a significant level (11
Holder,1988; 29
Schofield and Hackett,1993).
MSP-1 undergoes proteolytic processing at the time of merozoitematuration (20Lyon et al.,1986; 13
Holder et al.,1987; 11
Holder, 1988).The protein is cleaved into different sized N-terminal fragments(83-, 28-/30-, 38 kDa), and a 42 kDa C-terminal fragment.The cleaved fragments are held together as a noncovalent complexon the merozoite surface that is believed to serve as a receptorfor red cell recognition and/or adhesion during erythrocyticinvasion (21
McBride and Heidrich, 1987; 11
Holder, 1988). Before invasion of redblood cells, the C-terminus fragment is further processed to forma 33-kDa N-terminal fragment and a 19-kDa GPI-anchored C-terminalfragment (2
Blackman et al.,1990; 1
Blackman and Holder, 1992).While all the N-terminal fragments are shed during invasion of redblood cells, the 19 kDa C-terminal fragment remains membrane-boundthrough the GPI anchor moiety and is carried with the invading merozoite (2
Blackman et al., 1990),suggesting that the C-terminus of MSP-1 may have a role in the invasionprocess and/or subsequent adaptation of the parasite insidered blood cells.
Individuals living in malaria-endemic areas have anti-MSP-1 antibodies(12Holder and Freeman, 1982; 9
Hall et al., 1984; 11
Holder, 1988; 28
Riley et al., 1992). The antibodies raised against MSP-1inhibit red blood cell invasion in vitro, and providepartial or complete protection in vivo againstsubsequent challenge with parasites (15
Huiand Siddiqui, 1987; 31
Siddiqui et al., 1987; 11
Holder,1988; 8
Etlinger et al.,1991; 6
Cooper et al.,1992). Previous studies have shown that antibodies againstdisulfide bond-dependent epitopes within the 19-kDa C-terminus of MSP-1provide protective immunity to parasite infection (26
Murphyet al., 1990; 4
Chang et al., 1992; 16
Hui et al., 1993; 17
Kumar et al., 1995; 3
Chang et al., 1996).
MSP-1 has a glycophorin recognition region (32Suet al., 1993). A monoclonal antibody designatedas 2B10 that is directed against an N-terminal region of glycophorinwas shown to have the same recognition determinant on human erythrocytes asMSP-1 of P.falciparum, and the binding of bothligands to the erythrocyte was dependent on sialic acid. Rabbitpolyclonal anti-idiotype antibodies raised against 2B10 recognizeboth the glycophorin binding site on 2B10 and a peptide region within MSP-1(10471640 amino acid C-terminus). Since 2B10 is capableof both blocking the binding of MSP-1 to human erythrocytes andinhibiting the merozoite invasion of red blood cells, we reasonthat the C-terminal polypeptide encompassing the apparent red bloodcell recognition element and the 19 kDa C-terminus would comprisea potential vaccine.
A previous study reported the construction of cDNAs that encodea 70 kDa MSP-1 C-terminal polypeptide with or without the N-terminalsignal peptide and C-terminal GPI signal sequences, insertion ofthe cDNAs into vaccinia virus, expression of polypeptides in recombinantvirus-infected BSC-1 cells, and immune responses of denovo expressed polypeptides in mice and rabbits (34Yanget al., 1997). The polypeptides expressed fromthe cDNAs that contained the signal peptide sequence had significantlylower Mr than expected even though they were glycosylated.The polypeptides were not detectable in the culture medium. Furthermore,the polypeptide expressed de novo from cDNAs withthe signal peptide sequence elicited an order of magnitude higherimmune response in animals compared with those derived from cDNAswithout the signal peptide sequence (34
Yanget al., 1997). In view of the potentialsignificance of these results in using MSP-1 for malaria vaccinedevelopment and to understand the biochemical basis for the observed results,the polypeptides expressed from all four cDNA constructs in mammaliancells were characterized in detail; the results are presented here.
