J Bacteriol, 1995 May, 177(10), 2923 - 5 DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication; Sweasy JB et al.; We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair . We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E . coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication . Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E . coli polA mutant in the presence of mammalian DNA polymerase beta . Our results suggest that mammalian DNA polymerase beta can substitute for E . coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication. J Bacteriol, 1995 May, 177(10), 2878 - 86 Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli; Kumari S et al.; Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate . We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F . R . Blattner, V . Burland, G . Plunkett III, H . J . Sofia, and D . L . Daniels, Nucleic Acids Res . 21:5408-5417, 1993) . We constructed a mutant allele, delta acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome . Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (< or = 10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (> or = 25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested . Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes . Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested . Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence . This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii . The purified E . coli Acs then was used to raise anti-E . coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs . When purified in the presence, but not in the absence, of coenzyme A, the E . coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner . On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs. J Bacteriol, 1995 May, 177(10), 2798 - 803 Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli; Hopper S et al.; The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli . The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation . Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA . The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis . Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA . Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM . The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction . The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA . The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate . Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity. J Bacteriol, 1995 May, 177(10), 2769 - 80 Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order; Casjens S et al.; We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations . All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16- to 175-kbp size range . The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders . However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates . The 22 chromosomal maps currently available define eight classes of Lyme disease agents . Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica . In addition, the North American isolates 21038, DN127 c19-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above . These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria . In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes. J Bacteriol, 1995 May, 177(10), 2760 - 8 Regulation of 5-aminolevulinic acid synthesis in Rhodobacter sphaeroides 2.4.1: the genetic basis of mutant H-5 auxotrophy; Zeilstra-Ryalls JH et al.; Rhodobacter sphaeroides H-5 was isolated as a 5-aminolevulinic acid (ALA) auxotroph following treatment of wild-type cells with N-methyl-N-nitroso-N'-nitroguanidine (J . Lascelles and T . Altshuler, J . Bacteriol . 98:721-727, 1969) . The existence in R . sphaeroides 2.4.1 of the genes hemA and hemT, each encoding the enzyme 5-aminolevulinic acid synthase (EC 2.3.1.37), raised questions as to the genetic basis for the ALA auxotrophy in mutant H-5 . We therefore cloned both the hemA and hemT genes from mutant H-5 . The hemA gene has been sequenced in its entirety and bears four base pair substitutions which encode three amino acid changes relative to the sequence of wild-type strain 2.4.1 . Complementation analysis of an Escherichia coli ALA auxotroph has revealed that the loss of ALA synthase activity in the HemA mutant enzyme could be localized to two of the amino acid substitutions . On the other hand, the hemT gene from mutant H-5 was able to complement an E . coli mutant requiring ALA for growth . Complementation analyses were also carried out by introducing the cloned hemA or hemT gene of mutant H-5 or wild-type 2.4.1 in trans into H-5 and, in parallel, into our previously described HemA-HemT double mutant strain AT1 (E . L . Neidle and S . Kaplan, J . Bacteriol . 175:2304-2313, 1993) . This analysis revealed that while the complementation pattern of mutant AT1 parallels that for the E . coli ALA auxotroph, mutant H-5 could only be complemented by the wild-type hemA gene . The ability of the hemT gene of either mutant H-5 or wild-type 2.4.1 to complement the ALA auxotrophy of mutant AT1 but not mutant H-5 was consistent with beta-galactosidase activities obtained with hemT-lacZ transcriptional fusions . We conclude that the ALA auxotrophy of mutant H-5 arises from (i) a nonfunctional HemA protein containing multiple missense substitutions and (ii) an inability of the normal hemT gene to be expressed in the mutant H-5 genetic background, i.e., an additional mutation of unknown origin is required for hemT expression . These studies bear directly on the regulation of the expression of the hemA and hemT genes of R . sphaeroides 2.4.1. J Bacteriol, 1995 May, 177(10), 2713 - 20 The smaller of two overlapping cheA gene products is not essential for chemotaxis in Escherichia coli; Sanatinia H et al.; The cheA locus of Escherichia coli encodes two similar proteins, CheAL (654 amino acids) and CheAS (557 amino acids), which are made by initiating translation from different in-frame start sites {start(L) and start(S)} . CheAL plays an essential role in chemotactic signaling . It autophosphorylates at a histidine residue (His-48) and then donates this phosphate to response regulator proteins that modulate flagellar rotation and sensory adaptation . CheAS lacks the first 97 amino acids of CheAL, including the phosphorylation site at His-48 . Although it is unable to autophosphorylate, CheAS can form heterodimers with mutant CheAL subunits to restore kinase function and chemoreceptor control of autophosphorylation activity . To determine whether these or other activities of CheAS are important for chemotaxis, we constructed cheA lesions that abrogated CheAS expression . Mutants in which the CheAS start codon was changed from methionine to isoleucine (M98I) or glutamine (M98Q) retained chemotactic ability, ranging from 50% (M98Q) to 80% (M98I) of wild-type function . These partial defects could not be alleviated by supplying CheAS from a specialized transducing phage, indicating that the lesions in CheAL--not the lack of CheAS--were responsible for the reduced chemotactic ability . In other respects, the behavior of the M98I mutant was essentially normal . Its flagellar rotation pattern was indistinguishable from wild type, and it exhibited wild-type detection thresholds and peak positions in capillary chemotaxis assays . The lack of any substantive defect in this start(S) mutant argues that CheAS makes a negligible contribution to chemotactic ability in the laboratory . Whether it has functional significance in other settings remains to be seen. J Bacteriol, 1995 May, 177(10), 2695 - 706 Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase; Eraso JM et al.; Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides . prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA . Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities . Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis . Previously, J . K . Lee and S . Kaplan (J . Bacteriol . 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation . This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78) . Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression . Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes . Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA . The genetic organization of the photosynthesis response regulatory (PRR) region is discussed. J Bacteriol, 1995 May, 177(10), 2679 - 83 Identification of Lrp-regulated genes by inverse PCR and sequencing: regulation of two mal operons of Escherichia coli by leucine-responsive regulatory protein; Tchetina E et al.; We have used the technique of inverse PCR to identify Escherichia coli chromosomal genes carrying Lrp-regulated inserts . This technique revealed that malT, malEFG, and malB-lamB-malK are all activated two- to fivefold by Lrp and confirmed that Lrp regulates expression of the leuDBCA and livHJKG operons . lacZ transcription is also increased in the presence of Lrp . However, the growth rate of the Lrp mutant on maltose and lactose is not decreased by Lrp deficiency. J Bacteriol, 1995 May, 177(10), 2663 - 72 The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability; Liu MY et al.; The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T . Romeo, M . Gong, M.Y . Liu, and A.M . Brun-Zinkernagel, J . Bacteriol . 175:4744-4755, 1993; T . Romeo and M . Gong, J . Bacteriol . 175:5740-5741, 1993) . We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation . Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis . The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation . Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation . Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes . The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins . The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding. FEMS Microbiol Lett, 1995 May 1, 128(2), 189 - 94 Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli; Guzman CA et al.; Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1 . Two protease deficient E . coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot . The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient) . Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium . These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins. FEMS Microbiol Lett, 1995 May 1, 128(2), 185 - 8 The appearance of cytoplasmic membranes of Escherichia coli cells in freeze-fracture electron microscopy after stringent and relaxed response; Gitter B et al.; Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation . Studies of stringent controlled Escherichia coli CP78 (relA+) and relaxed controlled E . coli CP79 (relA-) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response . Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells . The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts. FEBS Lett, 1995 May 1, 364(1), 59 - 62 Time resolved measurements show that phosphate release is the rate limiting step on myofibrillar ATPases; Lionne C et al.; The myofibril is a good model to study the ATPase of the muscle fibre . When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of Pi formation, due to AM.ADP.Pi or Pi, as shown in the scheme: AM+ATP<-->A.M.ATP<-->AM.ADP.Pi<-->AM.ADP+Pi<-->AM+ADP . Therefore, in the steady state, either AM.ADP.Pi or AM.ADP or both predominate . To determine which, we studied the reaction using a Pi binding protein (from E . coli) labeled with a fluorophore such that it is specific and sensitive to free Pi {Brune, M . et al . (1994) Biochemistry 33, 8262-8271} . We show that the Pi bursts with myofibrillar ATPases (calcium-activated or not, or crosslinked) are due entirely to protein bound Pi . Thus, with myofibrillar ATPases the AM.ADP.Pi state predominates. FEBS Lett, 1995 May 1, 364(1), 55 - 8 Redox states of DsbA in the periplasm of Escherichia coli; Kishigami S et al.; DsbA is a periplasmic, disulfide bond formation factor of E . coli . We studied in vivo redox states of its active site cysteines . When periplasmic contents were prepared from iodoacetic acid-treated cells, according to the previously published procedures, variable but major proportions of DsbA were in the reduced form . We found that this was due to an artificial reduction that occurred after cell disruption; even purified and oxidized DsbA underwent reduction when incubated with cell extracts in the absence of any added reducing agent . Such DsbA-reducing activities were detected in both the periplasmic and the cytoplasmic fractions . To circumvent the artifact, we analyzed redox states of DsbA under denaturing conditions . Now virtually all the DsbA molecules were detected as oxidized or reduced in the dsbB+ background or in the dsbB- background, respectively . Using the improved method, we also examined redox states of DsbA when it was overproduced, and followed the oxidation/reduction pathway that DsbA follows after biosynthesis . It is suggested that newly synthesized DsbA is rapidly oxidized by pre-existing DsbA, while oxidation of mature (functional) DsbA requires DsbB, whose roles might include that of antagonizing the actions of DsbA-reducing enzyme(s). FEBS Lett, 1995 May 1, 364(1), 1 - 4 Ribosomal frameshifting at the Gag-Pol junction in avian leukemia sarcoma virus forms a novel cleavage site; Arad G et al.; The Gag and Gag-Pol precursors of avian sarcoma leukemia virus (ASLV) are translated from viral genomic-size mRNA at a molar ratio of about 20:1 . Translation of Gag is terminated at the stop codon UAG located at the carboxyl-terminus of the viral protease (PR), whereas a ribosomal frameshift occurring at the carboxyl-terminus of Gag allows translation of the Gag-Pol precursor . To determine how PR is released from the Gag-Pol precursor, a single base (A or T) was inserted at the Gag-Pol junction in order to adjust the translation into a single reading frame . These mutations allow processing of the viral precursor when expressed in bacterial cells, but cause cessation of viral production after transfection of avian cells . The viral PR released from the large precursor is one amino acid longer than PR cleaved from the Gag polyprotein and is terminated by an Ile instead of a Leu residue. DNA Cell Biol, 1995 May, 14(5), 445 - 50 Effect of mercury substitution of DNA on its susceptibility to cleavage by restriction endonucleases; Banfalvi G et al.; Mercurated DNA was synthesized in vitro by substituting Hg-dCTP or Hg-dUMP for