©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Chicken Guanylate-binding Protein
CONSERVATION OF GTPase ACTIVITY AND INDUCTION BY CYTOKINES (*)

(Received for publication, October 17, 1995; and in revised form, February 14, 1996)

Martin Schwemmle Bernd Kaspers (1) Andrea Irion Peter Staeheli (§) Ursula Schultz

From the Abteilung Virologie, Institut für Medizinische Mikrobiologie and Hygiene, University of Freiburg, Freiburg and Institut für Tierphysiologie, University of Munich, Munich, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

To gain further insights into the cytokine network of birds, we used polymerase chain reaction technology to clone a cDNA that codes for a chicken homolog of the interferon-induced guanylate-binding proteins (GBPs). In its N-terminal moiety, the 64-kDa chicken GBP contains two sequence blocks of 100 and 19 amino acids, respectively, that are about 70% identical to mammalian GBPs. The first region includes two motifs of the canonical GTP-binding consensus element. The other parts of chicken GBP are poorly conserved, except for a CAAX motif at the extreme C terminus which might signal isoprenylation. Like mammalian GBPs, recombinant chicken GBP specifically bound to agarose-immobilized guanine nucleotides and hydrolyzed GTP to both GDP and GMP. Regulation by interferons was also conserved: chicken GBP RNA was barely detectable in uninduced chicken cells. Low GBP RNA levels were found in cells treated with type I interferon, whereas very high levels were observed in cells treated with supernatant of a chicken T cell line that secretes a -interferon-like activity. Together with recent phylogenetic studies of interferon genes, these results suggest that in spite of low sequence conservation, the various components of the avian interferon system are functionally well conserved.


INTRODUCTION

An important role of interferons (IFNs) (^1)is to activate the virus defense system of vertebrate hosts(1) . Phylogenetic analysis of this presumably archetypal antiviral strategy has been hampered by the lack of information on the molecular nature of IFNs and their intracellular effector molecules in non-mammalian species. It was known for almost 40 years that chicken cells can be induced to secrete IFN(2) , but the molecular analysis of this activity proved difficult. The recent cloning of the first cDNA for a chicken IFN (3) showed that its primary sequence is poorly conserved. This finding raised the possibility that the avian IFN system has evolved independently and might use fundamentally different effector molecules(4) .

Among the IFN effector molecules of mammals, the guanylate-binding proteins (GBPs) are of particular interest due to their unusual biochemical properties and dramatic up-regulation in IFN-treated cells. Unlike other proteins with GTP-binding activity, GBPs bind to agarose-immobilized GTP, GDP, and GMP with very high affinity(5, 6, 7, 8) . Furthermore, GBPs not only hydrolyze GTP to GDP as conventional GTPases do, but they also degrade a significant fraction of this substrate to GMP(9) . Unlike most other GTPases, GBPs carry only the first two motifs of the tripartite GTP-binding consensus element(10) , while the third (N/T)XKD motif is missing(7) . All mammalian GBPs carry a CAAX isoprenylation signal motif at their C termini (7, 9) . Human GBP1 was recently shown to be farnesylated in vitro(9) and in vivo. (^2)In mouse and human cells, GBPs are strongly induced in response to IFN- and to a lesser extent by IFN-alpha and IFN-beta (type I IFNs)(5, 8) . In mouse cells, this induction process is mediated by the IFN-induced transcription factor IRF-1(11) . Mouse and human cells contain at least two functional GBP genes(7) , but the physiological roles of their encoded products in the IFN response remains unknown.

Here we demonstrate that GBPs also exist in birds. Analysis of a chicken cDNA clone showed that sequence conservation of GBPs is restricted to regions in the N-terminal moiety that harbor the GTP-binding consensus motifs. Nonetheless, the biochemical properties of recombinant chicken GBP closely resembled those of mammalian GBPs. Another conserved feature of GBPs was their regulation: chicken GBP gene was most strongly induced by an IFN--like activity present in chicken T cell supernatants.