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Results |
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[3H]GlcN labeling of cells expressingthe polypeptides and analysis by SDSPAGE and fluorographydemonstrated the presence of carbohydrate moieties only in polypeptidesfrom P1 and P2 but not in polypeptides from P3 and P4 (Figure 3).
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Cellular localization of the polypeptides
Polypeptides from all four cDNAs were not detectable on cell surfacesupon immunostaining using peroxidase-conjugated secondary antibody(Figure 7). However, upon permeabilization, cellsexpressing all four polypeptides were strongly stained (Figure 7). Previously, indirect immunofluorescencestaining suggested that the polypeptides from P1 and P2 were expressedon cell surfaces, whereas the polypeptides from P3 and P4 were exclusivelylocalized inside the cells (34Yang etal., 1997). The detection of polypeptides from P1and P2 on cell surfaces of intact cells by immunofluorescence staining(34
Yang et al., 1997)but not by immunoperoxidase staining (this study) suggests thatthese polypeptides are present on cell surfaces at levels belowthe detection limits of immunoperoxidase staining. Thus, these resultsdemonstrate that polypeptides from P3 and P4 are exclusively localizedinside the cells, whereas those from P1 and P2 are predominantlylocalized intracellularly with very low levels on cell surfaces.
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Discussion |
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This study demonstrates that the P.falciparum MSP-1signal peptide is efficiently recognized by the mammalian signalpeptide recognition particles, allowing for the translocation ofMSP-1 polypeptides to the lumen of the ER membranes. The signal peptidesequences of P.falciparum and P.vivax MSP-1are also recognized in Sf9 insect cells (26Murphyet al., 1990; 19
Longacreet al., 1994).
All four N-glycosylation sites of the P.falciparum MSP-1 70-kDaC-terminal portion are functional in the mammalian cells since majorportions of the polypeptides derived from P1 and P2 constructs contain14 N-linked oligosaccharide chains withfully glycosylated polypeptides predominating in the polypeptidefrom P1 (see Figures 2, 3).The N-glycosylation sites of 42-kDa P.falciparum and P.vivax MSP-1 C-terminal sequences are also glycosylatedwhen expressed in baculovirus-infected Sf9 cells (26Murphyet al., 1990; 19
Longacreet al., 1994). The N-glycosylationcapability of P.falciparum and P.vivax polypeptidesexpressed in mammalian and insect cells argue that the absence ornegligible N-glycosylation of native MSP-1 is notbecause of nonfunctional N-glycosylation sitesbut is due to very low levels of dolichol phosphate oligosaccharide donorsand/or oligosaccharyltransferase activity in the parasite,as was previously shown (7
Dieckmann-Schuppert et al., 1992).
The polypeptide derived from the cDNA P1 that contain the signalpeptide sequence but lack the GPI anchor signal sequence is expectedto be secreted if it normally passes through the secretory pathwayin the mammalian cells. However, the expressed polypeptide was retainedmainly intracellularly with a very low level on the cell surface.Furthermore, the polypeptide contained almost exclusively the highmannose-type N-linked oligosaccharides. These datasuggest that all or a major portion of the polypeptide has not traffickedthrough the Golgi and was likely retained in the Golgi or the pre-Golgi compartments.In contrast, it has been previously shown that the 42 kDa C-terminalpolypeptides of P.falciparum and P. vivax,and 19 kDa C-terminal polypeptide of P.vivax MSP-1 thatcontained the signal peptides expressed in Sf9 insect cells wereefficiently secreted (26Murphy etal., 1990; 19
Longacre et al., 1994). In Sf9 insect cells, N-glycosylationapparently facilitated passage through the secretory pathway sincethe inhibition of glycosylation with tunicamycin caused a markedreduction of extracellular secretion of the polypeptides (19
Longacre et al., 1994).
