Polypeptide Species Research Articles

Sequence of the Spike Srotein of the Porcine Epidemic Diarrhoea Virus

The complete sequence of the spike (S) gene of the Br1/87 isolate of porcine epidemic diarrhoea virus (PEDV) was determined from cDNA clones. The predicted polypeptide was 1383 amino acids long, contained 29 potential N-linked glycosylation sites and showed structural features similar to those of the coronavirus spike protein. The PEDV S protein, like that of the members of the transmissible gastroenteritis virus (TGEV)-related subset, lacks a proteolytic site to yield cleaved amino and carboxy subunits S1 and S2. Viral polypeptide species of the expected M(r), i.e. 170K/190K, were observed in PEDV-infected cells. Sequence comparison confirmed that, within the subset, PEDV was most closely related to the human respiratory coronavirus HCV 229E. However, PEDV S protein has an additional 250 residue N-terminal domain which is absent from HCV 229E and porcine respiratory coronavirus, the respiratory variant of TGEV. Alignment of the S1 regions revealed a second domain of about 90 residues with increased sequence divergence which might possibly express virus-specific determinants.

The Cellular U-Particle, Whose Synthesis Is Induced by Mengovirus Infection, Is Homologous to Apoferritin

Mengo virus infection of mouse L-cells results in induction of the synthesis of a cellular protein-containing particle, 12 nm in diameter, which was designated U (Boege et al. (1987) Virology 159,358-367). We have purified the U-particle from virus-infected cells by a series of chromatographic steps and found it to be composed of two polypeptide species (MW 23,000 and 25,000), present in a ratio of ∼7:3. Neither of these polypeptides is measurably glycosylated or phosphorylated and the U-particle contains no detectable nucleic acid. Several amino acid sequences obtained from CNBr fragments of the U-polypeptides identified them as the H- and L-chains of mouse apoferritin. This finding was supported by immunoblotting and electron microscopy. In terms of function, the U-particle/apoferritin effectively inhibits the translation of mRNAs in reticulocyte lysates. These experiments indicate that apoferritin may perform important functions in eukaryotic cells in addition to iron storage. Finally, we propose mechanisms to explain how Mengo virus infection could specifically induce the synthesis of apoferritin and how increasing amounts of cytoplasmic apoferritin could facilitate virus replication.

Subunit structure of ATP synthase from chloroflexus aurantiacus

An ATP synthase has been isolated from green nonsulfur photosynthetic bacterium Chloroflexus aurantiacus, a representative of a lower branch of eubacteria. The enzyme, reconstituted with the bacterial lipids into proteoliposomes, is shown to catalyze [ 32P]P i-ATP exchange (at a rate of 180 nmol [ 32P]ATP/ min mg ). The ATP synthase is composed of nine polypeptide species (60, 50, 33, 19, 16.5, 15.5, 14.5, 13, and 8 kDa as determined by urea-SDS-PAGE). The catalytic part of the ATP synthase (which is detached by chloroform treatment) contains the first four polypeptides. In the intact ATP synthase the 14.5 and 13 kDa polypeptides are connected by disulfide bonds to form a heterodimer of 25 kDa.

Purification of an endoproteinase that digests the wheat ‘Em’ protein in vitro, and determination of its cleavage sites

Germinating wheat embryos contain two endoproteolytic activities which digest the prominent ‘Em’ polypeptide. These are easily assayed in clarified embryonic homogenates and are distinguishable by the pattern of their peptide products and by their different pH optima. One activity has a pH optimum of 4.0; the second activity is a cysteine endoproteinase with a preference for the ‘Em’ protein as its substrate. It is maximally active between pH 5.5 and 6 at 25°C. Analysis of the early cleavage products of the cysteine proteinase indicates scissile bonds between residues Glu 32-Ala 33 and Asn 36-Leu 37 in the ‘Em’ polypeptide. This endoproteinase has been purified and identified as a single polypeptide species of ca. 38,000 kDa.

Further characterization of protective Trypanosoma cruzi-specific CD4+ T-cell clones: T helper type 1-like phenotype and reactivity with shed trypomastigote antigens

We previously reported the isolation from immune mice of a panel of murine clonal T-cell lines which specifically recognize antigens expressed by the trypomastigote stage of the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease. Our analysis indicated that distinct clones which recognize common as well as strain-specific antigenic determinants were represented. The immunoprotective potential of several of these T-cell clones was demonstrated by adoptive transfer of protection to naive syngeneic recipients. Here we report that these T-cell clones are all of the TH1 phenotype, as determined from their lymphokine secretion patterns. Significant levels of stimulatory activity for each clone were detected in trypomastigote supernatants, and the release of this activity was time and temperature dependent. Seven of 10 T-cell clones tested responded to nitrocellulose-immunoblotted trypomastigote proteins in the range of 90 to 47 kDa; no fewer than six distinct epitopes residing on at least five distinct polypeptide species were recognized by this panel of clones. Two clones (2G8 and 4B10) previously shown to protect in vivo responded to immunoblotted proteins in the range of 65 to 53 and 90 to 80 kD, respectively. Stimulatory activity for the latter clone was shown to be expressed on the surface of trypomastigotes and to bind specifically to wheat germ agglutinin, indicating that its target antigen is an 85-kDa trypomastigote surface glycoprotein.