EXPERIMENTAL PROCEDURES

Cell Culture

Chicken embryo cells were prepared from 10-day-old chicken embryos by trypsinization. They were cultured in Dulbecco's modified minimal essential medium (DMEM) supplemented with 7% newborn calf serum and 3% fetal calf serum (FCS) as described previously(12) . CEC-32 (13) and HD-11 (14) cells were propagated in DMEM supplemented with 2% chicken serum and 8% FCS. LMH cells (15) were grown in DMEM supplemented with 10% FCS. Chicken T cell lines were established after exposure of juvenile chicken bone marrow cells to avian reticuloendotheliosis virus strain T essentially as described elsewhere(16) . T cell lines were maintained in DMEM supplemented with 10% FCS. Supernatants of T cell cultures that had reached maximal density were cleared by centrifugation, before they were used for induction experiments. Supernatants were stored at -20 °C. Macrophage-activating factor activity was measured as described (17) .

Recombinant Chicken Interferon

Appropriate dilutions of culture supernatants from COS monkey cells transfected with a ChIFN expression construct (12) were used.

cDNA Library

The phage cDNA library that we used here was described previously(18) . It was generated from poly(A) RNA of poly(rIbulletrC)-treated chicken embryo cells.

Cloning of Chicken GBP

PCR was performed with degenerated primers deduced from highly conserved regions of human GBP1, human GBP2, and mouse GBP1(7) . Primer 5`-GG(C/T)AA(A/G)TC(C/T)TAC(C/T)TGATGAAC-3` corresponding to positions 216-236 of human GBP1 included parts of the first motif of the GTP-binding consensus element. Primer 5`-ACACCACATCCAGAT(G/T/A)CC(T/C)TT-3` was reverse complementary to positions 294-314 of human GBP1. PCR was performed for 35 cycles (94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min) in a total reaction volume of 100 µl with 500 ng of each primer, 1 µg of purified DNA of the cDNA library, and 2.5 units of Taq polymerase (U. S. Biochemical Corp.). A 100-base pair PCR product was cloned into the TA-cloning vector pCR (In Vitrogen, San Diego), and its sequence was determined. Translation of this sequence revealed that 11 out of 19 amino acids (positions 58-76 of chicken GBP; see Fig. 1) were identical to positions 57-75 of human GBP1. Approximately 3 times 10^5 phage clones of the chicken cDNA library were subsequently screened with the P-labeled PCR fragment. Hybridization was carried out in 10 mM Pipes, pH 6.8, 0.3 M NaCl, 10 mM EDTA, 50% formamide, 0.5% SDS, 1 times Denhardt's solution, and 100 µg/ml denatured herring sperm DNA. Of approximately 100 positive clones, two phages with long inserts were selected. The complete sequence of one clone was established using the T7 sequencing kit of Pharmacia Biotech Inc. (Freiburg, Germany).


Figure 1: Primary sequence of chicken GBP. Shown is the nucleotide sequence of a cDNA clone that codes for chicken GBP (GenBank accession no. X92112). Its deduced amino acid sequence is presented in the single-letter code. The two regions with high similarity to mammalian GBPs are shaded. Two motifs of the canonical GTP-binding consensus element (10) are highlighted by a solid line; the putative third motif is highlighted by a broken line. Asterisks mark a putative isoprenylation motif.



Plasmid Constructions

Using PCR, the complete open reading frame of chicken GBP was inserted between the BamHI and HindIII sites of the Escherichia coli expression vector pQE-9 (Diagen, Hilden, Germany). The sense primer 5`-ATTAAGGATCCGACACTCCGGTGCTCCCGATG-3` created a new restriction site for BamHI and corresponded to positions 19-39 of the chicken GBP cDNA. The antisense primer 5`-TACATTAAGCTTACTCCAATGGTGGCACAATG-3` created a new HindIII restriction site and was reverse complementary to positions 1748-1767 of the chicken GBP cDNA. The resulting plasmid, pHis-ChGBP, coded for a fusion protein between the oligopeptide Met-Arg-Glu-Ser-(His)(6)-Gly-Ser and chicken GBP from amino acid position 2 to position 573.