The data also demonstrate that the P.falciparum MSP-1GPI anchor signal sequence is either nonfunctional or very poorly functionalin mammalian cells. Several lines of evidence support this finding.(1) Partitioning of radioactive components obtained by the Pronasedigestion of [3H]GlcN-labeled polypeptideencoded by P2 showed the absence of lipid-bound carbohydrate moiety.(2) The expressed polypeptide could not be released by either PI-PLCor PI-PLD. (3) GlcN (non-N-acetylated) residueswere not detectable in the [3H]GlcN-labeled, N-glycanase-treated polypeptide encoded by P2.(4) Immunofluorescence (34Yanget al., 1997) and immunoperoxidase staining studiesshowed that the polypeptide is predominantly localized intracellularlywith only a very low level expression on the surfaces of mammaliancells. These data agree with the finding that the GPI anchor signalsequence of the P.falciparum circumsporozite proteinis poorly recognized by mammalian cells and that the requirementsfor the GPI anchor attachment are not identical in mammalian cellsand the malaria parasite (25
Moran and Caras,1994). Although the P.falciparum MSP-1 GPIanchor signal is nonfunctional in mammalian cells, the P.vivax MSP-1GPI anchor signal appears to be functional in insect cells. Thus,significant amounts of the 19 kDa and 42 kDa C-terminalpolypeptide of P.vivax MSP-1 expressed in insectcells have been shown to be GPI anchored (19
Longacreet al., 1994).
The results presented here show that the polypeptides derivedfrom P1 and P2 cDNAs were differentially truncated at their C-termini,apparently by proteases. Previous studies have shown that polypeptideswith uncleaved GPI anchor signals were retained inside the cellsand localized in the ER-Golgi intermediate compartments (24Moran and Caras, 1992). In agreement withthis finding, the polypeptide expressed from P2, which containsa noncleavable GPI anchor signal sequence, was found to be predominantlylocalized inside the cells. The polypeptide derived from cDNA P1,which lacks the GPI anchor signal, is also proteolytically processedand predominantly retained inside the cells. Accordingly, the intracellularretention and processing of the polypeptide derived from cDNA P2may not entirely be due to the presence of the uncleaved GPI anchor signalsequence. The small amount of polypeptide from P2 found on the cellsurface may be due to a low level of GPI anchor modification orthe inefficient transport of a membrane-bound truncated form ofP2. A similar low level cell surface localization of the polypeptidefrom P1 suggests the latter possibility. The 19- and 42-kDa P.vivax MSP-1C-terminal polypeptides expressed in Sf9 cells were not proteolytically processed(19
Longacre et al., 1994)indicating differences between the protein secretory pathways ofthe mammalian and insect cells.
Although the reason for the differential processing of polypeptidesfrom P1 and P2 is not known, N-glycosylation haslittle or no contribution since the molecular weights of polypeptidesof P1 and P2 expressed in the presence of tunicamycin were similarto those synthesized in the absence of tunicamycin after treatmentwith N-glycanase (see Figures 2, 4).
The results of the present study demonstrate that the MSP-1 C-terminalpolypeptides lacking the GPI anchor modification remain primarilyintracellular when expressed in mammalian cells. However, the polypeptidesexpressed from cDNAs that are engineered to contain mammalian GPIanchor signal sequences at their C-termini are expected to be modifiedwith GPI anchor moieties (25Moran and Caras,1994), and the GPI-anchored polypeptides are likely tobe transported to the cell surfaces. The GPI anchored proteins canelicit a far more effective immune response compared with correspondingproteins that lack GPI anchor moieties (30
Schofieldet al., 1999). Thus, cDNAs correspondingto the MSP-1 C-terminal polypeptides that are engineered to containa GPI anchor signal sequence functional in mammalian cells may proveto be effective immunogens.
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Material and methods |
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Preparation of recombinant vaccinia virus
Vaccinia viruses containing cDNA inserts corresponding to 70 kDaC-terminal polypeptide of MSP-1, with or without the peptide andGPI anchor signal sequences, were prepared as reported previously(34Yang et al., 1997).The plaque purified recombinant viruses were used for the preparationof high-titer stocks in HeLa S3 cells. All virus stocks were purifiedby ultracentrifugation through a 36% sucrose cushion. Viruseswere titered on monolayers of BSC-1 cells. Recombinant vaccinia virusesand wild type vaccinia virus (Western Reserve strain) were culturedin CV-1, HeLa, and Hu134TK cells.