dCTP in one strand of M13mp8DNA in a DNA polymerase I reaction . Restriction enzymes, including Sma I, Pst I, Bam HI, Hind III, and Hinc II, were completely inactive when their recognition sites were fully substituted with Hg-dCMP, while Hg-dUMP containing DNA was hydroxlyzed to some extent . Under conditions favoring star activities, or when DNA was substituted with a low level of mercury-nucleotide, DNA was cleaved by restriction enzymes . Susceptibility to degrading and synthesizing enzymes and insensitivity to restriction endonucleases of fully mercurated DNA makes mercuration an attractive molecular "tag" for in vitro manipulation and selective isolation of Hg-DNA. Am J Pathol, 1995 May, 146(5), 1207 - 19 Antiproteases modulate bronchial epithelial cell responses to endotoxin; Koyama S et al.; Escherichia coli endotoxin (0.1 to 1000 micrograms/ml) stimulated the release of neutrophil chemotactic activity (P < 0.001) and induced bronchial epithelial cell (BEC) cytotoxicity assessed by lactate dehydrogenase release (P < 0.001) . Endotoxin (100 micrograms/ml) inhibited BEC accumulation (P < 0.001) . In the present study, we investigated the role of proteolytic activity of BECs per se in response to endotoxin . Several structurally and functionally different antiproteases, alpha 1 protease inhibitor, soybean trypsin inhibitor, two chloromethyl ketone derivatives (N-tosyl-L-lysine chloromethyl ketone and methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone), and L-658,758, a neutrophil elastase inhibitor, attenuated the release of neutrophil chemotactic activity and lactate dehydrogenase (P < 0.01) . alpha 1-Protease inhibitor and N-tosyl-L-lysine chloromethyl ketone attenuated the inhibition of BEC accumulation by endotoxin (P < 0.001) . The proteolytic enzyme activity measured by synthetic substrates revealed that endotoxin significantly augmented the serine proteolytic activity in the cell layers . Culture supernatant fluids and cell lysates of BECs in the presence of endotoxin solubilized 14C-labeled casein . These data suggest that responses of BECs to endotoxin may involve activation of cellular proteolytic activity. Mol Pharmacol, 1995 May, 47(5), 934 - 9 Catalytic properties of NAD(P)H:quinone acceptor oxidoreductase: study involving mouse, rat, human, and mouse-rat chimeric enzymes; Chen S et al.; NAD(P):quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes . In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 {5-(aziridin-1-yl)-2,4-dinitrobenzamide} and EO9 {5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione} . By using Escherichia coli-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous finding showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities . Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage . To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated . MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxy-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase . In addition, MR-P and RM-P were found to be inhibited by flavones with Ki values similar to those for rat and mouse quinone reductases, respectively . Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones. Genes Dev, 1995 May 1, 9(9), 1123 - 36 Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons; Signon L et al.; Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant . Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host . Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important . We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes . First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process . Such negative regulation is prominent only for elements present on multicopy plasmid replicons . This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression . Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element . However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements . On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons. EMBO J, 1995 May 1, 14(9), 2121 - 31 The influence of the 2-amino group of guanine on DNA conformation . Uranyl and DNase I probing of inosine/diaminopurine substituted DNA; Bailly C et al.; The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small . Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs . We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width . At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement . The latter exerts the dominant effect . The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack . Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions . Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction. EMBO J, 1995 May 1, 14(9), 2112 - 20 Xer site-specific recombination in vitro; Arciszewska LK et al.; Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo . In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed . Recombination of this substrate is efficient and requires both XerC and XerD . However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed . Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC . The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region . Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA. EMBO J, 1995 May 1, 14(9), 2106 - 11 The DNA binding domain of the initiator protein DnaA; Roth A et al.; The 94 C-terminal amino acids of the initiator protein DnaA of Escherichia coli are required and sufficient for specific binding to the cognate DNA binding site . The binding domain contains two potential amphipathic alpha-helices and a third alpha-helix . It represents a new DNA binding motif so far not found in other DNA binding proteins . Temperature-sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA211, abolish DNA binding . In the solid-phase DNA binding assay, applicable to other DNA binding proteins, fusions of domains of DnaA protein to beta-galactosidase are reacted with biotinylated anti-beta-galactosidase antibody . These are coupled to streptavidin-coated magnetic beads . The DNA binding domain is able to selectively remove the DNA target (oriC) from the liquid phase . Alternatively, the DNA binding domain is fused to a peptide containing a target sequence which is naturally biotinylated in vivo in E.coli . This fusion protein can be coupled directly to streptavidin-coated magnetic beads . Homologies between DnaA protein and transcription factors of the NtrC family are discussed. Aust N Z J Surg, 1995 May, 65(5), 365 - 6 Psoas abscess following ileo-anal pouch surgery; Sanea O et al.; An unusual case of psoas abscess complicating one of the earliest performed ileo-anal pouch anastomoses is reported . The authors have been unable to find a similar previously published case. Mutat Res, 1995 May, 336(3), 257 - 67 Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli; Tajiri T et al.; 8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is a potent mutagenic substrate for DNA synthesis . The accumulation of 8-oxo-dGTP in the nucleotide pool induces G:C-->T:A transversion as well as A:T-->C:G transversion, and Escherichia coli cells possess mechanisms for preventing such mutations . The mutT gene product specifically hydrolyzes 8-oxo-dGTP to the monophosphate form while the mutM and the mutY gene products function