Purification of Recombinant Chicken GBP

Histidine-tagged chicken GBP (His-ChGBP) was purified from E. coli applying Ni-agarose chelate chromatography as described for Mx proteins(19, 20) . Partially purified His-ChGBP (350 µg in 200 µl) was applied to a Superose 12 HR (10/30) gel filtration chromatography column (Pharmacia) equilibrated in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl(2), 5% glycerol, 5 mM 2-mercaptoethanol) at a flow rate of 0.4 ml/min, and 0.5-ml fractions were collected. The bulk of His-ChGBP eluted with the void volume (>300 kDa). His-ChGBP was concentrated with a Centricon C30 cartridge (Millipore) according to the manufacturer's protocol at 8 °C.

GTPase Assay and HPLC Analysis

Concentrated His-ChGBP (5 µg in 20 µl of buffer A) was adjusted to 200 µM GTP and incubated for 1 h at 37 °C. GTPase activity was analyzed by applying 5 µl of the reaction mixture to HPLC analysis. Analysis of nucleotides was performed as described with slight modifications(20) . Briefly, a C-18 reversed phase column (0.4 times 25 cm filled with ODS Hypersil, 5 µm, Bischoff, Leonberg, Germany) was run at ambient temperature with a flow rate of 1 ml/min in 50 mM Na-phosphate buffer (pH 6.5) containing 0.2 mM tertiary butylammoniumbromide, 3% (v/v) acetonitrile, and 0.2 mM NaN(3). Under these conditions, GMP eluted with a retention of 4.7 min, GDP with 7.1 min, and GTP with 10.2 min. The absorption was measured at 252 nm with a VWM-2141 UV detector (Pharmacia), and the signals were quantified with a C-R5A integrator (Shimadzu, Kyoto, Japan).

Binding of His-ChGBP to Nucleotide Agarose

Binding assays were carried out with samples of about 3 µg of purified recombinant His-ChGBP as described elsewhere(9) . The nucleotide agarose was purchased from Sigma (Deisenhofen, Germany). Bound proteins were eluted with sample buffer, analyzed on a 10% SDS-PAGE, and visualized by Coomassie Blue staining.

Identification of GBPs in Chicken Cells by Affinity Chromatography

Affinity chromatography on immobilized GTP agarose beads was carried out essentially as described(5, 8) . CEC-32 cells in 90-mm dishes were induced for 15 h with a 1:50 dilution of a chicken T cell culture supernatant in 10 ml methionine- and cysteine-free medium containing 20 µl of [S]methionine and [S]cysteine (>1000 Ci/mmol, 14.3 mCi/ml) and 2% FCS. The cells from one 90-mm dish were resuspended in 100 µl of lysis buffer (10 mM Pipes, 1% Triton X-100, 100 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 300 mM sucrose and 100 µM phenylmethylsulfonyl fluoride) and lysed by incubation on ice for 5 min. Cell debris were removed by centrifugation for 15 min in a microcentrifuge. Samples of the extract were incubated with 100 µl of either GTP or CTP agarose beads equilibrated in washing buffer (40 mM Tris-HCl, pH 7.0, 300 mM NaCl, 10 mM MgCl(2) and 4 mM 2-mercaptoethanol) in a 15-ml Falcon tube for 20 min on ice with occasional shaking. The agarose beads were washed twice with 5 ml of ice-cold washing buffer, and matrix bound proteins were eluted by boiling in 40 µl of SDS gel sample buffer. Samples of 20 µl were analyzed by 10% SDS-PAGE. The gel was soaked in Amplify (Amersham Corp.), dried, and exposed to x-ray film.

RNA Analysis

Cells were treated for 15 h with either 500 units/ml recombinant ChIFN produced in COS cells(12) , 50-fold diluted supernatant of a T cell line that secretes a macrophage-activating factor (MAF) activity, or 50-fold diluted supernatant of a T cell line that does not secrete this activity. Total RNA was isolated by the guanidinium thiocyanate/acid phenol method(21) . RNAs were size-fractionated by electrophoresis through a 1.2% formaldehyde agarose gel before they were blotted onto nitrocellulose. The membranes were sequentially hybridized with a chicken GBP fragment that contained the entire coding region, the EcoRI-XhoI fragment of chicken IRF-1 cDNA(18) , an EcoRI-BamHI fragment of the chicken Mx cDNA(22) , and a PstI fragment of the chicken glyceraldehyde-3-phosphate dehydrogenase cDNA(23) . These hybridization probes were radiolabeled with P by nick translation.