Cell culture
The BSC-1 and Hu134TK cells were culturedin Eagles minimum essential medium supplemented with 10% fetalbovine serum. The CV-1 and HeLa cells were grown in Dulbeccosmodified minimum essential medium supplemented with 10% fetal bovineserum. Cells were cultured as monolayers at 37°Cin a humidified incubator.
Expression and analysis of the polypeptides incells infected with recombinant viruses
Confluent monolayers of cells in six-well plates, each well containing1 ml medium with 1% fetal bovine serum (in some experiments0.1% fetal bovine serum was used to avoid interferenceby serum albumin on the mobility of polypeptides during SDSPAGE),were infected with recombinant vaccinia virus at a multiplicityof 10 PFU. After 20 h, the medium from each well was removed, thecell layers were gently rinsed with fresh medium, and 50 µlof 0.5% SDS and 1% 2-mercaptoethanol in waterwas added to each well. The cell lysates were analyzed by Westernblotting either directly or after treatment with endoglycosidaseH or N-glycanase (see below). The spent mediumfrom each well was concentrated to 50 µl in Centricon 30 microconcentratorsand 10 µl was analyzed by Western blotting.
Inhibition of N-linked glycosylation with tunicamycin
Confluent monolayers of cells cultured in six-well plates were incubatedfor 4 h with medium containing 2.5 µgtunicamycin/ml. The cells were then infected separatelywith each recombinant vaccinia virus and wild type vaccinia virusat a multiplicity of 10 PFU. After 20 h, the medium was removed,the cell layers were rinsed with fresh medium, and 50 µl0.5% SDS and 1% 2-mercaptoethanol in water wasadded to each well. The lysates were mixed with 50 µlof 100 mM TrisHCl, pH 6.8, 200 mM dithiothreitol, 4% SDS,0.2% bromophenol blue and 20% glycerol. The solutionswere electrophoresed (Laemmli, 1970), and detected by antibody affinityblotting (see below).
Analysis of the polypeptides by Western blotting
Cells expressing polypeptides from cDNAs P1, P2, P3, and P4 were culturedin the absence (-) or presence of tunicamycin (+, 2.5 µg/ml), cell lysates preparedas described above, and 10 µl aliquots analyzedon 8% SDSpolyacrylamide gels (Laemmli, 1970). Theprotein bands on gels were electrotransferred to PVDF membranes.The membranes were blocked with 5% BSA in 200 mM TrisHCl,pH 7.5, for 2 h at room temperature and then treated with a 1:500dilution of antimouse serum against the 70-kDa C-terminal MSP-1polypeptide. After 2 h incubation, the membranes were washed andtreated with 1:7500 diluted alkaline phosphatase-conjugated goatantimouse IgG in TrisHCl, pH 7.5, for 90 min at room temperature.The protein bands on membranes were visualized with Western bluestabilized substrate for alkaline phosphatase according to manufacturers instructions.
Metabolic labeling of the polypeptides with [3H]GlcNand analysis by SDSPAGE/fluorography
Confluent monolayers of cells in six-well plates, each well containing1 ml medium, were separately infected with recombinant vacciniavirus and wild type virus at a multiplicity of 10 PFU.After 45 min, the medium in each well was replaced with 1 ml mediumcontaining 0.05% glucose and 50 µCi [3H]-GlcN,and further cultured for 18 h. The medium was removed, the cellswere rinsed three times with fresh medium, and 100 µl of50 mM TrisHCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenolblue and 10% glycerol added. About 10 µl celllysates were electrophoresed on 8% SDSpolyacrylamide gels(Laemmli, 1970). The gels were treated with MeOH, water, glacialHOAc (50:40:10, v/v) for 1 h, washed with water for 5 min,soaked in Amplify fluorographic solution for 1 h, dried, and exposedto x-ray film at 80°C.