A role for phosphorylation in the down-regulation of phenylalanine ammonia-lyase in suspension-cultured cells of french bean

A role for the phosphorylation of phenylalanine ammonia-lyase (PAL) which catalyses a key metabolic step in the biosynthesis of plant phenolics has been identified. Using immunoprecipitation of proteins radiolabelled with [ 32P]orthophosphate in cells in vivo, the M r 70 000 subunit degradation product was found to be phosphorylated. This phosphorylation was amplified by treatment of the cells with high concentrations of forskolin, but not elicitor alone, relative to the level of [ 35S]methionine labelled M r 77 000 and 70 000 subunit forms newly synthesised and turned-over as a consequence of elicitor action. Forskolin treatments bring about a correspondingly decreased specific activity of the extracted enzyme. Although a high level of phosphorylation was observed for a large number of species of polypeptides in cell-free extracts in vitro, including one of M r 69 000, this was not immunoprecipitated. The results are consistent with phosphorylation of the M r 70 000 PAL degradation product being specific and completed in vivo. Since forskolin brings about the down-regulation of elicitor-induced PAL, it is probable that the transient appearance of the enzyme activity and its turnover is in part regulated by phosphorylation, which is possibly cAMP dependent, in addition to other inactivating processes.

Molecular genetic analysis of a class B periplasmic-flagellum gene of Treponema phagedenis.

Treponema phagedenis is a host-associated spirochete with multiple polypeptides making up its periplasmic flagella (PFs). Each PF has a 39-kDa polypeptide making up the sheath (class A PF polypeptide) and two to four antigenically similar 33- to 34-kDa polypeptide species making up the core (class B PF polypeptides). A genetic analysis of the PF-deficient mutants T-40 and T-55 has shown that the PFs are involved in motility. To better understand the synthesis and assembly of these complex organelles and to compare the PF genes with those of other spirochetes, we cloned and characterized the T. phagedenis flaB2 gene, which encodes one class B polypeptide. The flaB2 gene consists of an open reading frame of 858 nucleotides capable of encoding a protein of 31.5 kDa. The predicted amino acid sequence of the FlaB2 polypeptide was 92% identical to that of T. pallidum FlaB2, with a 76% identity at the nucleotide level. These results confirm previous immunological and N-terminal-sequence analyses which suggested that the PF genes are well conserved in the spirochetes. Primer extension analysis of T. phagedenis flaB2 indicated that the start site of transcription was 127 nucleotides upstream from the ATG initiation codon. Preceding the start site is a DNA sequence similar to the sigma 28 consensus promoter sequence commonly found associated with motility genes. Northern (RNA) blots probed with a segment of flaB2 DNA revealed a 1,000-nucleotide monocistronic transcript in the wild type and in PF-deficient mutants T-40 and T-55. DNA sequencing of most of the flaB2 gene of the mutants revealed no differences from the wild-type gene. Because the mutants fail to synthesize detectable class B PF polypeptides yet synthesize extensive amounts of flaB2 mRNA, PF synthesis in T. phagedenis is likely to involve regulation at the translational level.

An active covalently linked dimer of human interferon-gamma. Subunit orientation in the native protein.

We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.

Purification of an active species of recombinant K-bungarotoxin

The expression of κ-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide. These include not only biologically active κ-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species. Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies. This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation. The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure-function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor. In addition, the κ-bungarotoxin produced in this manner has the distinct advantage over venompurified κ-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations.

Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: Immunochemical and functional characterization

Glucuronidation of three substrates prototypical for different UDP-glucuronosyltransferase (UDPGT) isoforms in hepatic, renal, intestinal, and branchial microsomes of corn oil, 3-methylcholanthrene, Aroclor 1254, and clofibrate-pretreated plaice was investigated. The differential expression of UDPGT in the four tissues clearly demonstrated for the first time that multiple isoforms with differing substrate specificities were present in fish. The liver was quantitatively the most important site for the glucuronidation of all three compounds studied. Phenol UDPGT activity was ubiquitous to all tissues and was induced by 3-methylcholanthrene and Aroclor 1254 in hepatic tissue and by Aroclor 1254 in renal tissue. The glucuronidation of testosterone was restricted to liver and intestinal tissue, while conjugation of bilirubin was expressed solely in hepatic tissue. The biotransformation of the endogenous compounds was not induced in the xenobiotic-treated animals. The presence of immunoreactive UDPGTs in the four tissues was demonstrated by immunoblot analysis using sheep anti-plaice UDPGT antibodies. Hepatic tissue displayed a range of immunoreactive polypeptides of 52 to 57 kDa, while a 55-kDa polypeptide was detected in extrahepatic tissues. An increased intensity of the latter polypeptide species was demonstrated in liver and kidney microsomes in which there was a concomitant induction of phenol UDPGT activity in xenobiotic-treated fish. The results indicate that the 55-kDa polypeptide was the major polyaromatic hydrocarbon-inducible UDPGT isoenzyme in hepatic and renal microsomes.