RESULTS

Cloning of a Chicken Guanylate-binding Protein

In order to amplify GBP-related sequences of the chicken by PCR, we used a cDNA library prepared from poly(rIbulletrC)-treated chicken embryo cells (18) and degenerated primers from highly conserved regions of mammalian GBPs. Sequencing of a resulting 100-base pair fragment revealed an open reading frame with significant homology to human GBP1. To isolate a full-length cDNA clone, we screened the cDNA library with the PCR fragment. Sequencing of one positive phage clone showed the presence of a long open reading frame capable of coding for a polypeptide of 573 amino acids (Fig. 1) with a calculated molecular mass of 64,465. Alignment with known GBP sequences revealed two conserved sequence blocks between amino acid positions 36-135 and 165-183 (Fig. 1, shaded areas). These two regions showed about 70% sequence identity to mammalian GBPs. The first two motifs of the tripartite GTP-binding consensus element (GXXXXGKS, DXXG, (N/T)KXD) (10) are localized in the first conserved sequence block (Fig. 1). Like mammalian GBPs(7) , the chicken GBP lacks the third motif of the consensus element. Due to the fact that conservation is poor in the remaining parts of the molecules, the overall sequence identity of mammalian and chicken GBPs is only about 40%. The extreme C terminus of the chicken GBP carries a CAAX motif, which serves as signal sequence for isoprenylation in various other proteins, including human GBP1(9) .

Chicken GBP Has GTP-binding and GTP-hydrolyzing Activity

To determine the biochemical properties of chicken GBP, we expressed a variant of chicken GBP in E. coli that carries a histidine tag at the N terminus (His-ChGBP), and purified it by nickel-chelate agarose chromatography. Subsequent purification was facilitated by the fact that His-ChGBP formed oligomers and that it eluted with the void volume of a Superose 12 column (molecular mass >300 kDa) (data not shown). His-ChGBP purified in this manner was virtually pure and migrated on 10% SDS-PAGE with an apparent molecular mass of 65 kDa (Fig. 2, panel A, lane 1). First evidence that His-ChGBP was active came from binding studies with agarose beads carrying immobilized nucleotides: it efficiently bound to GTP agarose (Fig. 2, panel A, lane 4) but failed to bind to CTP agarose (Fig. 2, panel A, lane 3). Other unrelated proteins that we expressed and purified by similar means in the past (19, 20) (^3)never showed such binding activity. Preferential binding to immobilized guanine nucleotides thus seems to be a unique feature of GBPs from both mammals (5, 6, 7, 8) and birds.


Figure 2: Guanine nucleotide binding and GTPase activity of chicken GBP. A, purified recombinant His-ChGBP (lane 1) was allowed to react with CTP agarose (lane 3) or GTP agarose (lane 4), and bound material was analyzed by 10% SDS-PAGE and Coomassie Blue staining. Three times more His-ChGBP was used for the binding experiments than loaded in lane 1. Protein size marker (M). B, purified recombinant His-ChGBP (0.3 µg/µl) was incubated for 1 h at 37 °C in the presence of 200 µM GTP, and the reaction product was analyzed for the presence of GDP and GMP by HPLC. A control reaction lacking His-ChGBP was performed under identical conditions. The chromatograms were monitored at 252 nm. The identity of the nucleotide peaks and their retention times are indicated. The asterisks mark signals of an unknown buffer contaminant.



Since human GBP1 was recently shown to be an unorthodox GTPase that hydrolyzes GTP to GDP as well as to GMP(9) , we examined whether the chicken GBP would exhibit similar biochemical activity. Incubation of His-ChGBP with 200 µM GTP for 60 min resulted in partial hydrolysis of this substrate to GDP and GMP (Fig. 2, panel B). The reaction product consisted of approximately 87% GDP and 13% GMP. ATP was not hydrolyzed by purified His-ChGBP at a detectable rate under our reaction conditions (data not shown), indicating that the preparation was virtually free of contaminating phosphatases. Furthermore, histidine-tagged dehydrofolate reductase or a variant of human GBP1 with a mutation in the first element of the GTP-binding motif (^4)that were both expressed and purified by similar means failed to hydrolyze GTP at a detectable rate (data not shown). The fact that GMP was a product of the chicken GBP-catalyzed GTP hydrolysis reaction demonstrated that, like human GBP1, the chicken homolog has unusual GTPase properties. However, the two recombinant proteins differed clearly in their fidelity by which they synthesized GMP. Human GBP1 preferentially hydrolyzed GTP to GMP (9) , whereas chicken GBP degraded GTP predominantly to GDP. Kinetic studies showed that the specific GTPase activity of our best preparations of His-ChGBP were about 3 nmol of GTP/min/mg, a value that was about 10-fold lower than that determined for human GBP1(9) .

