Biotechnol Prog, 1995 Sep-Oct, 11(5), 533 - 9 Improved homogenization of recombinant Escherichia coli following pretreatment with guanidine hydrochloride; Bailey SM et al.; Pretreatment of recombinant Escherichia coli, expressing human growth hormone inclusion bodies, with guanidine hydrochloride and Triton X-100 prior to high-pressure homogenization has been investigated . Homogenates were analyzed for protein release, viscosity, and particle size . We were able to reduce the number of passes required for cell disruption and the number of downstream processing steps required for the recovery of protein from inclusion bodies by pretreating cells with guanidine HCl and Triton X-100 . Pretreatment of exponential growth phase cells with 1.5 M guanidine HCl and 1.5% Triton X-100 gave adequate disruption after one pass at 41 MPa with a particle size distribution similar to that for untreated cells disrupted after one pass at 62 MPa . This combination of guanidine HCl and Triton X-100 was also selected so as to wash the inclusion bodies without solubilization of the human growth hormone . Pretreatment of cells with 4 M guanidine HCl produced cell debris that was substantially smaller than the debris from untreated cells and partially solubilized the inclusion bodies . Cells harvested in the stationary growth phase were more resistant to high-pressure homogenization and pretreatment. Vet Microbiol, 1995 Sep, 46(1-3), 21 - 8 Recent isolates of Newcastle disease virus in Australia; Spradbrow PB et al.; Forty-five recently isolated strains of Newcastle disease virus and the V4 vaccine strain of Newcastle disease virus were used to infect experimental chickens . Neither V4 nor any of the new strains produced detectable clinical disease . All the viruses produced an antibody response and spread by contact . Some of the newly isolated viruses produced a more rapid serological response than V4 virus did . Dual or multiple infections with one of the new strains of Newcastle disease virus, infectious bronchitis virus and Escherichia coli did not enhance the pathogenicity of any of the agents. Vet Microbiol, 1995 Sep, 46(1-3), 181 - 91 Expression of small regions of equine herpesvirus 1 glycoprotein C in Escherichia coli; Crabb BS et al.; A series of truncated equine herpesvirus 1 (EHV1) glycoprotein C (gC) molecules was examined for use as serodiagnostic antigens for EHV1 and EHV4 . Small regions of EHV1 glycoprotein C, an immunodominant EHV1 glycoprotein, were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using the bacterial expression vector pGEX-2T . Sera obtained from horses, including sera from specific-pathogen-free (SPF) foals, following exposure to either EHV1, EHV4 or both viruses were used . Several of the fusion proteins were shown to encompass EHV1 specific epitopes while others encompassed strong, cross-reactive epitopes . One clone, termed pEC-3, produced a soluble and stable fusion protein which encompassed amino acids 107-275 of EHV1 gC . Strong cross-reactive epitopes on pEC-3 were localised to a region encompassed by amino acids 137 to approximately 152 while EHV1 specific epitope(s) were identified downstream of this region, i.e., approximately amino acids 152 to 275 . E . coli expressed EHV1 gC polypeptides showed clear potential for use as diagnostic reagents for the detection of cross-reactive and type-specific EHV1 and EHV4 antibodies present in convalescent equine sera. J Med Genet, 1995 Sep, 32(9), 680 - 8 The molecular basis of ornithine transcarbamylase deficiency: modelling the human enzyme and the effects of mutations; Tuchman M et al.; Human ornithine transcarbamylase is a trimer with 46% amino acid sequence homology to the catalytic subunit of E coli aspartate transcarbamylase . Secondary structure predictions, distributions of hydrophilic and hydrophobic regions, and the pattern of conserved residues suggest that the three dimensional structures of the two proteins are likely to be similar . A three dimensional model of ornithine transcarbamylase was generated from the crystal structure of the catalytic subunit of E coli aspartate transcarbamylase in the holoenzyme, by aligning the sequences, building in gaps, and minimising the energy . The binding sites for carbamyl phosphate in both enzymes are similar and the ornithine binding site in ornithine transcarbamylase appears to be in the same location as the L-aspartate binding site in aspartate transcarbamylase, with negatively charged side chains replaced by positively charged residues . Mutations in the ornithine transcarbamylase gene found in patients with hyperammonaemia of the "neonatal type" are clustered in important structural or functional domains, either in the interior of the protein, at the active site, or at the interchain interface, while mutations found in patients with milder "late onset" disease are located primarily on the surface of the protein . The predicted effects of all known missense mutations and in frame deletions in the ornithine transcarbamylase gene on the structure and function of the mature enzyme are described. Hokkaido Igaku Zasshi, 1995 Sep, 70(5), 743 - 53 {Analysis of the human acidic epididymal glycoprotein-like molecule: isolation of cDNA and tissue localization}; Hayashi M; Acidic epididymal glycoprotein (AEG) is an androgen-dependent, epididymal secretory protein assumed to play a major role in sperm maturation . In the present study, we isolated cDNA clones encoding the human AEG-like molecule and determined their nucleotide sequences . The deduced human AEG-like molecule was made up of 230 amino acids, excluding a signal peptide, and contained one potential N-linked glycosylation site . All cysteinyl residues were conserved between the human AEG-like molecule and the AEG molecules of rats and mice . The human AEG-like molecule was equally similar to the AEG molecules of rats and mice and a related testis-specific protein known as TPX1 of human and mice (approximately 40% amino acid sequence similarity) . Northern blot analysis showed that the human AEG-like gene is expressed specifically in the epididymis . To identify the product of the human AEG-like gene, polyclonal antibody was produced by immunizing rabbits with a recombinant human AEG-like protein expressed in E . coli . This antibody detected a major band of 30 kD and a minor band of 26 kD in the caput, corpus, and cauda regions of the epididymis, the ductus deferens, the sperm, and the seminal plasma . Immunohistochemical analysis showed that the human AEG-like molecule is located in the lumen and epithelium of distal ductus efferentes and epididymal ducts, and on the postacrosomal region of the sperm head. Curr Biol, 1995 Sep 1, 5(9), 1036 - 46 Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site; Blake DG et al.; BACKGROUND: The inverted repeat is a common feature of protein-binding sites in DNA . The two-fold symmetry of the inverted repeat corresponds to the two-fold symmetry of the protein that binds to it . In most natural inverted-repeat binding sites, however, the DNA sequence does not have perfect two-fold symmetry . Our study of how a site-specific recombinase recognizes an inverted-repeat binding site indicates that such sequence asymmetry can be functionally important . RESULTS: Tn3 resolvase forms two complexes with the 34 base-pair binding site II of its recombination region, res . A resolvase monomer first binds at the left end of the site; a second monomer then binds cooperatively at the right end . In both complexes, the DNA is bent by resolvase . In contrast, the closely related gamma delta resolvase binds to site II mainly as a dimer . Insertion of 5 or 10 base pairs at the centre of the site does not prevent cooperative binding of two Tn3 resolvase subunits . The fully occupied site II has a very asymmetric structure . Reversal of the orientation of site II in res blocks recombination; thus, its asymmetric properties are functionally important . We propose a structure for the two-subunit complex formed with site II, based on our results and by analogy with the co-crystal structure of gamma delta resolvase bound to res site I . CONCLUSIONS: Deviations from perfect inverted-repeat symmetry in a resolvase-binding site lead to ordered binding of subunits, structural asymmetry of resolvase-DNA complexes, and asymmetric function. Hum Mol Genet, 1995 Sep, 4(9), 1493 - 8 Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli; Goodman SI et al.; We have cloned, sequenced, and expressed cDNAs encoding wild type human glutaryl-CoA dehydrogenase subunit, and have expressed a mutant enzyme found in a patient with glutaric acidemia type I . The mutant protein is expressed at the same level as the wild type in Escherichia coli, but has less than 1% of the activity of wild-type dehydrogenase . We also present evidence that the glutaryl-CoA dehydrogenase transcript is alternatively spliced in human fibroblasts and liver; the alternatively spliced mRNA, when expressed in E.coli, encodes a stable but inactive protein . Purified expressed human glutaryl-CoA dehydrogenase has kinetic constants similar to those of the previously purified porcine dehydrogenase . The primary translation product from in vitro transcribed glutaryl-CoA dehydrogenase mRNA is translocated into mitochondria and processed in the same manner as most other nuclear-encoded mitochondrial proteins . Human glutaryl-CoA dehydrogenase shows 53% sequence similarity to porcine medium chain acyl-CoA dehydrogenase, and these similarities were utilized to predict structure-function relationships in glutaryl-CoA dehydrogenase. Hum Mol Genet, 1995 Sep, 4(9), 1475 - 83 Cloning and characterization of alternatively spliced isoforms of Dp71; Austin RC et al.; Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues . In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes . The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78 . RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver . To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins . These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B) . The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values . Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues . This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78 . Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Surg, 1995 Sep, 161(9), 635 - 8 Effects of naloxone on glucagon and insulin concentrations after injection of endotoxin in rats; Johansen O et al.; OBJECTIVE: To find out how infusions of endotoxin and endotoxin plus naloxone affected glucose, glucagon, and insulin concentrations in rats . DESIGN: Random control study . SETTING: University hospital, Norway . MATERIAL: 27 Male Wistar rats . INTERVENTIONS: 8 Rats were given Escherichia coli endotoxin 0.25 mg in saline; 5 were given a naloxone infusion (0.4 ml of 0.04 mg/ml for the first half hour and continued at a rate of 0.4 ml/hour for a total of 80 minutes) starting 10 minutes before the same dose of endotoxin; 6 were given saline alone for 70 minutes, and 8 saline alone for 10 minutes . Blood was taken for analysis after 70 minutes in the first three groups and at the end of the infusion in the 10 minute group . MAIN OUTCOME MEASURES: Serum concentrations of glucose, glucagon, and insulin compared with baseline (10 min group) . RESULTS: Median (interquartile) concentrations of all three substances rose significantly 70 minutes after the injection of endotoxin compared to basal values: 9.8 (8.2-10.9) compared with 6.0 (4.8-7.5) mmol/l for glucose (p < 0.01); 53.1 (44.6-56.0) compared with 3.6 (3.1-5.1) ng/l for glucagon; and 39.4 (38-50.4) compared with 16.7 (11.2-25.5) pmol/l for insulin . When naloxone was combined with endotoxin glucagon and insulin concentrations were significantly lower: 24.3 (23.9-37.5) ng/l (p < 0.01), and 30.6 (24.5-31.6 pmol/l (p < 0.05), respectively . The concentration of glucose in venous blood was unchanged . CONCLUSIONS: The rise in glucagon and insulin concentrations after endotoxin infusions may be partly mediated by opioids . Naloxone in the dose given did not abolish the increase in glucagon after endotoxin . The high glucagon:insulin ratio after both endotoxin alone and endotoxin plus naloxone may be important in the aetiology of the hyperglycaemia seen. Antimicrob Agents Chemother, 1995 Sep, 39(9), 2000 - 7 Anti-human immunodeficiency virus (HIV) activities of halogenated gomisin J derivatives, new nonnucleoside inhibitors of HIV type 1 reverse transcriptase; Fujihashi T et al.; Halogenated gomisin J (a derivative of lignan compound), represented by the bromine derivative 1506 {(6R, 7S, S-biar)-4,9-dibromo-3,10-dihydroxy-1,2,11,12-tetramethoxy-6, 7-dimethyl-5,6,7,8- tetrahydrodibenzo{a,c}cyclo-octene}, was found to be a potent inhibitor of the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) on MT-4 human T cells (50% effective dose, 0.1 to 0.5 microM) . Gomisin J derivatives were active in preventing p24 production from acutely HIV-1-infected H9 cells . The selective indices (toxic dose/effective dose) of these compounds were as high as > 300 in some systems . 1506 was active against 3'-azido-3'-deoxythymidine-resistant HIV-1 and acted synergistically with AZT and 2',3'-ddC . 1506 inhibited HIV-1 reverse transcriptase (RT) in vitro but not HIV-1 protease . From the time-of-addition experiment, 1506 was found to inhibit the early phase of the HIV life cycle . A 1506-resistant HIV mutant was selected and shown to possess a mutation within the RT-coding region (at position 188 {Tyr to Leu}) . The mutant RT expressed in Escherichia coli was resistant to 1506 in the in vitro RT assay . Some of the HIV strains resistant to other nonnucleoside HIV-1 RT inhibitors were also resistant to 1506 . Comparison of various gomisin J derivatives with gomisin J showed that iodine, bromine, and chlorine in the fourth and ninth positions increased RT inhibitory activity as well as cytoprotective activity. Proteins, 1995 Sep, 23(1), 126 - 7 Crystallization and preliminary crystallographic analysis of recombinant abrin-a A-chain with ribosome inactivating activity; Kohno T et al.; Crystals have been obtained for a recombinant abrin-a A-chain produced by E . coli . The crystals were grown using PEG6000 as the precipitating agent . The crystals belong to an orthrhombic space group (P2(1)2(1)2(1)) and diffract to 1.7 A. Proteins, 1995 Sep, 23(1), 122 - 5 Crystallization of a soluble, catalytically active form of Escherichia coli leader peptidase; Paetzel M et al.; Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins . It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space . Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (delta 2-75) . Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant . Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis . The crystals belong to the tetragonal space group P4(2)2(1)2, with unit cell dimensions of a = b = 115 A and c = 100 A, and contain 2 molecules per asymmetric unit . This is the first report of the crystallization of a leader (or signal) peptidase. J Cell Sci, 1995 Sep, 108 ( Pt 9), 3013 - 28 Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin; Levilliers N et al.; Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity . The location, the nature and the subcellular distribution of the epitopes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin fusion peptides generated in E . coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references . AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxyl-terminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in alpha-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various alpha- and beta-tubulin isoforms displaying different apparent molecular masses . These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa . This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal alpha- and beta-tubulin. Genetics, 1995 Sep, 141(1), 15 - 24 Correlation of Rhs elements with Escherichia coli population structure; Hill CW et al.; The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection . The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection . This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element . A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements . The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation . Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups . In the course of this analysis, two new Rhs elements absent from E . coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily. Baillieres Clin Haematol, 1995 Sep, 8(3), 499 - 514 Transcobalamin II and the membrane receptor for the transcobalamin II-cobalamin complex; Rothenberg SP et al.; Transcobalamin II is a plasma protein that binds vitamin B12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues . The circulating transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis . A number of genetic abnormalities are characterized either by a failure to express transcobalamin II or by synthesis of an abnormal protein . These disorders result in cellular cobalamin deficiency and megaloblastic anaemia . In this chapter we review the structural and functional properties of transcobalamin II, the receptor for the transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating transcobalamin II . In addition, we provide emerging data about the molecular genetics of transcobalamin II which has emanated from our own and other laboratories. Vet Immunol Immunopathol, 1995 Sep, 48(1-2), 77 - 88 Effect of bovine leukemia virus infection on bovine peripheral blood monocyte responsiveness to lipopolysaccharide stimulation in vitro; Werling D et al.; The effects of bovine leukemia virus (BLV) infection on cytokine activity of bovine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined . Compared to supernatants of LPS-stimulated monocytes from BLV-negative cows, supernatants from BLV-positive cows contained about four times more interleukin-1 beta (IL-1 beta) (as measured by an enzyme-linked immunosorbent assay (ELISA) for bovine IL-1 beta) . Despite their higher IL-1 beta concentration, supernatants from BLV-positive cows stimulated proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide) assay similar to supernatants from BLV-negative cows, but showed about 30% less IL-1 activity than supernatants from BLV-negative cows on the IL-1-dependent cell line LBRM-33 1 A-5, and about five times more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929 . These results demonstrate that BLV infection changes the cytokine response of bovine monocytes to LPS stimulation in vitro . The results are consistent with the assumption that BLV infection leads to the production and secretion of a soluble IL-1 inhibitor by LPS-stimulated peripheral blood monocytes. Br J Pharmacol, 1995 Sep, 116(2), 1757 - 60 The in vivo effect of lipopolysaccharide on the spontaneous release of transmitter from motor nerve terminals; Liu SH et al.; 1 . The in vivo effect of E . coli lipopolysaccharide (LPS) on the spontaneous release of transmitter was studied in the isolated phrenic nerve-diaphragm preparation of the mouse . 2 . The resting membrane potential was decreased and frequency of miniature endplate potentials (m.e.p.ps) was increased by treatment with LPS . 3 . Pretreatment of diaphragms with ouabain markedly increased the frequency of m.e.p.ps in control group but not in the LPS group . 4 . When mice were treated with polymyxin B (a LPS neutralizer), pentoxifylline (an inhibitor of tumor necrosis factor-alpha formation) and NG-nitro-L-arginine (an inhibitor of nitric oxide (NO) synthase) the effects of LPS were reversed . 5 . These results suggest that LPS increases the spontaneous transmitter release through, at least in part, the pathways of tumour necrosis factor-alpha and NO followed by an inhibition of the Na(+)-pump activity in the endplate area. Protein Sci, 1995 Sep, 4(9), 1914 - 9 Influence of divalent cations in protein crystallization; Trakhanov S et al.; We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells . Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality . This comes about 15 years after the ligand-free proteins were crystallized . Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology . Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals . Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms . The concentration of Cd2+ is critical . The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM . Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source. Protein Sci, 1995 Sep, 4(9), 1758 - 67 Structure and function of microplasminogen: I . Methionine shuffling, chemical proteolysis, and proenzyme activation; Wang J et al.; We have cloned and expressed microplasminogen (mPlg), consisting of the N-terminal undecapeptide of human glu-Plg spliced to its proenzyme domain . This truncated (approximately 28 kDa) proenzyme retained the distinctive catalytic activities of the larger parent . Replacement of M residues followed by M shuffling permitted subsequent scission by site-directed chemical proteolysis (in CNBr/formic acid) without impairing any of the protein's characteristic properties . Activation of chymotrypsinogen-related zymogens occurs by limited proteolysis; the newly liberated, highly conserved N-terminus (VVGG) forms a salt bridge with an aspartyl residue immediately upstream of the active site serine . The role of both of these elements in mPlg activation was probed using protein engineering and site-directed proteolysis to alter the length and amino acid composition of the N-terminus, and to replace the aspartate . All modifications affected both Km and Kcat . The results identify some structural parameters of the N-terminus required for proenzyme activation. Protein Sci, 1995 Sep, 4(9), 1750 - 7 Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis; Onuffer JJ et al.; Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E . coli tyrosine aminotransferase (eTATase) have been unsuccessful . Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions . The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures . Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase . Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297) . The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69) . These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase . Five combinations of the individual mutations were obtained from mutagenesis reactions . The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase . Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Sci, 1995 Sep, 4(9), 1743 - 9 The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases; Onuffer JJ et al.; The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants . However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase . A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes . A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes . This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner . The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes . A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase . The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid. Protein Sci, 1995 Sep, 4(9), 1718 - 29 The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism; Sukumar M et al.; The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans . It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity . Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods . Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures . The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44 . The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb . The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix . The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity . CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function . Reduced STb adopts a predominantly random-coil conformation . Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative . Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds. Hum Gene Ther, 1995 Sep, 6(9), 1185 - 93 Regenerating cells in human airway surface epithelium represent preferential targets for recombinant adenovirus; Dupuit F et al.; To investigate the efficiency of adenovirus-mediated gene delivery in regenerating human respiratory epithelium, we have performed infections with an E1- and E3-deleted type 5 recombinant adenovirus containing the Escherichia coli LacZ reporter gene on different culture models of regenerating human nasal polyp surface epithelium . These models included: (i) an ex vivo organ culture of nasal polyp tissue, (ii) an explant outgrowth cell culture, and (iii) an in vitro wound repair model, on dissociated cells . In ex vivo nasal polyp tissue, transduced cells were not detected in normal pseudostratified areas, but were found in areas of the surface epithelium with a morphology reminiscent of regenerating airway tissue . In the explant outgrowth cell culture, adenovirus-infected cells were preferentially detected at the periphery of the outgrowth . These transducible epithelial cells, representative of epithelial cells present in vivo during the process of surface airway epithelium regeneration, were shown to be migrating and poorly differentiated cells, which were proliferating or not . In the in vitro wound repair model, the efficiency of cell transduction was much higher in cells present in the wound area than in those far from the wound area . These results indicate that regenerating cells from human airway surface epithelium represent preferential targets for transgene expression, and suggest that efficiency of CFTR gene transfer by recombinant adenovirus vectors may be higher in regenerating CF airway mucosa than in normal tissue . However, since these cells do not show endogenous CFTR expression, the relevance of their preferential transduction for the functional correction of the ion transport defect in cystic fibrosis needs further investigations. J Rheumatol, 1995 Sep, 22(9), 1776 - 8 Emphysematous septic arthritis: case report and review of the literature; Roy R et al.; We describe a patient with Escherichia coli infectious arthritis associated with intraarticular emphysema . The implications of this finding and previous experience with this entity are reviewed. Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1750 - 2 Characterization of rat N-acetylglucosaminyltransferase I expressed in Escherichia coli; Nishiu J et al.; The catalytic domain (sGnT-I) of rat liver N-acetylglucosaminyl-transferase I (GnT-I) was expressed in Escherichia coli . Lysates from pETsGnT-I transformants contained a prominent protein species of 46 kDa with which a significant GnT-I activity was associated . To purify the relevant enzyme, we constructed cDNAs encoding sGnT-ICH and sGnT-INH, which had six additional histidine residues as an affinity tag at the C-terminal and the N-terminal of sGnT-I, respectively, and introduced them into E . coli cells for expression . sGnT-INH was purified and its enzymatic properties were examined. Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1707 - 11 Isolation and characterization of the IS3-like element from Thermus aquaticus; Utsumi R et al.; We have cloned and characterized a genetic element (1187 bp) that is responsible for the induction of thermotolerance as well as ompC expression in E . coli . This element (ISLtaq1) was isolated from Thermus aquaticus . DNA and protein data bases were searched with this element (ISLtaq1), which suggested it to be very similar to IS150 belonging to the IS3 family . ORF1, found on ISLtaq1, which encodes 100 amino acids (aa), had a DNA-binding motif: a helix-turn-helix and a leucine zipper . In fact, when the ORF1 protein was overproduced in E . coli, thermotolerance as well as ompC expression was induced. Biophys J, 1995 Sep, 69(3), 754 - 66 Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli; Reusch RN et al.; The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi) . The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels . Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E . coli . Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine . The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order . The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi . This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E . coli . This is the first report of a biological non-proteinaceous calcium channel . We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels. Biophys J, 1995 Sep, 69(3), 1036 - 45 Analysis of the structure of dimeric DNA catenanes by electron microscopy; Levene SD et al.; We analyzed the structure of open-circular and supercoiled dimeric DNA catenanes generated by site-specific recombination in vitro . Electron microscopy of open-circular catenanes shows that the number of duplex crossings in a plane is a linear function of the number of catenane interlinks (Ca/2), and that the length of the catenane axis is constant, independent of Ca . These relationships are similar to those observed with supercoiled DNA . Statistical analyses reveal, however, that the conformations of the individual rings of the catenanes are similar to those of unlinked circles . The distribution of distances between randomly chosen points on separate rings depends strongly on Ca and is consistent with a sharp decrease in the center-of-mass separation between rings with increasing Ca . Singly linked supercoiled catenanes are seen by microscopy to be linked predominantly through terminal loops in the respective superhelices . The observations suggest that chain entropy is a major factor determining the conformation of DNA catenanes. Ann Thorac Surg, 1995 Sep, 60(3), 593 - 7; discussion 597-8 Pleural-based mesothelioma in immune competent rats: a model to study adenoviral gene transfer; Kucharczuk JC et al.; BACKGROUND . Despite multimodality approaches, pleural-based malignant mesothelioma remains a disease with a very poor prognosis . Novel therapeutic strategies such as gene therapy clearly are needed to improve the survival of patients with this neoplasm . To aid in the evaluation of new treatment strategies, animal models that closely mimic human disease are required . This article describes the establishment of a pleural-based model of malignant mesothelioma in immune-competent Fischer rats . METHODS . Via a modified left anterior lateral thorocotomy, a syngeneic malignant mesothelioma cell line, called II-45, was placed into the pleural cavity of Fischer rats . RESULTS . Placement of II-45 cells into the pleural cavity of Fischer rats results in a model of pleural mesothelioma that closely resembles the disease seen in patients and is highly reproducible, with animals dying within 1 month . We also demonstrate the feasibility of adenoviral-mediated gene transfer to normal mesothelial cells lining the pleural cavity, as well as to malignant cells deep within the substance of pleural-based malignant mesothelioma . CONCLUSIONS . The model described here offers the opportunity to study a variety of new treatment modalities, especially somatic gene transfer, against pleural-based malignant mesothelioma in an immune competent setting. Am J Pathol, 1995 Sep, 147(3), 707 - 17 Hepatocytic differentiation of cultured rat pancreatic ductal epithelial cells after in vivo implantation; Chen JR et al.; We have investigated the differentiation potential of propagable cultured rat pancreatic duct epithelial cells after in vivo implantation in isogeneic Fischer-344 rats . Cells genetically labeled with Escherichia coli beta-galactosidase (lacZ) reporter gene were embedded in a mixture of collagen and Matrigel (basement membrane matrix) and implanted either subcutaneously or intraperitoneally . Tissues from the two locations were harvested 4 to 8 weeks later . The great majority of the lacZ-labeled epithelial cells colonizing both sites phenotypically resembled hepatocytes, although they demonstrated different degrees of hepatocytic differentiation . Less than 5% of lacZ-labeled cells formed ductular structures . The hepatocyte-like cells from the subcutaneous implantation site expressed mixed phenotypes of both hepatocyte and ductal cell, including the expression of alpha-fetoprotein, tyrosine amino-transferase, gamma-glutamyl transpeptidase, carbonic anhydrase II, and cytokeratin 19 . In contrast, the hepatocyte-like cells colonizing the mesentery showed the phenotype of mature hepatocytes, including an abundant glycogen storage and a lack of alpha-fetoprotein and carbonic anhydrase II expressions . Neither acinar cell nor endocrine differentiation was seen . These findings demonstrate that pancreatic ductal cells can be the progenitor cell for transdifferentiated hepatocytes. J Magn Reson B, 1995 Sep, 108(3), 220 - 34 Investigation of protein amide-proton exchange by 1H longitudinal spin relaxation; Zheng Z et al.; The general theory of the Linderstrom-Lang model, which, in simplified form, is widely used for the interpretation of NMR data on backbone-proton exchange in proteins, is systematically discussed . An experimental protocol for testing the applicability of the customary simplifications is described and experimental data which require the application of the general, rather than of the simplified, theory are presented . The appearance of pH-dependent biexponential magnetization recovery for any amide group in a protein is an unequivocal indication that the conventional simplified versions of the model do not apply. Ann Neurol, 1995 Sep, 38(3), 429 - 36 An adenoviral vector can transfer lacZ expression into Schwann cells in culture and in sciatic nerve; Shy ME et al.; Although a number of genetic defects in the P0, peripheral myelin protein-22, and connexin-32 genes recently were shown to cause the demyelinating forms of Charcot-Marie-Tooth disease, there is yet no effective treatment for these patients . Recent studies showed that replication defective adenoviral vectors can efficiently introduce genes into muscle, brain, lung, and other tissues, suggesting that this vector system may be useful for the treatment of a number of genetic diseases . In this work, we demonstrated that a replication deficient adenovirus expressing the Escherichia coli beta-galactosidase gene (AdCMVLacZ) can introduce genes into Schwann cells, in culture as well as in sciatic nerve . Schwann cells cultured at a multiplicity of infection of 250:1 did not demonstrate cytopathic effects . Following injection of AdCMVLacZ into sciatic nerve of rats, lacZ-expressing, myelinating Schwann cells could be detected for at least 45 days . These data suggest that in the future, these vectors may be useful both in perturbing Schwann cell gene expression and in designing therapies for the treatment of Charcot-Marie-Tooth disease. J Leukoc Biol, 1995 Sep, 58(3), 359 - 64 Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen; Driscoll KE et al.; Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses . To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide . Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence . A cDNA containing the coding region of rat MIP-2 was cloned and sequenced . Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level . The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity . The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes . In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro. J Bacteriol, 1995 Sep, 177(18), 5381 - 2 Pyridine carboxylic acids as inhibitors and substrates of the Escherichia coli gab permease encoded by gabP; King SC et al.; Although considered selective for its natural substrate, 4-aminobutyrate, gab permease was inhibited by 1,2,3,6-tetrahydro-3-pyridinecarboxylate and 1,2,3,6-tetrahydro-4-pyridinecarboxylate . The former is a transported substrate, since its preloading into metabolically poisoned cells stimulated transient accumulation of 4-aminobutyrate via counterflow. J Bacteriol, 1995 Sep, 177(18), 5368 - 9 The distance between Escherichia coli genes is related to gene expression levels; Eyre-Walker A; It is shown that the distance between Escherichia coli genes oriented in the same direction on the chromosome is positively correlated to the expression level of the downstream gene . It is suggested that this could be a strategy to avoid interference between ribosomes translating two different genes. J Bacteriol, 1995 Sep, 177(18), 5364 - 7 The MalK protein of the ATP-binding cassette transporter for maltose of Escherichia coli is accessible to protease digestion from the periplasmic side of the membrane; Schneider E et al.; The ATP-hydrolyzing subunit MalK of the ATP-binding cassette transporter for maltose of Escherichia coli is demonstrated to be accessible to digestion by proteinase K in right-side-out membrane vesicles . This finding suggests a partial transmembrane orientation of the protein. J Bacteriol, 1995 Sep, 177(18), 5355 - 7 Genetic analysis suggests functional interactions between the N- and C-terminal domains of the TetA(C) efflux pump encoded by pBR322; McNicholas P et al.; Genetic analysis of the tetA(C) gene of pBR322 indicates the importance of two-cytoplasmic loops in the TetA(C) protein (P . McNicholas, I . Chopra, and D . M . Rothstein, J . Bacteriol . 174:7926-7933, 1992) . In this study, we characterized second-site suppressor mutations that suggest a functional interaction between these two cytoplasmic regions of the protein. J Bacteriol, 1995 Sep, 177(18), 5338 - 41 Promoter 7 of the Escherichia coli pfl operon is a major determinant in the anaerobic regulation of expression by ArcA; Drapal N et al.; The anaerobically inducible pfl operon of Escherichia coli has a regulatory sequence comprising 494 bp, which includes two anaerobically regulated promoters, termed P6 and P7 . In this study, we show that in its normal context the activity of P7 is constrained and that one important function of the promoter is to mediate controlled ArcA-dependent regulation of the operon. J Bacteriol, 1995 Sep, 177(17), 5189 - 92 Effects of temperature and of heat shock on the expression and action of the colicin A lysis protein; Cavard D; At low temperature, the synthesis of the colicin A lysis protein in Escherichia coli was slowed down, and consequently its functioning was retarded . The rates were restored when the bacteria were shifted for 10 min to 42 degrees C, except in an rpoH mutant, suggesting that one or more proteins regulated by sigma 32 is necessary for expression of colicin A lysis protein. J Bacteriol, 1995 Sep, 177(17), 5186 - 8 The C-terminal region of the alpha subunit of Escherichia coli RNA polymerase is required for transcriptional activation of the flagellar level II operons by the FlhD/FlhC complex; Liu X et al.; A number of transcription activators have been found to activate transcription via protein-protein contact between RNA polymerase alpha subunits and transcription factors; they are classified as class I factors . In this report, we demonstrate that the FlhD/FlhC complex, a transcription activator of the Escherichia coli flagellar regulon, requires the C-terminal domain of the RNA polymerase alpha subunit for transcription activation . We conclude that FlhD/FlhC is a class I transcription factor. J Bacteriol, 1995 Sep, 177(17), 5155 - 7 Identity of the Escherichia coli cls and nov genes; Tropp BE et al.; cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene . A comparison of the DNA sequences of both genes further verifies their identity. J Bacteriol, 1995 Sep, 177(17), 5035 - 9 MalY of Escherichia coli is an enzyme with the activity of a beta C-S lyase (cystathionase); Zdych E et al.; The Escherichia coli maltose system consists of a number of genes whose products are involved in the uptake and metabolism of maltose and maltodextrins . MalT is the central positive gene activator of the regulon and is, together with the cyclic AMP-catabolite gene activator protein system, necessary for the expression of the maltose genes . Expression of malY, a MalT-independent gene, leads to the repression of all MalT-dependent genes . We have purified MalY to homogeneity and found it to be a pyridoxal-5-phosphate-containing enzyme with the enzymatic activity of a beta C-S lyase (cystathionase) . MalY is a monomeric protein of 42,000 to 44,000 Da . Strains expressing MalY constitutively abolish the methionine requirement of metC mutants . The enzymatic activity of MetC, the cleavage of cystathionine to homocysteine, ammonia, and pyruvate, can be catalyzed by MalY . However, the cystathionase activity is not required for the function of MalY in repressing the maltose system . By site-directed mutagenesis, we changed the conserved lysine residue at the pyridoxal phosphate binding site (position 233) of MalY to isoleucine . This abolished beta C-S lyase activity but not the ability of the protein to repress the maltose system . Also, the overexpression of plasmid-encoded metC did not affect mal gene expression, nor did the deduced amino acid sequence of MetC show homology to that of MalY. J Bacteriol, 1995 Sep, 177(17), 5016 - 27 Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluster of genes on the IncHI2 plasmid R478; Whelan KF et al.; A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Ter), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced . Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F . Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Ter operon from plasmid pMER610 . There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides . There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments . The three phenotypes were expressed in Escherichia coli DH5 alpha by an 8.4-kb EcoRI insert subcloned from a cosmid of R478 . The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells . This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment . Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked . Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes . Insertions in terA reduced phage inhibition levels only . The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Ter. J Bacteriol, 1995 Sep, 177(17), 4980 - 4 Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression; Ghrist AC et al.; The Escherichia coli glycine cleavage enzyme system catalyzes the cleavage of glycine, generating CO2, NH3, and a one-carbon unit . Expression of the operon encoding this enzyme system (gcv) is induced in the presence of glycine and repressed in the presence of purines . In this study, a mutant with high-level constitutive expression of a gcvT-lacZ gene fusion was isolated . The mutation in this strain was designated gcvR1 and was mapped to min 53.3 on the E . coli chromosome . A single-copy plasmid carrying the wild-type gcvR gene complemented the mutation, restoring normal regulation of a gcvT-lacZ fusion, while a multicopy plasmid carrying gcvR led to superrepression under all growth conditions . Negative regulation of a gcvT-lacZ fusion by GcvR was shown to require GcvA, a LysR family protein known to both activate gcv in the presence of glycine and repress gcv in the presence of purines . Models explaining how GcvR and GcvA might interact to regulate gcv expression are proposed. J Bacteriol, 1995 Sep, 177(17), 4969 - 73 Multicopy suppression of cold-sensitive sec mutations in Escherichia coli; Danese PN et al.; Mutations in the secretory (sec) genes in Escherichia coli compromise protein translocation across the inner membrane and often confer conditional-lethal phenotypes . We have found that overproduction of the chaperonins GroES and GroEL from a multicopy plasmid suppresses a wide array of cold-sensitive sec mutations in E . coli . Suppression is accompanied by a stimulation of precursor protein translocation . This multicopy suppression does not bypass the Sec pathway because a deletion of secE is not suppressed under these conditions . Surprisingly, progressive deletion of the groE operon does not completely abolish the ability to suppress, indicating that the multicopy suppression of cold-sensitive sec mutations is not dependent on a functional groE operon . Indeed, overproduction of proteins unrelated to the process of protein export suppresses the secE501 cold-sensitive mutation, suggesting that protein overproduction, in and of itself, can confer mutations which compromise protein synthesis and the observation that low levels of protein synthesis inhibitors can suppress as well . In all cases, the mechanism of suppression is unrelated to the process of protein export . We suggest that the multicopy plasmids also suppress the sec mutations by compromising protein synthesis. J Bacteriol, 1995 Sep, 177(17), 4940 - 6 DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli; Wilson RL et al.; The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA . Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions . DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from -28 to +20 . Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions -271 to -242, a 28-bp region extending from -242 to -214, and a 35-bp region covering positions -69 to -34 relative to the transcription initiation site . PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv. J Bacteriol, 1995 Sep, 177(17), 4921 - 6 Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product; Mori H et al.; We attempted to clone an inosine kinase gene of Escherichia coli . A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning . A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine . The fragment was sequenced, and one protein of 434 amino acids long was found . This protein was overexpressed . The overexpressed protein was purified and characterized . The enzyme had both inosine and guanosine kinase activity . The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively . The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively . This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate . These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine . The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W . Harlow, P . Nygaard, and B . Hove-Jensen, J . Bacteriol . 