Identification of a 9 kDa γ- crystallin fragment in human lenses

The degraded polypeptides ( M r < 14 kDa) were isolated by a preparative SDS-PAGE method from water soluble (WS) and water insoluble (WI) proteins of human lenses from donors of ages between 5 and 75 years. SDS-PAGE analysis showed the presence of a major 9 kDa polypeptide species that showed an agerelated increase in levels in WS-polypeptide preparations. In order to identify the parent crystallin of the 9 kDa polypeptide, the immunoreactivities of the WS- and WI-degraded polypeptides to immuno-affinitypurified anti-human α-, β- and γ-crystallin antibodies were determined by the Western blot method. The WS- and WI-9 kDa polypeptides showed immunoreactivity to only the anti-γ-crystallin antibody suggesting it to be a fragment of γ-crystallin. A 9 kDa species was purified by Sephadex G-50 chromatography from the WS-protein fraction of lenses from 20–30-year-old donors. The purified polypeptide showed a single protein band during SDS-PAGE and also an apparent single spot on two-dimensional gel-electrophoresis (IEF followed by SDS-PAGE). The purified preparation also showed a single major peak during reverse phase HPLC chromatography. The purified 9 kDa polypeptide showed immunoreactivity to only the anti-γ-crystallin antibody. A polyclonal antibody raised against the purified 9 kDa polypeptide showed immunoreactivity only to a 20 kDa γ-crystallin species. The partial N-terminal sequence analysis of the 9 kDa polypeptide showed it to be a fragment of γD-crystallin. Together these results show that a 9 kDa γD-crystallin fragment exists in increasing quantities in human lenses during aging.

Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus.

Simian varicella virus (SVV) causes a varicella-like disease in non-human primates. In this study, SVV virions were purified from SVV-infected BSC-1 cells by zonal and differential gradient centrifugation and the virion polypeptide composition was analysed by SDS-PAGE. SVV virions had a buoyant density of 1.21 g/ml, identical to the value obtained for varicella-zoster virus (VZV) virions purified by the same method. Electron microscopy of the concentrated SVV virions revealed characteristic herpesvirus morphology. SVV virions consisted of at least 30 polypeptide species ranging from 16K to greater than 200K. The electrophoretic profiles of radiolabelled SVV and VZV virion polypeptides were very similar. Immunoprecipitations of solubilized SVV-infected cell preparations using SVV immune sera revealed at least 18 viral polypeptides with an Mr range of 12K to 142K and six glycoproteins ranging from 46K to 115K. In addition, extensive cross-reactivity between SVV and VZV proteins and glycoproteins was demonstrated by immunoprecipitation with heterologous immune sera. The high degree of antigenic relatedness between SVV and VZV provides further support for simian varicella as a model for VZV infections.

Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.

The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H+ pumps: a vacuolar ("V-type") H(+)-ATPase (EC 3.6.1.3) and a H(+)-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1). The H(+)-PPase, like the V-type H(+)-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast H(+)-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H(+)-PPase of Arabidopsis thaliana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H(+)-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the Mr 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H(+)-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H(+)-PPase and any other characterized H+ pump or PPi-dependent enzyme implies a different evolutionary origin for this translocase.

The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle.

Herpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein. In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle. Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species. Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher Mr species, and a minor population encoding the lowest Mr species alone. DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only. The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro. Following subcellular fractionation of infected cells, they are found predominantly associated with membranes. Within the virus particle, they appear to reside within the tegument. An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype. Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.

Molecular cloning of the alpha-globin transcription factor CP2.

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Molecular Cloning of the a-Globin Transcription Factor CP2

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Expression of a synthetic gene coding for the amino acid sequence of Clostridium pasteurianum rubredoxin.