to correct mispairs caused by incorporation of 8-oxoguanine into DNA . From analyses of forward mutations induced in cells lacking 8-oxo-dGTPase (MutT protein) and/or repair enzymes that suppress mutations caused by 8-oxoguanine in DNA (MutM and MutY proteins), cooperative functions of these proteins in control of the spontaneous mutagenesis became evident . In mutator strains lacking MutT and/or MutM proteins, 8-oxoguanine of DNA increased to a concentration expected from the increased rate of mutation. Mol Biol Evol, 1995 May, 12(3), 451 - 8 On the use of nucleic acid sequences to infer early branchings in the tree of life; Yang Z et al.; Simplifying assumptions made in various tree reconstruction methods--notably rate constancy among nucleotide sites, homogeneity, and stationarity of the substitutional processes--are clearly violated when nucleotide sequences are used to infer distant relationships . Use of tree reconstruction methods based on such oversimplified assumptions can lead to misleading results, as pointed out by previous authors . In this paper, we made use of a (discretized) gamma distribution to account for variable rates of substitution among sites and built models that allowed for unequal base frequencies in different sequences . The models were nonhomogeneous Markov-process models, assuming different patterns of substitution in different parts of the tree . Data of the small-subunit rRNAs from four species were analyzed, where base frequencies were quite different among sequences and rates of substitution were highly variable at sites . Parameters in the models were estimated by maximum likelihood, and models were compared by the likelihood-ratio test . The nonhomogeneous models provided significantly better fit to the data than homogeneous models despite their involvement of many parameters . They also appeared to produce reasonable estimation of the phylogenetic tree; in particular, they seemed able to identify the root of the tree. Am Surg, 1995 May, 61(5), 427 - 30 Contaminated wounds: the effect of initial management on outcome; Smilanich RP et al.; Delayed primary closure has been advocated as the optimal method of management in the presence of wound contamination . The present study was performed to determine whether surgeons have accepted this standard . A total of 918 surgical wounds were evaluated and classified according to the level of contamination and type of wound management used . We found that 150 patients had a Class III or Class IV contaminated wound; however, only 21 per cent were treated with delayed primary closure . The 118 patients treated with primary closure and antibiotics had an aggregate wound infection rate of 27 per cent (Class III-29%; Class IV-24%) . Only one (3%) of the wounds managed by delayed primary closure developed an infection . If infection did not occur, there was no difference in the length of stay between patients managed with primary closure and delayed primary closure . However, there was a significantly longer length of stay in the primary closure group if infection occurred . Benefit risk analysis of the patients with contaminated wounds confirmed that in this clinical setting, delayed primary closure remains the optimal method of management for the wound. Am Surg, 1995 May, 61(5), 407 - 11 Pyogenic hepatic abscess: results of current management; Hashimoto L et al.; From 1980 to 1991, 56 cases of pyogenic liver abscess were treated at the Cleveland Clinic . The most frequently used treatment was percutaneous catheter drainage of the abscess under computed tomography (CT) guidance (39 patients), followed by CT-guided aspiration without catheter drainage (10 patients) . Six patients were initially treated by open operative drainage; another five were operated upon after CT guided drainage had failed . One patient with advanced pancreatic cancer was treated with antibiotics only . The overall mortality rate was 12.5% (7/56) . It is clear that the preferred method of treatment for pyogenic hepatic abscess is now CT guided catheter drainage . Operative drainage is reserved for patients who fail to respond to percutaneous drainage or in whom surgery is indicated for other purposes . Aspiration without catheter drainage is a modality that needs further evaluation to define its indications. J Gen Virol, 1995 May, 76 ( Pt 5), 1165 - 72 Mapping of neutralization epitopes on infectious pancreatic necrosis viruses; Frost P et al.; We have characterized and mapped variable and conserved neutralization epitopes of serogroup A strains of aquatic birnaviruses . Epitope mapping using monoclonal antibodies (MAbs) and Escherichia coli-expressed deletion fragments of VP2 of the N1 strain of infectious pancreatic necrosis virus (IPNV) demonstrated that two variable epitopes, H8 and B9, depend on the variable region between amino acid 204-330 . A conserved neutralization epitope, F2, was shown to depend on the same region as epitopes H8 and B9 but was additionally dependent on amino acids between 153-203 . The neutralization epitopes H8, B9 and F2 were also shown to overlap by a competitive binding assay . One conserved neutralization epitope, AS-1, was not exposed on any of the recombinant VP2 deletion fragments and was therefore not possible to map . However, the MAbs AS1 and F2 were partly competitive indicating that these epitopes are overlapping . All neutralization epitopes were independent of a conserved non-neutralization epitope, E4 . Our results demonstrate that the central third of VP2 contains several partly overlapping neutralization epitopes, both variable and conserved among serogroup A strains of IPNV. J Bacteriol, 1995 May, 177(9), 2592 - 3 icdB mutants of Escherichia coli; Helling RB; icdB mutations map at 16 min, lead to the specific loss of citrate synthase, and are complemented by a prophage containing a gltA+ gene . Thus, they are allelic with gltA. J Bacteriol, 1995 May, 177(9), 2564 - 6 High-level expression of soluble recombinant RNase P protein from Escherichia coli; Rivera-Leon R et al.; We have expressed recombinant RNase P protein from Escherichia coli in high yield . A hexahistidine sequence at the amino terminus allowed protein purification in a single step . Mass spectrometry confirmed the molecular weight of the purified protein and indicated a purity of > 95% . Protein functionality was demonstrated by reconstitution of active holoenzyme. J Bacteriol, 1995 May, 177(9), 2524 - 9 Nucleoside diphosphate kinase from Escherichia coli; Almaula N et al.; Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized . Gel filtration analysis of the purified enzyme indicated that it forms a tetramer . The enzyme was phosphorylated with {gamma-32P}ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated . Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated . A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated . All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated . In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists . These results are discussed in light of the recent report of N . J . MacDonald et al . on the autophosphorylation of human NDP kinase (J . Biol . Chem . 