177:2236-2240, 1995). J Bacteriol, 1995 Sep, 177(17), 4900 - 7 Hsc66, an Hsp70 homolog in Escherichia coli, is induced by cold shock but not by heat shock; Lelivelt MJ et al.; Hsc66 is the second identified Hsp70 protein in Escherichia coli . Mutations in hscA, the gene encoding Hsc66, compensate for some phenotypic effects of a mutation in hns, a gene encoding the cold-inducible, nucleoid-associated protein H-NS . Expression of hscA was not induced upon heat shock but was induced approximately 11-fold 3 h after a shift from 37 to 10 degrees C . Furthermore, hscA was induced upon chloramphenicol addition, which induces the synthesis of other cold-inducible genes . Mapping of the transcription initiation site showed that hscA was cotranscribed with an upstream dnaJ-like gene, hscB; thus, hscB was also cold inducible . The hscBA promoter did not contain a Y-box element found in some cold-inducible promoters . Using two-dimensional electrophoresis, we identified Hsc66 under static 37 degrees C growth conditions and showed that Hsc66 was induced, as well as hscA, 3 h after a cold shock . Growth of an hscA mutant following cold shock was monitored relative to that of an isogenic wild-type strain . While cold shock adaptation as a function of growth rate was not significantly impaired in an hscA mutant, the expression of at least five other proteins was altered in this mutant following cold shock . On the basis of the homology to Hsp70 proteins and the induction following cold shock, we speculate that Hsc66 functions as a cold shock molecular chaperone. J Bacteriol, 1995 Sep, 177(17), 4865 - 71 Boundaries of the pSC101 minimal replicon are conditional; Miller CA et al.; The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon . We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning . Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein . However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required . One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA . The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T . T . Stenzel, T . MacAllister, and D . Bastia, Genes Dev . 5:1453-1463, 1991), also affect both replication and partitioning . Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon . Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division. J Bacteriol, 1995 Sep, 177(17), 4829 - 35 Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function; Kadner RJ et al.; Vitamin B12 (CN-Cbl) and iron-siderophore complexes are transported into Escherichia coli in two energy-dependent steps . The first step is mediated by substrate-specific outer membrane transport proteins and the energy-coupling TonB protein complex, and the second step uses separate periplasmic permeases for transport across the cytoplasmic membrane . Genetic and biochemical evidence suggests that the TonB-dependent outer membrane transporters contact TonB directly, and thus they might compete for limiting amounts of functional TonB . The transport of iron-siderophore complexes, such as ferrichrome, causes a partial decrease in the rate of CN-Cbl transport . Although CN-Cbl uptake does not inhibit ferrichrome uptake in wild-type cells, in which the amount of the outer membrane ferrichrome transporter FhuA far exceeds that of the cobalamin transporter BtuB, CN-Cbl does inhibit ferrichrome uptake when BtuB is overexpressed from a multicopy plasmid . This inhibition by CN-Cbl is increased when the expression of FhuA and TonB is repressed by growth with excess iron and is eliminated when BtuB synthesis is repressed by CN-Cbl . The mutual inhibition of CN-Cbl and ferrichrome uptake is overcome by increased expression of TonB . Additional evidence for interaction of the Cbl and iron transport systems is provided by the strong stimulation of the BtuB- and TonB-dependent transport of CN-Cbl into a nonexchangeable, presumably cytoplasmic pool by preincubation of cells with the iron(II) chelator 2,2'-dipyridyl . Other metal ion chelators inhibited CN-Cbl uptake across the outer membrane . Although the effects of chelators are multiple and complex, they indicate competition or interaction among TonB-dependent transport systems. Crit Care Med, 1995 Sep, 23(9), 1519 - 27 Dysregulation of the veno-arterial response in the superior mesenteric artery during endotoxic shock; Revelly JP et al.; OBJECTIVE: To investigate whether the vascular dysfunction in endotoxic shock is associated with inhibition of the veno-arterial response of the superior mesenteric artery . DESIGN: Prospective, concurrent trial . SETTING: Animal laboratory . SUBJECTS: Domestic pigs . INTERVENTIONS: Two groups of pigs were anesthetized with ketamine and pentobarbital, mechanically ventilated, and hemodynamically monitored . One group (n = 8) was challenged with Escherichia coli endotoxin (30 micrograms/kg iv), while the other group (n = 4) served as time controls . Portal vein pressure was transiently increased in a series of steps from baseline to 25 mm Hg by partially obstructing portal venous flow . MEASUREMENTS AND MAIN RESULTS: The effects of increases in portal pressure on superior mesenteric artery resistance, superior mesenteric artery fractional flow, and cardiac output were assessed . Under pre-endotoxin conditions, raising portal pressure induced an increase in superior mesenteric artery resistance, a decrease in superior mesenteric artery fractional flow, and no significant change in cardiac output (i.e., a normally regulating veno-arterial response) . After endotoxin administration, raising portal pressure induced a decrease in superior mesenteric artery resistance, no change in superior mesenteric artery fractional flow, and a decrease in cardiac output (i.e., a dysregulated veno-arterial response) . CONCLUSIONS: Under baseline conditions, a normally regulating veno-arterial response in the mesenteric vascular bed should minimize intestinal blood pooling with acute portal hypertension . Under conditions of endotoxemic shock, the dysregulation of the veno-arterial response could substantially contribute to blood pooling and edema formation in the intestinal vascular bed during septic shock . This phenomenon may account for many of the macro- and microcirculatory manifestations of septic shock. Biophys Chem, 1995 Sep-Oct, 56(1-2), 63 - 70 Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin; Liu XM et al.; Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin and other retinal proteins . However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique . While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments . A new approach has recently been reported (Sonar et al., Nature Struct . Biol . 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL) . We have now used this method to re-examine bands assigned in the bR-->M difference spectrum to tyrosine residues . Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active . This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins. Arch Ophthalmol, 1995 Sep, 113(9), 1120 - 3 Diagnosis and successful medical treatment of Acanthamoeba keratitis; D'Aversa G et al.; OBJECTIVE: To identify the methods that result in timely diagnosis and effective treatment of Acanthamoeba keratitis . METHODS: We retrospectively reviewed the medical records of 12 consecutive patients whom we treated for culture-proved Acanthamoeba keratitis in 14 eyes . RESULTS: Contact lenses were worn in 13 of 14 affected eyes and substandard methods were often used to care for them . The diagnosis was established in all patients by laboratory analysis of corneal scrapings; corneal biopsies were not required . Acanthamoeba organisms were identified on smears from 12 of 14 eyes with use of standard, nonfluorescent stains and recovered in culture from all patients by inoculating scrapings on nonnutrient agar overlaid with Escherichia coli . Eleven of 14 eyes were medically cured with a combination of antiamebic drugs, most commonly propamidine isethionate, neomycin sulfate, and clotrimazole . Topical corticosteroids were used in only one patient . Two of the three eyes that required therapeutic keratoplasty were not treated before surgery according to our usual protocol; the third required keratoplasty for treatment of a severe bacterial superinfection . Twelve of 14 eyes recovered 20/50 or better visual acuity . Bacterial superinfections were a serious problem, with a total of six superinfections occurring in three treated eyes . CONCLUSION: With timely diagnosis and medical treatment with a combination of antiamebic drugs and avoidance of topical corticosteroids, most cases of Acanthamoeba keratitis can be cured, with an excellent prognosis for visual recovery. Am J Gastroenterol, 1995 Sep, 90(9), 1511 - 3 Strangulation of the colon complicating acute pancreatitis; Tenner S et al.; We report a patient with alcohol-induced necrotizing pancreatitis who developed a severe ileus followed by incarceration of a portion of the transverse colon within a ventral hernia . Laparotomy 9 days after the onset of symptoms revealed infarction of the transverse colon and infection of the pancreas . This is the first report of a case of acute pancreatitis that led to a strangulated ventral hernia of the colon . We believe that the enteric organisms that infected the pancreas originated in the incarcerated transverse colon. J Biol Chem, 1995 Sep 1, 270(35), 20862 - 9 Human DNA repair excision nuclease . Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies; Matsunaga T et al.; Human DNA repair excision nuclease removes DNA damage by incising on both sides of the lesion in a precise manner . The activity requires participation of 16-17 polypeptides . Of these, the XPF.ERCC1 complex and XPG were predicted to carry the nuclease active sites based on studies with the recombinant proteins and the yeast homologs of these proteins . Furthermore, recent work with model (undamaged) substrates have led to predictions of the roles of these proteins in incising 5' or 3' to the lesion . We have used damaged DNA substrates and antibodies to XPG and ERCC1 to test these predictions . Our results reveal that anti-XPG antibodies change the site of 3' incision and at high concentration inhibit the 3' incision without significantly affecting the 5' incision, indicating that XPG makes the 3' incision and further that under this condition 5' incision can occur without 3' incision . In contrast, anti-ERCC1 antibodies inhibit both the 3' and 5' incisions . Using a defined system for excision repair we also demonstrate that the 3' incision can occur without the 5' incision, leading us to conclude that under certain conditions the two incisions can occur independently. J Biol Chem, 1995 Sep 1, 270(35), 20781 - 6 Role of glycine 1 and lysine 2 in the glycation of bovine gamma B-crystallin . Site-directed mutagenesis of lysine to threonine; Casey EB et al.; To determine the role of Gly-1 and Lys-2 of bovine gamma B-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis . A polymerase chain reaction was used to perform site-directed mutagenesis on the third codon (AAG-->ACG) of bovine gamma B-crystallin cDNA . The wild type and mutant cDNAs were cloned into pMON5743 and expressed in JM101 Escherichia coli cells, and the identity of gamma B-crystallin was confirmed by Western blotting after purification by cation exchange high performance liquid chromatography . Glycation of gamma B-crystallin by {14C}glucose was reduced significantly due to the mutation of Lys-2, supporting the view that Lys-2 is a major glycation site . Peptide mapping showed the presence of two major labeled peptides containing N-terminal sequences, and in the mutant these peptides had longer retention times and reduced radioactivity . Amino acid analysis, after glycation with {14C}glucose, revealed N-terminal glycine as the most predominant glycation site . Lys-2 was glycated slower than Gly-1 but faster than Lys-163 . Glycation with DL-glyceraldehyde showed an important role for both Gly-1 and Lys-2 in the glycation-mediated gamma B-crystallin cross-linking. J Biol Chem, 1995 Sep 1, 270(35), 20730 - 6 Inactivation of Escherichia coli phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP . Identification of active site lysines; Hilden I et al.; The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2',3'-dialdehyde ATP (oATP) . The reaction displayed complex saturation kinetics with respect to oATP with an apparent KD of approximately 0.8 mM . Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit . oATP served as a substrate in the presence of ribose-5-phosphate, and the enzyme could be protected against inactivation by ADP or ATP . Isolation of radioactive peptides from the enzyme modified with radioactive oATP, followed by automated Edman sequencing allowed identification of Lys181, Lys193, and Lys230 as probable sites of reaction with the analog . Cysteine 229 may also be labeled by oATP . Of these four residues, Lys193 is completely conserved within the family of PRPP synthetases, and Lys181 is found at a position in the sequence where the cognate amino acid (Asp181) in human isozyme I PRPP synthetase has been previously implicated in the regulation of enzymatic activity . These results imply a functional role for at least two of the identified amino acid residues. J Biol Chem, 1995 Sep 1, 270(35), 20615 - 20 Molecular cloning and expression of a pituitary gland protein modulating intestinal fluid secretion; Johansson E et al.; Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin . cDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced . The sequence contained 1309 base pairs plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed this size . One large open reading frame was found to code for 382 amino acids . The protein was expressed in pGEX-lambda 1T/Escherichia coli and purified . The recombinant AF was extremely potent, 9 ng (2.10(-13) mol), giving a significant antisecretory activity against cholera toxin-induced fluid secretion in rat . Antiserum against recombinant AF was used in immunohistochemical and Western blot analysis . Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part . Both recombinant AF and AF extracted from pituitary gland appeared in SDS-polyacrylamide to have a molecular mass of 60 kDa, although the renal value was 41 kDa . The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function. J Biol Chem, 1995 Sep 1, 270(35), 20568 - 74 Asymmetry of Escherichia coli F1-ATPase as a function of the interaction of alpha-beta subunit pairs with the gamma and epsilon subunits; Haughton MA et al.; The asymmetry of Escherichia coli F1-ATPase (ECF1) has been explored in chemical modification experiments involving two mutant enzyme preparations . One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant beta G149C:V198A) . The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the epsilon subunit (mutant beta G149C:V198A:E381C/epsilon S108C) . On CuCl2 treatment of this second mutant, there is cross-linking of one copy of the beta subunit to gamma via the Cys at 381, a second to the epsilon subunit (between beta Cys381 and epsilon Cys108), while the third beta subunit in the ECF1 complex is mostly free (some cross-linking to delta); thereby distinguishing the three beta subunits as beta gamma, beta epsilon, and beta free, respectively . Both mutants have ATPase activities similar to wild-type enzyme . Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys149, dicyclohexyl carbodiimide (DCCD) which reacts with Glu192, and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr297 . Thus, beta gamma reacted with DCCD but not NEM or NbfCl; beta free was reactive with all three reagents; beta epsilon reacted with NEM, but was poorly reactive to DCCD or NbfCl . There was a strong nucleotide dependence of the reaction of Cys149 in beta epsilon (but not in beta free) with NEM, indicative of the important role that the epsilon subunit plays in functioning of the enzyme. J Biol Chem, 1995 Sep 1, 270(35), 20556 - 9 Overexpression, purification, and crystallization of the DNA binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1; Barwell JA et al.; The Epstein-Barr virus nuclear antigen (EBNA) 1 binds to and activates DNA replication from the latent origin of Epstein-Barr virus . Six different fragments of EBNA1 that retain DNA binding activity were expressed in bacteria, purified, and crystallized . Two fragments, EBNA470-619 and EBNA470-607, formed well ordered crystals that diffracted beyond 2.5-A resolution . Two different EBNA470-619 crystals were grown from sodium formate, pH 6-6.5 . One crystal belonged to the trigonal space group P3 with unit cell dimensions a = b = 86.5 A and c = 31.8 A and with two molecules in the asymmetric unit . The other crystal, which appeared only twice and was likely related to the P3 crystal form, belonged to the trigonal space group P312 with cell dimensions a = b = 86.7 A and c = 31.8 A . Crystals of EBNA470-607 were grown by lowering the salt concentration to 0-100 mM NaCl at pH 6.0 . These crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and had cell dimensions a = 59 A, b = 66.9 A, and c = 69.8 A with two molecules in the asymmetric unit. J Biol Chem, 1995 Sep 1, 270(35), 20550 - 5 The flavin reductase activity of the flavoprotein component of sulfite reductase from Escherichia coli . A new model for the protein structure; Eschenbrenner M et al.; Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups . It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates . The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction . A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction . The overproducer allows a fast and simple preparation of large amounts of the flavoprotein . Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism . NADP+ inhibition studies show that both substrates, NADPH and free flavins, bind to the same site . While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity . In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit . This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit. J Biol Chem, 1995 Sep 1, 270(35), 20543 - 9 Carboxypeptidase A isoforms produced by distinct genes or alternative splicing in brain and other extrapancreatic tissues; Normant E et al.; The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the pancreatic enzyme had not been reported so far . Transcripts of two known CPA genes, CPA1 and CPA2, were identified in extremely low abundance in brain and several other extrapancreatic tissues using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis . Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases) . Northern blot analysis using various probes, RT-PCR, and 5'-rapid amplification of cDNA 5'-end (5' RACE analysis) all indicated that this smaller size of the brain transcript was attributable to production by alternative splicing of the pro-mRNA . This process corresponds to deletion of the first four exons, leading to a mRNA encoding a protein in which the signal peptide and activation peptide of prepro-CPA2 are absent but the active site remains . The prediction that the shorter CPA2 isoform, designated CPA2(S), should correspond to a cytoplasmic metallopeptidase that does not require tryptic activation was verified by characterization of the recombinant protein and comparing it with the native CPA-like activity in brain . Both recombinant CPA2(S) generated in Escherichia coli and a soluble protein from brain displayed similar sizes on Western blots (32 kDa to be compared to 34 kDa for pancreatic CPA2) . Recombinant CPA2(S) and a soluble CPA-like activity from brain displayed similar sensitivity to a series of inhibitors, contrasting with that of the pancreatic enzyme . It is concluded that alternative splicing produces a truncated CPA2 with distinct subcellular localization and modified catalytic activity . In spite of the presence of the CPA1 mRNA, no corresponding CPA activity could be detected in brain extracts, even after tryptic activation . This apparent discrepancy seems attributable to the presence of an endogenous peptide inhibitor which remains to be identified. J Biol Chem, 1995 Sep 1, 270(35), 20491 - 6 Removal of feedback inhibition of delta 1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants; Zhang CS et al.; delta 1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps in proline biosynthesis in plants . The Vigna aconitifolia P5CS cDNA was expressed in Escherichia coli, and the enzyme was purified to homogeneity . The Vigna P5CS exhibited two activities, gamma-glutamyl kinase (gamma-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase . The gamma-GK activity of the P5CS was detected by the hydroxamate assay and by a {14C}glutamate assay . The native molecular mass of the P5CS was 450 kDa with six identical subunits . The Vigna P5CS showed a Km of 3.6 mM for glutamate, while the Km for ATP was 2.7 mM . The gamma-GK activity of the P5CS was competitively inhibited by proline, while its GSA dehydrogenase activity was insensitive to proline . In addition, a protein inhibitor of the P5CS was observed in the plant cell . Western blot showed that the level of the P5CS was enhanced in Vigna root under salt stress . A single substitution of an alanine for a phenylalanine at amino acid residue 129 of the P5CS resulted in a significant reduction of proline feedback inhibition . The 50% inhibition values of gamma-GK activity of the wild type and the mutant P5CS were observed at 5 mM and 960 mM proline, respectively . The other properties of the mutant P5CS remained unchanged . These results may allow genetic manipulation of proline biosynthesis and overproduction of proline in plants for conferring water stress tolerance. J Biol Chem, 1995 Sep 1, 270(35), 20453 - 8 Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides . Analysis of the cis-acting downstream regulatory sequence; Lee JK et al.; Both site-directed and spontaneous mutagenesis have been used to investigate the role of the cis-acting regulatory region between -92 and -1 base pair (bp) of the puc operon of Rhodobacter sphaeroides . The DNA sequence from -84 to -66 bp upstream of the 5' end of the start site of puc operon transcription is essential for normal puc operon expression . This regulatory effect was exerted irrespective of the presence or absence of additional upstream regulatory sequences extending from -629 to -93 bp . It is likely that this region is involved in activator binding . Additionally, two regions of dyad symmetry centered at -42 and -17 bp are shown to be involved in oxygen repression of puc operon expression . Mutations within these regions of dyad symmetry were further subdivided on the basis of whether or not the upstream regulatory region was required to observe the mutant phenotype . Based upon these observations we conclude that these regions of dyad symmetry possessing the motif TGT-N12-ACA (where N represents any nucleotide) are involved in repressor binding with the puc operon promoter overlapping each of these dyad symmetries. J Biol Chem, 1995 Sep 1, 270(35), 20447 - 52 Cloning and characterization of a heterologously expressed bifunctional chorismate synthase/flavin reductase from Neurospora crassa; Henstrand JM et al.; Activities of all chorismate synthases (CS) so far analyzed are absolutely dependent upon reduced flavin . For monofunctional CSs, which represent the only class of CSs that have yet been