Chicken GBP Is Strongly Induced by Supernatant of a Chicken T Cell Line

Mammalian GBPs are expressed in a variety of cell types in response to IFN- and, to a lesser extent, in response to type I IFNs(5, 8) . To determine whether transcription of the chicken GBP gene is controlled similarly, various chicken cell lines were treated with either recombinant type I IFN, supernatant of a chicken T cell line that secretes antiviral and MAF activity, (^5)or supernatant of a control T cell line that lacks these activities. Circumstantial evidence suggests that the activity secreted by the former T cell line is chicken IFN-(17) .^5

In a first series of experiments, RNAs from the various cell lines were tested for chicken GBP expression by Northern blot analysis (Fig. 3). Untreated cells showed virtually no GBP RNAs. CEC-32 cells treated with supernatant of the MAF-secreting T cell line contained high levels of three GBP RNAs of 2-3.5-kilobase pair length, which are either the products of multiple GBP genes or transcription variants of a single GBP gene (Fig. 3). Similarly treated HD-11 cells contained high levels of a single GBP RNA of about 2 kilobase pairs. Treatment of CEC-32 and HD-11 cells with supernatant of the control T cell line did not result in the accumulation of detectable levels of GBP RNAs (Fig. 3). Recombinant chicken type I IFN induced the GBP genes of CEC-32 and HD-11 cells only very weakly (signals hardly visible on the short exposure shown in Fig. 3). LMH cells and primary chicken embryo cultures did not contain detectable levels of GBP RNAs under all induction conditions studied (Fig. 3, and data not shown). In all cell lines, the IRF-1 gene was induced very strongly by supernatant of the MAF-secreting T cell line, to a lower extent by recombinant type I IFN, and not detectably by supernatant of the control T cell line (Fig. 3). In LMH and HD-11 cells, the Mx gene was strongly induced by type I IFN but not by the T cell supernatants, whereas in CEC-32 cells the Mx gene seemed inert to induction by the various chicken cytokines (Fig. 3). Taken together, these results suggested a strong conservation of GBP regulation in response to cytokines in mammals and birds.


Figure 3: Detection of GBP transcripts in established chicken cell lines under various induction conditions. LMH, CEC-32 and HD-11 cells were treated for 15 h with either plain culture medium (), 500 U/ml of recombinant chicken type I IFN (ChIFN1), 50-fold diluted supernatant of a chicken T cell line that secretes antiviral and MAF activity (MAF), or 50-fold diluted supernatant of a chicken T cell line that fails to secrete MAF (no MAF). The cells were lysed, the RNAs were extracted, and samples (20 µg/lane) of total RNA were subjected to Northern blot analysis. The membrane was sequentially hybridized to radiolabeled chicken GBP, chicken IRF-1(18) , chicken Mx (22) and chicken glyceraldehyde-3-phosphate dehydrogenase (23) cDNA probes.



To determine whether the induced GBP RNAs were translated into functional proteins, CEC-32 cells were incubated with either MAF-containing T cell supernatant or plain medium in the presence of radiolabeled amino acids, before cell lysates were prepared and samples were analyzed for proteins capable of binding to agarose-immobilized GTP. Two GTP-binding proteins of M(r) 65,000 and 70,000 were detected in the MAF-induced cells that were absent from the uninduced control cells (Fig. 4). These proteins most likely represent bona fide chicken GBPs.


Figure 4: Induction of endogenous guanine nucleotide-binding proteins in CEC-32 cells. Cells maintained in the presence of [S]methionine and [S]cysteine were treated for 15 h with either plain medium () or with 50-fold diluted supernatant of a T cell line that secretes antiviral and MAF activity (MAF). Cell lysates were prepared and allowed to react with GTP-agarose, before the bound proteins were eluted and separated by 10% SDS-PAGE. The gel was soaked in Amplify (Amersham), dried, and exposed to x-ray film. The gel positions of two induced GTP-binding proteins are marked.