268:25780-25789, 1993). J Bacteriol, 1995 May, 177(9), 2505 - 12 Purification from Fusobacterium mortiferum ATCC 25557 of a 6-phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase that hydrolyzes maltose 6-phosphate and related phospho-alpha-D-glucosides; Thompson J et al.; 6-Phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (6-phospho-alpha-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557 . p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity . The O2-sensitive, metal-dependent phospho-alpha-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn2+ ion in chromatography buffers . Various 6-phosphoryl-O-alpha-linked glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalose 6-phosphate, and sucrose 6-phosphate, were hydrolyzed by the enzyme to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ratio . 6-Phospho-alpha-glucosidase (M(r) of approximately 49,000; pI of approximately 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maximum rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within the pH range 7.0 to 7.5 . The sequence of the first 32 amino acids of 6-phospho-alpha-glucosidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-beta-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli . Western blots involving highly specific polyclonal antibody against 6-phospho-alpha-glucosidase and spectrophotometric analyses with pNP alpha Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F . mortiferum . Synthesis of 6-phospho-alpha-glucosidase increased dramatically during growth of the organism on alpha-glucosides, such as maltose, alpha-methylglucoside, trehalose, turanose, and palatinose. J Bacteriol, 1995 May, 177(9), 2416 - 24 The NarX and NarQ sensor-transmitter proteins of Escherichia coli each require two conserved histidines for nitrate-dependent signal transduction to NarL; Cavicchioli R et al.; The NarX, NarQ, and NarL proteins of Escherichia coli constitute a two-component regulatory system that controls the expression of a number of anaerobic respiratory pathway genes in response to nitrate . NarX and NarQ are sensor-transmitter proteins that can independently detect the presence of nitrate in the cell environment and transmit this signal to the response regulator, NarL . Upon activation, NarL binds DNA and modulates the expression of its target genes by the repression or activation of transcription . NarX and NarQ each contain a conserved histidine residue that corresponds to the site of autophosphorylation of other sensor-transmitter proteins . They also contain a second conserved histidine residue that is present in the NarX, NarQ, UhpB, DegS, and ComP subfamily of sensor-transmitter proteins . The second histidine is located near a universally conserved asparagine residue, the role of which in signal transduction is unknown . To investigate the role of these conserved amino acids in the NarX and NarQ proteins, we mutated the narX and narQ genes by site-directed mutagenesis . In vivo, each mutation severely impaired NarL-dependent activation or repression of reporter gene expression in response to nitrate . The in vivo data suggest that the environmental signal nitrate controls both the kinase and phosphatase activities of the two sensor-transmitter proteins . The altered NarX and NarQ proteins were purified and shown to be defective in their ability to autophosphorylate in the presence of {gamma-32P}ATP . The NarX and NarQ proteins with amino acid substitutions at the first conserved histidine position were also unable to dephosphorylate NarL-phosphate in vitro . In contrast, the proteins containing amino acid substitutions at the second conserved histidine or at the conserved asparagine residue retained NarL-phosphate dephosphorylation activity . The conserved histidine and asparagine residues are essential for NarX and NarQ function, and this suggests that other two-component sensor-transmitter proteins may function in a similar fashion. J Bacteriol, 1995 May, 177(9), 2315 - 20 Sequences determining the cytoplasmic localization of a chemoreceptor domain; Seligman L et al.; The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions . We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions . The principal findings were as follows . (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence . (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion . (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence . The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure. J Bacteriol, 1995 May, 177(9), 2305 - 14 Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase; Touati D et al.; The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity . Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants . We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions . fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis . Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity . Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection . Functional recombinational repair was necessary for protection, but SOS induction was not involved . Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants . Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions . Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN . Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity . Furthermore, overexpression of SOD partially protected fur recA mutants . We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope. Infect Immun, 1995 May, 63(5), 2062 - 9 A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice; Wilson ME et al.; Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis . Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host . To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library . One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L . chagasi infection . The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene . Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa . Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L . chagasi infection . Immunization with Lcr1 partially protected BALB/c mice against challenge with L . chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens. Infect Immun, 1995 May, 63(5), 1927 - 32 Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin; L'Hote C et al.; Known glycoproteins were used to determine the differences occurring in the binding specificities of the three variants of the K88 lectin in an approach essentially based on lectin blotting . During the screening, it was demonstrated that each variant of the K88 lectin biotinylated via its amino groups (NbioK88) exhibited a characteristic binding to the three chains of porcine fibrinogen . NbioK88ab weakly bound to A alpha chains, NbioK88ac bound to B beta and gamma chains, and NbioK88ad bound only to the gamma chain . To validate this model, the oligosaccharide moieties of porcine fibrinogen were analyzed with glycosidases and by lectin blotting and sugar composition . Both the B beta chain and gamma chain carry biantennary N-glycans of the N-acetyllactosamine type that are not recognized by K88 lectins . A alpha chains are substituted by sialylated T antigen . O-glycans were also detected on B beta and gamma chains of porcine fibrinogen and contribute to the recognition of these chains by K88ac and K88ad fimbriae. Infect Immun, 1995 May, 63(5), 1617 - 23 Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity; Dickinson BL et al.; The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin . A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity . These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations . In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants . Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity . Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit . This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2 . This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity . Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone . Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice. Obstet Gynecol, 1995 May, 85(5 Pt 1), 651 - 5 Ability of normal vaginal flora to produce detectable phosphatidylglycerol in amniotic fluid in vitro; Lambers DS et al.; OBJECTIVE: To determine if bacteria are capable of producing phosphatidylglycerol in amniotic fluid (AF) and the number of colony forming units (CFU) of bacteria necessary to produce this result . METHODS: Eleven species of bacteria and one species of yeast, common to the female genital tract and implicated in chorioamnionitis, were selected . Amniotic fluid was collected from 21 women and inoculated with 10(8) CFU/mL of each isolate . Aliquots of AF were tested at 0, 4, 12, and 24 hours for colony counts and the presence of phosphatidylglycerol by thin-layer chromatography . RESULTS: The mean gestational age (+/- standard deviation) of the 21 study patients was 33 weeks and 1 day (+/- 4 weeks) . Among the 12 species studied, Escherichia coli produced phosphatidylglycerol, at a concentration of 1.75 x 10(8) CFU/mL, beginning 12 hours after incubation . CONCLUSION: Escherichia coli is capable of producing phosphatidylglycerol in AF in vitro and is present in the vagina in 24% of normal pregnant patients . Our findings question the validity of using vaginal pool AF specimens for phosphatidylglycerol determination . Therefore, we recommend that patients presenting with preterm premature rupture of membranes be evaluated by amniocentesis to determine fetal lung maturity with phosphatidylglycerol and the lecithin-sphingomyelin ratio. Fertil Steril, 1995 May, 63(5), 1077 - 82 Inability of human sperm to change their orientation in response to external chemical stimuli; Makler A et al.; OBJECTIVE: To investigate whether human sperm can respond to external chemical stimuli by orienting themselves toward chemoattractants or withdrawing from hostile environments . DESIGN: Controlled laboratory assays . SETTING: Normal human sperm and two other flagellated micro-organisms were exposed to various potential chemoattractant or chemorepellent substances . INTERVENTION: Human sperm, Euglena viridis, and Escherichia coli were exposed to various substances from the female reproductive system or to various toxic agents by placing them within tiny wells in a sealed minichamber . They were followed by microscopic observation and by intermittent photography . MAIN OUTCOME MEASURE: Images of photographed micro-organisms were analyzed for signs of attraction to or withdrawal from the test substances . RESULTS: Human sperm neither changed their orientation toward nor accumulated next to the well that contained cervical mucus, uterine cavity and follicular fluid, cumulus cells, or intact nonfertilized human eggs . Contrary to other micro-organisms that turned away from sources of hydrochloric acid, sodium hydroxide, ethanol, or glutaraldehyde, human sperm did not withdraw from these solutions . They swam along the ascending chemical gradient, facing ahead while becoming immobilized by these agents . CONCLUSION: It may be implied from the observation that they did not turn away from a hostile environment when expected to do so or turn toward chemoattractants that human sperm do not respond to external chemical stimuli and, most probably, chemotaxis between human sperm and ova in nature does not exist. J Virol, 1995 May, 69(5), 2858 - 62 Localization of antigenic sites of the S glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement; Corapi WV et al.; The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement . To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation . Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs) . A region spanning residues 509 to 673 reacted with most MAbs tested . Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs . To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced . Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus . Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus . Site A2 contains residues 643, 649, and 656 . Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing . The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement. J Virol, 1995 May, 69(5), 2745 - 50 Oligomerization of the hydrophobic heptad repeat of gp41; Bernstein HB et al.; The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix . To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T . The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41 . Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer . Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A . Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography . A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization . Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization . The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion . A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed. Vopr Virusol, 1995 May-Jun, 40(3), 103 - 5 {Stability and virus-inhibiting effect of recombinant receptor toxins based on the human CD4 receptor and diphtheria toxin}; Sidorov AV et al.; The causes of differences in the stability of CD4 receptor and diphtherial toxin based recombinant receptorotoxins synthesized in E.coli and differing by their primary structure were under study . Insertion of a CD4 receptor fragment, responsible for HIV binding, on the N terminus of hybrid protein was found to lead to a drastic reduction of the stability of the hybrid polypeptide in E.coli and to impossibility of obtaining full-size protein product . In vitro experiments on a model of human T lymphocyte culture demonstrated that recombinant receptorotoxin stably expressed in E.coli inhibited the cytopathic effect of type 1 HIV and the effect of syncytium formation induced by the virus. Proteins, 1995 May, 22(1), 67 - 72 Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: an application of reverse screening; Stura EA et al.; X-ray quality crystals of class I-deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate . The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate . The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 183.1 A, b = 61.4 A, c = 49.3 A and a = 179.2 A, b = 60.5, A, c = 49.1 A, respectively . Two molecules in the asymmetric unit are related by a noncrystallographic 2-fold axis . The crystals are stable in the X-ray beam and diffract to at least 2.6 A . A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions. Proteins, 1995 May, 22(1), 55 - 7 Purification and crystallization of a