cloned, the flavin must be reduced either (photo-)chemically or by a separable flavin reductase (FR) for in vitro activity . Neurospora crassa CS, in contrast, possesses an intrinsic FR activity and represents the only firmly established member of a bifunctional class of CSs . To better understand this bifunctional protein, a cDNA from an N . crassa expression library encoding a 46.4-kDa protein was cloned by complementation of the CS-deficient Escherichia coli strain AB2849 . The deduced amino acid sequence was highly similar (79%) to a previously isolated Saccharomyces cerevisiae CS . The N . crassa sequence was unequivocally shown to encode the bifunctional CS/FR by analysis of the purified protein expressed in E . coli . Based on sequence comparisons with known monofunctional CSs, two regions of 18 internal residues and 29 C-terminal residues unique to N . crassa CS were deleted, and the constructs were also expressed in E . coli . The presence of these regions was found not essential for complementation of the CS- phenotype of E . coli strain AB2849 . Although a 3.5-fold decline in specific activity of the purified CS from cells expressing the C-terminal deletion construct was observed, bifunctional activity was not eliminated . These data strongly suggest that the domain(s) responsible for reduction of flavin lie(s) within regions in which homology is also shared among monofunctional CSs. J Biol Chem, 1995 Sep 1, 270(35), 20404 - 9 A monomeric variant of GroEL binds nucleotides but is inactive as a molecular chaperone; White ZW et al.; The heat shock protein GroEL from Escherichia coli is a tetradecameric oligomer that facilitates the refolding of nonnative polypeptides in an ATP-hydrolysis dependent reaction . A mutant in GroEL was prepared in which lysine 3 was substituted with glutamate, which destabilizes the oligomeric structure of GroEL (Horovitz, A., Bochkareva, E.S., and Girshovich, A.S . (1993) J . Biol . Chem . 268, 9957-9959) . The highly expressed and purified GroELK3E was judged to be monomeric by sedimentation equilibrium, yielding a molecular weight of 54,500, despite a weak tendency of the mutant to reversibly form higher order aggregates above 4 mg ml-1 . The monomeric variant appears to be folded based on the far UV circular dichroism spectrum, which shows significant alpha-helical content, but with slight differences in conformation relative to wild-type GroEL . The increase in exposed hydrophobic surface of the monomer was probed with the dye 4,4'-bis-1-anilino-3-naphthalenesulfonate (bis-ANS) . The fluorescence of bis-ANS increases approximately 150-fold in the presence of the mutant, and about 4 mol of bis-ANS bind per mol of monomer, with a binding constant of 1.6 microM . Adenosine nucleotide binding to monomeric GroELK3E resulted in considerable quenching of bis-ANS fluorescence, correlating with significant structural changes as seen in the far UV circular dichroism, and permitted the measurement of binding isotherms for ATP and ADP . Hyperbolic ATP binding isotherms yield a dissociation constant of 82 microM, about 4-fold weaker than the K0.5 for ATP seen in steady-state kinetics assays of the wild-type GroEL ATPase.A similar difference was seen for ADP binding . These results suggest that the mutation disrupts the native tetradecameric quaternary structure through conformational changes that may also weaken nucleotide binding . The monomeric mutant exhibited no chaperone activity as evidenced by a filure to inhibit or facilitate the refolding of chemically denatured enolase, an inability to refold denatured rhodanese above spontaneous levels, and a lack of binding to alpha-casein, a competitor in many chaperonin-promoted refolding reactions . Thus, the formation of assembly incompetent monomers by the lysine 3 to glutamate mutation results in a dramatic decrease in the affinity for nonnative polypeptide chains and suggests that the oligomeric nature of GroEL is crucial for its molecular chaperone function. J Biol Chem, 1995 Sep 1, 270(35), 20273 - 7 Expression of Desulfovibrio gigas desulforedoxin in Escherichia coli . Purification and characterization of mixed metal isoforms; Czaja C et al.; The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity . The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D . gigas . These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands . Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8 . Interestingly, E . coli produced two forms of desulforedoxin containing iron . One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc. J Biol Chem, 1995 Sep 1, 270(35), 20250 - 3 Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli; Hackett M et al.; Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity) . When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B . pertussis . In contrast to the native protein from B . pertussis, which is exclusively palmitoylated, recombinant toxin from E . coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated . Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860 . These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function. Invest Ophthalmol Vis Sci, 1995 Sep, 36(10), 1949 - 59 Endotoxin-induced uveitis . Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body; McMenamin PG et al.; PURPOSE . Footpad injection of bacterial lipopolysaccharide (LPS) causes pronounced anterior uveitis in susceptible species and strains . Recent studies using wholemount techniques have demonstrated the presence of rich networks of major histocompatibility complex (MHC) class II-positive dendritic cells (DC) and resident tissue macrophages in the iris and ciliary body . The aim of this investigation was to determine the immunophenotype and dynamics of the inflammatory cell infiltrate during LPS-induced anterior uveitis using the wholemount method and to examine the response of the resident tissue macrophages and DC to an acute inflammatory episode in the anterior segment . METHODS . Female Lewis rats (8 to 12 weeks old, n = 49) received a single footpad injection of 100 micrograms of LPS and were killed at various times up to 6 weeks after injection . The iris-ciliary body complex from each eye was removed intact and subdivided into segments and immunostained using a panel of monoclonal antibodies to a variety of immune cell types . RESULTS . The wholemount method clearly illustrates that during endotoxin-induced uveitis (EIU), the earliest cellular infiltrate includes small, round ED1+ mononuclear cells marginating in the iris vasculature approximately 2 hours after injection . Marginating Ox42+ polymorphonuclear leukocytes were detectable in the iris vessels approximately 4 to 6 hours after injection and were especially numerous in the ciliary body base approximately 24 hours after injection . The overall density of resident tissue macrophages (ED2+) remained largely unchanged in the course of EIU . In contrast, the total number of MHC class II-bearing (Ox6+) cells (putative dendritic cells) increased 30% in the first 6 hours and 200% by 72 hours . During the acute phase of the inflammatory response (up to 24 hours), the proportion of these cells with a dendritiform morphology decreased (93% to 50%) . The number of T cells showed a biphasic response peaking at 4 to 6 hours and again at 24 hours (290 cells/mm2); however, their numbers had resumed normal low density (4 cells/mm2 to 25 cells/mm2) by 6 weeks . CONCLUSIONS . The results suggest that the neutrophilic infiltration in EIU occurs predominantly in the base of the ciliary body, whereas the monocytic and lymphocytic infiltrate occurs in the iris vasculature . Resident tissue macrophages do not undergo marked changes in density or morphology in the early course of the disease . Recruitment of T cells into the anterior segment in EIU may suggest a previously unsuspected role for these cells in the immunopathology of this disease . Changes in density and morphology of MHC class II+ DC in the iris, which persisted for at least 6 weeks, were interpreted as an increase in recruitment and migration of these cells that may serve to enhance the efficiency of immune surveillance in the anterior segment at crucial times of bacterial infection. Biochem J, 1995 Sep 1, 310 ( Pt 2), 507 - 16 Isocitrate dehydrogenase from bovine heart: primary structure of subunit 3/4; Zeng Y et al.; Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx . 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties {Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346} . In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability . Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing . Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues . The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme . Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit . Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information . The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da . Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme {Huang and Colman (1990) Biochemistry 29, 8266-8273} suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme . An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated . One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated . Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known {Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316} shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function. Biochem J, 1995 Sep 1, 310 ( Pt 2), 483 - 90 Retinoid receptors cause distortion of the retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter; Scott DK et al.; Functional retinoic acid response elements (RAREs) have been described wherein the direct repeats are separated by 1, 2 or 5 bp (termed DR1, DR2 and DR5 respectively) . We have previously shown that retinoic acid receptor/retinoid X receptor (RAR/RXR) binds a DR1 RARE within the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and is the trans-acting complex that mediates the retinoic acid (RA) response . However, the mechanism of trans-activation is unknown . The consequences of RAR/RXR binding to the PEPCK RARE were examined using a circular permutation analysis as a first step to explore the possible role of DNA conformational changes in the RA response . The RAR/RXR heterodimer produced a distortion angle of 78 degrees . The DNA distortion was shown to be at the centre of the PEPCK RARE; RA did not affect the severity of the distortion angle or the location of the distortion centre . Monomers and homodimers of RAR also distorted the DNA, but to a lesser extent than did RAR/RXR . The results of a phasing analysis demonstrated that RAR/RXR heterodimers did not induce a static DNA bend, in either the presence or the absence of RA . A cyclization kinetics assay was employed to show that RAR/RXR binding affected DNA ring closure in a phase-sensitive, RA-insensitive, manner . Taken together, these observations support the idea that RAR/RXR heterodimers distort the structure of the PEPCK RARE, at least in part, by altering DNA flexibility . The conformational change in the PEPCK RARE upon RAR/RXR binding has implications for how RAR/RXR heterodimers recognize various RARE structures. Biochem J, 1995 Sep 1, 310 ( Pt 2), 389 - 94 Evidence that His110 of the protein FadL in the outer membrane of Escherichia coli is involved in the binding and uptake of long-chain fatty acids: possible role of this residue in carboxylate binding; Black PN et al.; The binding of exogenous fatty acids to the outer-membrane protein FadL of Escherichia coli is specific for long-chain fatty acids (C14-C18) . Oleoyl alcohol {(Z)-9-octadecen-1-ol} and methyl oleate were unable to displace FadL-specific binding of {3H}oleate (C18:1), suggesting that the carboxylate of the long-chain fatty acid was required for binding . Therefore the binding of exogenous fatty acids to FadL is governed, in part, by the carboxy group of the long-chain fatty acid . Treatment of whole cells with 1 mM diethyl pyrocarbonate (DEPC) depressed binding by 43-73% over the range of oleate concentrations used (10-500 nM) . On the basis of these results and the notion that histidine residues often play a role involving proton transfer and charge-pairing, the five histidine residues within FadL (His110, His226, His327, His345 and His418) were replaced by alanine using site-directed mutagenesis . Altered FadL proteins were correctly localized in the outer membrane at wild-type levels and retained the heat-modifiable property characteristic of the wild-type protein . Initial screening of these fadL mutants revealed that the replacement of His110 by Ala resulted in a decreased growth rate on minimal oleate/agar plates . The rates of long-chain fatty acid transport for delta fadL strains harbouring each mutation on a plasmid, with the exception of fadLH110A, were the same, or nearly the same, as those for the wild-type . fadLH110A was also defective in binding, arguing that the functional effect of this mutation was at the level of long-chain-fatty-acid binding.