Analysis of N-glycanase and endoglycosidaseHtreated polypeptides by Western blotting
Lysates of CV-1, Hu134TK and HeLa cellsexpressing polypeptides from cDNAs P1 and P2 (see above) were heated at100°C for 10 min, cooled, and mixedwith equal volumes of 100 mM sodium phosphate, 2% NP-40,pH 7.5, and 10 µl of the solution incubatedwith 1 IUB milliunit of N-glycanase at 37°Cfor 2 h. For digestion with endoglycosidase H, the lysates weremixed with equal volumes of 100 mM sodium citrate, pH 5.5,and 10 µl of this solution incubatedwith 1 IUB unit of endoglycosidase H at 37°Cfor 2 h. Untreated and enzyme-treated cell lysates were eletrophoresedon 8% SDSpolyacrylamide gels, the proteinbands were electrotransferred onto PVDF membranes and detected withmouse antiserum against the 70 kDa recombinant MSP-1 polypeptide.Alkaline phosphataseconjugated goat antimouse IgG wasused as the secondary antibody.
Carbohydrate analysis
Cells expressing polypeptides from cDNAs P1 and P2 were metabolicallylabeled with [3H]GlcN and then lysedwith 50 µl of water containing 0.5% SDS,1% 2-mercaptoethanol (see above). Portions of lysates weretreated with N-glycanase. The untreated and N-glycanase-treatedcell lysates were electrophoresed on SDSpolyacrylamidegels, transferred onto PVDF membranes, and stained with Coomassieblue. In some experiments, untreated and N-glycanase-treatedcell lysates were diluted to 0.5 ml with 50 mM TrisHCl,150 mM NaCl, pH 7.5, and the polypeptides immunoprecipitatedwith antimouse IgG against the 70-kDa MSP-1 C-terminal polypeptide. Theimmunoprecipitated polypeptides were recovered by protein A affinityabsorption, analyzed by SDSPAGE, and transferred to PVDFmembranes.
The PVDF membranes were thoroughly washed with water, the membranescontaining polypeptide bands excised, and hydrolyzed with 400 µl of 3.5 M HCl at 100°Cfor 6 h. For sialic acid analysis, the membranes containing polypeptide bandswere hydrolyzed with 400 µl of 2 M HOAcat 80°C for 4 h. The hydrolysates wererecovered and dried in a Speed-Vac. The residues were dissolvedin water, mixed with appropriate standard sugars, and analyzed byhigh-pH anion-exchange chromatography with a Dionex BioLC HPLC coupledto pulsed amperometric detection using a CarboPac PA1 column (4 x 250 mm) (10Hardyand Townsend, 1994). The eluents were: (1) 20 mM NaOHat a flow rate of 0.8 ml/min for hexosamines, (2) 100 mMNaOH, 150 mM NaOAc at a flow rate of 0.8 ml/min for sialicacids. Elution of radioactivity was monitored by liquid scintillationcounting of fractions collected at 0.30.4 min intervals. [3H]-labeledsugars were identified by either coelution or comparison of elutiontime with standard sugars. Nonradioactive standard sugars were detectedby pulsed amperometric detection.
Analysis of the polypeptide for GPI anchor moieties
Monolayers of CV-1 cells infected with recombinant vaccinia viruscontaining cDNA P2 inserts were metabolically labeled with [3H]GlcN.The cell lysates were electrophoresed on 8% polyacrylamidegels under reducing conditions and the protein bands were transferredon PVDF membranes. The radiolabeled protein band corresponding tothe polypeptide from P2 was excised and analyzed for GPI moietiesby the following methods.