schistosomal glutathione S-transferase; McTigue MA et al.; The 26-kDa glutathione S-transferase from Schistosoma japonica (Sj26), a potential antischistosomal vaccine antigen, has been crystallized in an unligated form . Sj26 was recombinantly produced in E . coli without using a glutathione affinity column to facilitate preparation of unligated enzyme . The recombinant protein contains all 218 residues of Sj26 and an additional 13 residues linked to the C-terminus . Crystals of recombinant Sj26 were obtained by the vapor diffusion method using ammonium sulfate as the precipitant at pH 5.6 . The crystals belong to the hexagonal space group P6(3)22 with unit cell dimensions a = b = 125.2 A and c = 72.0 A and contain one Sj26 monomer per asymmetric unit . A complete native diffraction data set has been obtained to 2.4 A resolution. Proteins, 1995 May, 22(1), 41 - 4 NMR study of the reconstitution of the beta-sheet of thioredoxin by fragment complementation; Tasayco ML et al.; The study of complementary protein fragments is thought to be generally useful to identify early folding intermediates . A prerequisite for these studies is the reconstitution of the native-like structure by fragment complementation . Structural analysis of the complementation of the domain-sized proteolytic fragments of E . coli thioredoxin, using a combination of H-exchange and 2D NMR experiments as a fingerprint technique, provide evidence for the extensive reconstitution of a native beta-sheet, with local conformational adjustments near the cleavage site . Remarkably, the antiparallel beta-strand between the fragments shows a native-like protection of the amide protons to solvent exchange . Our results indicate that these fragments can be useful to study the early events in the still little understood formation of beta-sheets. J Neurosci Res, 1995 May 1, 41(1), 15 - 26 Bacterially expressed F1-20/AP-3 assembles clathrin into cages with a narrow size distribution: implications for the regulation of quantal size during neurotransmission; Ye W et al.; F1-20/AP-3 is a synapse-specific phosphoprotein . In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages . We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain . This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3 . It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3 . Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution . This may be important for the regulation of quantal size during neurotransmission . We also express the 33 kD NH2-terminus of F1-20/AP-3 in E . coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages . It has been suggested that the 33 kD NH2-terminus of F1-20/AP-3 constitutes a clathrin binding domain . We find that the bacterially expressed 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly . The finding that the 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies . The finding that the 33 kD NH2-terminus of F1-20/AP-3 fails to bind to clathrin cages is novel and potentially important . It is clear from these experiments that the 33 kD NH2-terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated. J Chem Neuroanat, 1995 May, 8(4), 267 - 82 Critical re-examination of the distribution of aromatase-immunoreactive cells in the quail forebrain using antibodies raised against human placental aromatase and against the recombinant quail, mouse or human enzyme; Foidart A et al.; Mouse and quail aromatase cDNAs were isolated from libraries of mouse ovary and quail brain by using a human aromatase cDNA fragment (hA-24) as a probe . These three cDNAs were inserted into plasmid vectors and expressed in Escherichia coli . Antisera against these purified recombinant proteins were raised in rabbit and purified by ammonium sulfate fractionation and affinity chromatography . The three antibodies directed against recombinant human, mouse and quail proteins were used to visualize aromatase-immunoreactive cells in the quail brain . They were compared with the antibody raised against human placental aromatase used in previous experiments and with another antibody recently developed by similar methods . The signal obtained with all antibodies was completely abolished by preadsorption with the homologous recombinant antigens and the signal produced by the two antibodies raised against placental aromatase was similarly abolished by a preadsorption with recombinant quail aromatase . The antibodies raised against recombinant proteins identified the major groups of aromatase cells previously described in the quail brain . The antibodies directed against the mouse and quail antigen identified more positive cells and stained them more densely than the antibodies raised against human recombinant antigen or purified placental aromatase . The new cell groups identified by the antibody raised against quail recombinant aromatase were located in an area ventral to the fasciculus prosencephali lateralis, the nucleus accumbens, the paleostriatum ventrale, the nucleus taeniae, the area around the nucleus ovoidalis, the caudal tuber and the mesencephalic central gray . A critical re-examination of the distribution and nomenclature of the aromatase-positive cells is proposed based on these new findings. Anal Biochem, 1995 May 1, 227(1), 168 - 75 Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin; Chen LL et al.; The human immunodeficiency virus-1 Tat protein can efficiently enter cells when added exogenously in tissue culture . Using the transactivation activity of Tat as a measure of intracellular delivery, we found that the addition of hydrophobic groups to Tat potentiated its uptake . Biotin was the most promising of the reagents tested and we characterized this effect in more detail . When coupled through a cysteine thiol, the addition of a single biotin to Tat increased activity by about six-fold . Increased activity was only seen with reducible biotin analogs, as modification with noncleavable analogs is known to block Tat transactivation activity . Biotin had no effect on Tat uptake when mixed with Tat without cross-linking . Recently, Tat was used as a carrier to direct the uptake of heterologous proteins into cells . We have used RNase as a model system for studying Tat-mediated uptake and found that biotin also increased the delivery of a Tat37-58-RNase conjugate . The increased uptake of Tat and Tat conjugates by addition of hydrophobic groups may significantly enhance the usefulness of Tat as a delivery vehicle, and the approach may be applicable to other systems. Anal Biochem, 1995 May 1, 227(1), 148 - 55 A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate; Holskin BP et al.; The 28-kDa protease from human cytomegalovirus (hCMV) has been successfully cloned, expressed, and purified to homogeneity . An internally quenched fluorescent substrate (4-4'-dimethylaminophenazo)benzoyl-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser -Arg-Leu-Ala-5-{(2'-aminoethyl)-amino}-naphthalene-1-sulfonic acid; DABCYL-CMV-EDANS) based on the maturational cleavage site (M-site) junction was synthesized in an effort to develop a fluorescence-based assay . This substrate is cleaved specifically between the Ala-Ser peptide bond thereby liberating