A synthetic gene based on the published amino acid sequence for Clostridium pasteurianum rubredoxin was constructed, cloned in Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promoter system in E. coli HMS273. UV/visible spectroscopy and metal analyses indicated that the as-isolated synthetic gene product is a mixture of holo-(i.e. iron-containing) rubredoxin and zinc-substituted rubredoxin, with the latter amounting to approximately 70% of the total rubredoxin. The UV/visible absorption and resonance Raman spectra of the cloned holorubredoxin are characteristic of the native rubredoxin-type iron site. N-terminal amino acid sequencing suggests that the gene product consists of at least three polypeptide species with the initial sequences (approximate relative abundances): Met-Met-Lys-... (63%), blocked (30%) and Met-Lys-... (7%). The blocked portion presumably consists of a mixture of nMet-Met-Lys-... and nMet-Lys-..., where nMet represents an amino-blocked methionine residue.

Spirochete chemotaxis, motility, and the structure of the spirochetal periplasmic flagella

Spirochetes have a unique motility system that is characterized by flagellar filaments contained within the outer membrane sheath. Direct evidence using video microscopy has recently been obtained which indicates that these periplasmic flagella (PF) rotate in several spirochetal species. This rotation generates thrust. As shown for one spirochete, Spirochaeta aurantia, motility is driven by a proton motive force. Spirochete chemotaxis has been most thoroughly studied in S. aurantia. this spirochete exhibits three distinct behaviours, runs of smooth swimming, reversais and flexing. These behaviours are modulated by addition of attractants such that S. aurantia swims towards higher concentrations of attractants in a spatial gradient. Unlike the prototypical bacterium, Escherichia coli, chemotaxis in S. aurantia involves fluctuations in membrane potential. The PF of a number of spirochetes have been examined in considerable detail. For most species, the PF filaments are complex, consisting of an assembly of several different polypeptides. There are several antigenically related core polypeptides surrounded by an outer layer consisting of a different polypeptide. Borrelia burgdorferi and Spirochaeta zuelzerae represent exceptions where the filaments are composed of a single major polypeptide species. The genes encoding the filament polypeptides from several spirochete species have been cloned and analysed. Apparently, the outer layer polypeptides of S. aurantia, Treponema pallidum and Serpulina hyodysenteriae are transcribed from sigma-70-like promoters, whereas the core polypeptide genes are transcribed from sigma-28-like promoters. A gene encoding the hook polypeptide in Treponema phagedenis has been cloned and analysed. The product of this gene shows significant similarity to the E. coli hook protein, FlgE, and homologs have been identified in T. pallidum and B. burgdorferi. Besides their direct involvement in motility, the PF of T. phagedenis show a complex interaction with the cell cylinder that results in bending of the ends of the cells. Furthermore, as best documented in T. phagedenis, flagellar filaments can protrude from some cells in culture and generate motion. These protruding filaments are PF surrounded by a membrane. In conclusion, significant progress has been made in the last decade towards understanding the basis of motility at a molecular, biochemical and cellular level. This progress enables us to present an overall scheme for spirochete PF filament structure, assembly, and motion.

Human eosinophil cytotoxicity-enhancing factor. II. Multiple forms synthesized by U937 cells and their relationship to thioredoxin/adult T cell leukemia-derived factor.

Recently, our laboratory reported the purification and partial amino acid sequence of a 10-kDa eosinophil cytotoxicity-enhancing factor (ECEF) polypeptide from the U937 cell source. This cytokine enhanced human eosinophil antibody-dependent cytotoxic function by greater than 200% and was half-maximally effective at a concentration of approximately 1 ng/ml. In this study, we describe the conditions required for ECEF synthesis and the use of rabbit antibody raised to 10-kDa ECEF to investigate the existence of related polypeptide species. Unstimulated U937 cells released an immunoreactive 14-kDA species. Cells stimulated with 7.5 micrograms/ml of LPS also released a 13-kDa species. Cells stimulated with 400 ng/ml of PMA also synthesized a 10-kDa species (equivalent in size to the form we had purified). This 10-kDa species remained primarily cell associated, but detectable amounts were released into the supernatant by 48 h of culture. In washed cell pellets, the location of the 10-kDa species was found to be in the plasma membrane, externally oriented, as determined by FACS analysis, iodination with the membrane impermeable reagent 125I-sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, and by its removal with brief trypsin treatment. Partial amino acid sequence data suggested that the 14-, 13-, and 10-kDa species all share the same N-terminal. The 14- and 10-kDa ECEF species were recovered by electroelution from SDS-PAGE gels and tested for activity in the assay of eosinophil cytotoxic function. Because of the amino acid sequence similarities between the ECEF species and thioredoxin (TRX), rTRX (synthesized in Escherichia coli and purified) was also tested for activity. The 14-kDa ECEF and rTRX induced a slight, but consistent and statistically significant enhancement of eosinophil cytotoxic function. By comparison, lower doses of the 10-kDa ECEF induced a major increase in cytotoxic function. Thus the forms of ECEF differ in size, conditions required for synthesis, trafficking by the U937 cell after synthesis, and biologic activity. It is likely that these considerations bear on the involvement of ECEF in the pathophysiology of eosinophilia in vivo.