The [3H]GlcN-labeled polypeptidebands (50006000 c.p.m.) on PVDF membranes were suspendedin 500 µl of 100 mM TrisHCl,1 mM CaCl2, pH 8.0, containing 0.05% SDS and 0.5% NP-40,and incubated with pronase (1.0 mg, added 0.5 mg aliquotsat 0 and 24 h) at 55°C for 48 h. Theenzyme digests were centrifuged, the membranes washed with water (3 x 100 µl),and the combined supernatants and washings dried in a Speed Vac.The residues were dissolved in water (0.5 ml) and extractedwith water-saturated 1-butanol (see Figure 5).The aqueous and organic phases were separately dried in a rotaryevaporator and dissolved in water, and the radioactivity was measuredby liquid scintillation counting.
The [3H]GlcN-labeled polypeptideband (derived from cDNA P2, ~10,000 c.p.m.) on PVDF membrane wassuspended in 400 µl of 0.2M NaOAc, pH 3.8, containing 1 M NaNO2 (28). After 18h at room temperature, the pH of the solution was adjusted to 7with 2 M NaOH, centrifuged and the membrane washed with water. Thecombined supernatant and washings was measured for radioactivityby liquid scintillation counting. The membrane was hydrolyzed with400 µl of 3.5 M HCl at 100°Cfor 6 h. The polypeptide band not treated with HNO2 wassimilarly analyzed as a control. The hydrolysates were dried ina Speed Vac and analyzed by high-pH anion-exchange chromatography(10Hardy and Townsend, 1994).
Analysis for GPI anchor moieties in the polypeptidesby treatment of cells with PI-PLC and phosphatidylinositol-specific phospholipaseD (PI-PLD)
The monolayers of cells (in a 6-well plate) expressing the polypeptidesfrom P2 were gently rinsed with 25 mM HEPES, 150 mM NaCl, pH 7.4,and then treated with 1 unit of PI-PLC in 300 µlof the above buffer at 37°C for 1 h.Equivalent control cells were incubated in parallel under the similarconditions but without the enzyme. The buffers were carefully removed, centrifugedat 4000 r.p.m. to remove any detached cells, concentrated on Centricon30 tubes and analyzed by Western blotting as described above. ForPI-PLD treatment, the cell layers were incubated in the above buffercontaining 2 mM CaCl2 and 10 µlof fresh rabbit serum (as source of PI-PLD) (23Menon,1994). The supernatants were analyzed by Western blotting.
Analysis of the polypeptides for proteolytic processing
Lysates of cells expressing polypeptides from cDNAs P1, P2, P3,and P4 were electrophoresed on 8% SDSpolyacrylamide gels.The protein bands on gels were electrotransferred onto PVDF membranes.Detection of polypeptides on the membranes using 5.2 monoclonalantibody specific to the C-terminal region of MSP-1 was carriedout as described above. Alkaline phosphataseconjugatedgoat antimouse IgG was the secondary antibody and Western blue stabilizedsubstrate for alkaline phosphatase was the color developing reagent.
Immunostaining of cell expressing the polypeptides
Confluent monolayers of cells in six-well plates, each well containing1 ml medium with 2% bovine fetal serum, were infected withrecombinant vaccinia virus at a multiplicity of 10 PFU.After 20 h, the medium from each well was removed and the cell layerswere gently rinsed with fresh medium. Immunostaining using peroxidase-conjugatedsecondary antibody was carried out according to Sutter etal., 1994. The cells were fixed with acetone,methanol (1:1 v/v) for 2 min, washed with PBS, pH 7.4,and then incubated at room temperature with 1:200 diluted mousepolyclonal antiserum against 70 kDa MSP-1 polypeptide. After 1 h,the cells were washed three times with 1 ml PBS, pH 7.4, and thenincubated at room temperature with 1:1000 diluted peroxidase-conjugatedgoat antimouse IgG in PBS, pH 7.4, containing 2% fetalbovine serum. After 45 min, the plates were incubated with 0.5 mlof PBS, pH 7.4, containing 80 µg/mldianisidine hydrochloride and 0.03% hydrogen peroxide atroom temperature until color developed.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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b Towhom correspondence should be addressed at: Department of Biochemistryand Molecular Biology, Georgetown University Medical Center, 3900Reservoir Road, NW, Washington, DC 20007
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References |
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