DISCUSSION

Our experiments showed that, although the primary sequences of chicken and mammalian GBPs differ greatly, their biochemical properties and their regulation by IFNs are remarkably well conserved. The previously characterized chicken homologs of IRF-1(18) , ICSBP(18) , and Mx protein (22) had revealed a different picture: the coding sequences of these genes are well conserved over most regions. The situation reported here for the GBPs is reminiscent to that reported for type I IFNs of birds and mammals, which show minimal sequence conservation in spite of strong functional similarities(3) .

Sequence conservation between chicken and mammalian GBPs is prominent in two regions (Fig. 1, shaded areas), one of which is 100 amino acids long and harbors two motifs of the tripartite GTP-binding consensus element(10) . The second region is only 19 amino acids long and starts 25 residues downstream. Since GBPs are unique among GTP-binding proteins in that they lack the classical third motif ((N/T)KXD) of the tripartite GTP-binding element(7) , this constellation suggests that the missing motif is hidden in the second conserved region. Considering the chemical nature of the various amino acids, we speculate that the sequence TVRD (amino acid positions 175 to 178 in ChGBP; Fig. 1) is required for guanine nucleotide binding of GBPs. Substitution of the (N/T)KXD motif by TVRD might explain the unorthodox nucleotide binding properties of GBPs. Site-directed mutagenesis and structural studies of the GBP nucleotide binding pocket will be necessary to evaluate this hypothesis.

Although the characteristic biochemical features of mammalian GBPs are conserved in the chicken GBP, we still observed differences between the GTPase activities associated with chicken GBP and human GBP1. The most important difference was that GMP represented only a minor product of the chicken GBP-catalyzed hydrolysis reaction, whereas it was the predominant product of the human GBP1-catalyzed reaction(9) . In this respect, chicken GBP closely resembles GBP2, the product of a second human GBP gene(7) : hydrolysis of GTP by GBP2 yielded about 7-fold more GDP than GMP under standard reaction conditions. (^6)It thus seems that although all GBPs can form GMP, they do it to variable extents.

Chicken GBP RNA was not found in uninduced cells, but it was abundantly present in cells treated with supernatant of a T cell line that secretes antiviral and MAF activity. Based on other results (B. Kaspers, unpublished results), we previously speculated that these two activities might result from the avian homolog of IFN-(17) . The results described here seem to support this view and further provide a new method for measuring this activity with very high sensitivity. Interestingly, the GBP gene(s) of CEC-32 and HD-11 cells were strongly inducible, whereas those of LMH and chicken embryo cells were not. By monitoring the induction of IRF-1 RNA we could demonstrate that all these cell lines responded otherwise equally well to the inducing agents, indicating that the corresponding cytokine receptors and signal transduction pathways are functional. The fact that primary embryo cells failed to express the GBP genes, whereas two of three established cell lines expressed them strongly is a puzzling finding. It suggests that genetic differences may exist between the chickens used to establish the various cell lines. Genetic differences could also explain the fact that the Mx gene could not be induced in CEC-32 cells, although it was strongly induced in type I IFN-treated LMH and HD-11 cells. The situation in the chicken may thus resemble that of mice, where inbred strains with genetic defects of Mx and GBP genes were described(8, 24) .


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Ernst and Berta Grimmke-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79008 Freiburg, Germany. Tel.: 49-761-203-6579; Fax: 49-761-203-6562; staeheli{at}sun1.ukl.uni-freiburg.de.

(^1)
The abbreviations used are: IFN, interferon; GBP, guanylate-binding protein; FCS, fetal calf serum; MAF, macrophage-activating factor; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid.

(^2)
D. E. Nantais, M. Schwemmle, and J. E. Buss, submitted for publication.

(^3)
M. Schwemmle and P. Staeheli, unpublished results.

(^4)
M. Schwemmle, unpublished results.

(^5)
B. Kaspers, unpublished results.

(^6)
R. Neun and M. Schwemmle, unpublished results.


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