(ABSTRACT TRUNCATED AT 250 WORDS) Radiat Res, 1995 Sep, 143(3), 281 - 5 Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity; Lawrence TS et al.; It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated . We have shown that HT29 human colon cancer cells transfected with the E . coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP . We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd . The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which had been transfected with only the expression vector for the E . coli gene (con clones) . We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity . Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd . Flow cytometry showed that all clones progressed past the G1/S-phase boundary and into early S phase during FdUrd treatment . These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked . These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G1/S-phase boundary in the presence of FdUrd. Mol Cell Biol, 1995 Sep, 15(9), 5007 - 16 The transcriptional repressor even-skipped interacts directly with TATA-binding protein; Um M et al.; The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription . However, the mechanism of repression is not known . Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP) . Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement . Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain . Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells . Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP . The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions . We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro . Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP. Mol Cell Biol, 1995 Sep, 15(9), 4683 - 93 A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking; Austin RJ et al.; We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription . A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain . This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter . Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding . Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur . Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter . Binding to some of these low-affinity sites was shown to contribute to repression . Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism . We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking. Infect Immun, 1995 Sep, 63(9), 3459 - 66 A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease; Feng S et al.; We have identified a 55-kDa antigen encoded by a gene on a 49-kb plasmid of Borrelia burgdorferi . The screening of a B . burgdorferi DNA expression library (N40 strain) with rabbit anti-B . burgdorferi serum and then with serum from a patient with Lyme disease arthritis revealed a clone that synthesized an antigen that was reactive with both sera . DNA sequence analysis identified an operon with two genes, s1 and s2 (1,254 and 780 nucleotides), that expressed antigens with the predicted molecular masses of 55 and 29 kDa, respectively . Pulsed-field gel electrophoresis showed that the s1-s2 operon was located on the 49-kb plasmid . Recombinant S1 was synthesized as a glutathione S-transferase fusion protein in Escherichia coli . Antibodies to recombinant S1 bound to a 55-kDa protein in lysates of B . burgdorferi, indicating that cultured spirochetes synthesized S1 . Thirty-one of 100 Lyme disease patients had immunoglobulin G (IgG) and/or IgM antibodies to S1 . IgG antibodies to S1 were detected by enzyme-linked immunosorbent assay and immunoblots in the sera of 21 (21%) of 100 patients with Lyme disease; 11 (27.5%) of the S1-positive samples were from patients (40) with early-stage Lyme disease, and 10 (16.7%) were from patients (60) with late-stage Lyme disease . Fifteen (38.5%) of 40 serum samples from patients with early-stage Lyme disease had IgM antibodies to S1 . These data suggest that the S1 antigen encoded by a gene on the 49-kb plasmid is recognized serologically by a subset of patients with early- or late-stage Lyme disease. Infect Immun, 1995 Sep, 63(9), 3417 - 21 Characterization of an invasive phenotype associated with enteroaggregative Escherichia coli; Benjamin P et al.; Enteroaggregative Escherichia coli (EAggEc) strains are associated with persistent diarrhea in children in the developing world and exhibit a classic aggregative phenotype . We have demonstrated that EAggEc strains isolated from children with persistent diarrhea in Brazil, Bangladesh, and Pakistan also have the potential to be internalized by HeLa cells in the gentamicin protection assay . We have confirmed this phenomenon with transmission electron micrographs of bacteria engulfed by HeLa cells . We examined the mechanisms by which this process occurs . Staurosporine inhibited internalization of EAggEc strain 162 by 50% at a concentration of 0.1 microM . Genistein inhibited internalization of this same organism by 50% at a concentration of 50 microM . Cytochalasin D inhibited internalization by 50% at a concentration of 1 microgram/ml . Staurosporine, genistein, and cytochalasin D inhibited the internalization of EAggEc strain 162 by HeLa cells in a dose-dependent manner . These data suggest that active cell processes such as signal transduction by protein kinase and/or tyrosine kinase may be involved in the internalization of EAggEc strain 162 by HeLa cells and that actin filaments and cytoskeletal structure may be important for this process. Infect Immun, 1995 Sep, 63(9), 3327 - 35 Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo; Wallich R et al.; Previously we have found that sera from immunocompetent mice infected either naturally by ticks or experimentally with low numbers of Borrelia burgdorferi ZS7 bacteria lack OspA- and OspB-specific antibodies but confer optimal protection on severe combined immunodeficiency mice against challenge with spirochetes (U.E . Schaible, L . Gern, R . Wallich, M . D . Kramer, M . Prester, and M . M . Simon, Immunol . Lett . 36:219-226, 1993) . We have now used the latter immune sera to identify new spirochetal structures with relevance for protection from an expression library of the virulent European strain B . burgdorferi ZS7 . Here we report the cloning and characterization of a novel lipoprotein, designated pG, the gene for which is located on a 48-kb linear plasmid . Sequence analysis of the pG gene revealed an open reading frame encoding a putative lipoprotein of 196 amino acids with a calculated molecular mass of 22 kDa and a consensus cleavage sequence (Leu-X-Y-Z-Cys) recognized by signal peptidase II . Restriction fragment length polymorphism analyses of pG derived from independent B . burgdorferi isolates from different geographic areas revealed that the gene is species specific, with, however, extensive genotypic heterogeneity . Comparison of the protein sequence of pG with those of other known B . burgdorferi outer surface lipoproteins (OspA to OspF and P27) demonstrated that pG is most related to OspF . Furthermore, the upstream region of pG exhibited extensive sequence homology (> 94%) with the ospEF promoter region . Mouse immune sera to recombinant pG did not recognize a corresponding molecule in lysates of in vitro-propagated ZS7 spirochetes . However, experimental or natural infection of mice with ZS7 resulted in the induction of antibodies with reactivity for pG and the potential to delay the development of clinical arthritis . Together with the finding that sera from Lyme disease patients also contain antibodies to pG, our data suggest that the pG gene is preferentially expressed in the mammal environment. J Virol, 1995 Sep, 69(9), 5631 - 9 Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro; Muller B et al.; We have prepared and characterized several monoclonal antibodies (MAbs) against the Rous sarcoma virus integrase protein (IN) with the aim of employing these specific reagents as tools for biochemical and biophysical studies . The interaction of IN with the purified MAbs and their Fab fragment derivatives was demonstrated by Western blot (immunoblot), enzyme-linked immunosorbent assay, and size exclusion chromatography . A series of truncated IN proteins was used to determine regions in the protein important for recognition by the antibodies . The MAbs described here recognize epitopes that lie within the catalytic core region of IN (amino acids 50 to 207) and are likely to be conformational . A detailed functional analysis was carried out by investigating the effects of Fab fragments as well as of intact MAbs on the activities of IN in vitro . These studies revealed differential effects which fall into three categories . (i) One of the antibodies completely neutralized the processing as well as the joining activity and also reduced the DNA binding capacity as determined by a nitrocellulose filter binding assay . On the other hand, this MAb did not abolish the cleavage-ligation reaction on a disintegration substrate and the nonspecific cleavage of DNA by IN . The cleavage pattern generated by the IN-MAb complex on various DNA substrates closely resembled that produced by mutant IN proteins which show a deficiency in multimerization . Preincubation of IN with substrate protected the enzyme from inhibition by this antibody . (ii) Two other antibodies showed a general inhibition of all IN activities tested . (iii) In contrast, a fourth MAb stimulated the in vitro joining activity of IN . Size exclusion chromatography demonstrated that IN-Fab complexes from representatives of the three categories of MAbs exhibit different stoichiometric compositions that suggest possible explanations for their contrasting effects and may provide clues to the relationship between the structure and function of IN. Immunology, 1995 Sep, 86(1), 18 - 24 In vivo modulation of murine serum tumour necrosis factor and interleukin-6 levels during endotoxemia by oestrogen agonists and antagonists; Zuckerman SH et al.; Oestrogen has been reported to modulate tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6 cytokine levels in human mononuclear cell cultures . In the present study, the effects of exogenous oe