the C-terminal peptide-EDANS fragment from the proximity quenching effect of the DABCYL group . This results in greater than a 10-fold increase in fluorescence that is observed at the EDANS emission wavelength of 495 nm . Human CMV protease efficiently cleaved this synthetic substrate permitting continuous assay at peptide concentrations lower than 10 microM . At substrate concentrations greater than 10 microM, linearity was lost due to the "inner filter effect." This represents the first fluorescence-based assay for any of the herpes virus proteases . Additionally, a peptidyl inhibitor, H-Arg-Gly-Val-Val-Asn-Ala-psi{CH2NH}-Ser-Ser-Arg-Leu-Ala-OH, was prepared . This inhibitor was also based on the same M-site cleavage junction with a nonhydrolyzable reduced peptide bond incorporated at the cleavage site . Using the fluorescence-based assay, this reduced peptide bond analog was observed to be an inhibitor of hCMV protease with an inhibition constant of > 500 microM. Clin Diagn Lab Immunol, 1995 May, 2(3), 263 - 7 Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes; Hemmen F et al.; A thus far unknown gene encoding a Brucella abortus protein has been isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep . Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa (calculated molecular mass) . The recombinant B . abortus protein, expressed in Escherichia coli, and the corresponding Brucella melitensis protein migrated at the same apparent molecular masses as shown by Western blotting (immunoblotting) . Among a series of serum samples from B . melitensis- or B . abortus-infected sheep and cows, 51 and 39%, respectively, showed a signal at 17 kDa on Western blot analysis of total protein extract from Brucella bacteria . These figures amount to 70 and 61% for sheep and cattle, respectively, in a competitive enzyme-linked immunosorbent assay with a specific monoclonal antibody . These data indicate that the 17-kDa antigen may be useful for serological diagnosis of Brucella infection. Biochem Mol Biol Int, 1995 May, 36(1), 77 - 85 The interaction of glutamate decarboxylase from Escherichia coli with substrate analogues modified at C-3 and C-4; Khristoforov RR et al.; The interaction of glutamate decarboxylase with the aspartate analogues 3-arsonoalanine and 3-phosphonoalanine, with the glutamate analogues 2-amino-4-arsonobutyric acid and 2-amino-4-phosphonobutyric acid, and with 4-(methylthio)-L-glutamic acid, both as a mixture of diastereoisomers and as the (2S,4R)-form, was studied . All these analogues were poor substrates for the enzyme and only weak inhibitors . Their decarboxylation was accompanied by transamination of the enzyme-bound pyridoxal phosphate (PLP) to pyridoxamine phosphate (PMP), thus inactivating the decarboxylase . With arsonoalanine only part of the PLP was converted into PMP. Biochem Mol Biol Int, 1995 May, 36(1), 209 - 18 Cloning and nucleotide sequence of the gene cluster encoding ribosomal proteins S12 and S7 from Mycobacterium bovis BCG; Iwanaga S et al.; A pair of oligonucleotide primers, based on the experimentally determined amino terminal sequence of Mycobacterium bovis BCG ribosomal protein S12 (MboS12) and a highly conserved sequence found in all mycobacterial ribosomal S12 proteins, was used for polymerase chain reaction (PCR) with M . bovis genomic DNA as template . The nucleotide sequence of the 338 bp fragment thus produced confirmed its origin in MboS12 gene . A 5.0 kb EcoRI fragment of M . bovis DNA hybridizing to this fragment was cloned . Its sequencing analysis revealed the presence of two open reading frames in the same strand . Their amino acid sequences deduced from DNA sequence showed high homology with Escherichia coli ribosomal proteins S12 and S7 . However, the intercistronic region between S12 and S7 genes, which plays an important role for autoregulation for the str operon in E . coli, is completely absent in M . bovis. Biochem Mol Biol Int, 1995 May, 36(1), 145 - 54 Nucleoids of pea chloroplasts: microscopic and chemical characterization . Occurrence of histone-like proteins; Yurina NP et al.; The chloroplast genome is highly condensed and packed into discrete structures called "nucleoids" . Each pea chloroplast contains 16-20 spherical nucleoids randomly located in the matrix of organelle . Nucleoids are shown to contain DNA, RNA and proteins (1:0.62:2.3) . Approximately 30 polypeptides (mol . masses 94 to 12 kD) have been found in nucleoids . Chloroplast DNA is associated with acidic as well as basic proteins, two of which are electrophoretically similar to histones H2A+H2B and H3 of pea cell nuclei . Amino acid composition of these proteins demonstrates high similarity with "HU" proteins of E . coli. Protein Sci, 1995 May, 4(5), 983 - 93 Amide exchange rates in Escherichia coli acyl carrier protein: correlation with protein structure and dynamics; Andrec M et al.; The acyl carrier protein (ACP) of Escherichia coli is a 77-amino acid, highly negatively charged three-helix protein that plays a central role in fatty acid biosynthesis . Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary structural elements . The stability of these elements is now examined by monitoring amide exchange in apo-ACP using NMR-based methods . Because ACP exhibits many rapid exchange rates, application of traditional isotope exchange methods is difficult . In one approach, heteronuclear correlation experiments with pulsed field-gradient coherence selection have reduced the time needed to collect two-dimensional 1H-15N correlation spectra to the point where measurement of exchange of amide protons for deuterium on the timescale of minutes can be made . In another approach, water proton selective inversion-exchange experiments were performed to estimate the exchange rates of protons exchanging on timescales of less than a second . Backbone amide protons in the region of helix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesting a dynamic disruption of the second helix . Highly protected amides occur on faces of the helices that may pack into a hydrophobic core present in a partially disrupted state. Protein Sci, 1995 May, 4(5), 925 - 35 On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N; Gondry M et al.; Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups . At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN . Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis . Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column . The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms . Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop . In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond . Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site . Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E . coli; as a result, the synchrony between folding and flavin insertion is lost . Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme. Protein Sci, 1995 May, 4(5), 1007 - 9 Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans; Roach PL et al.; Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system . The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant . The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A . The asymmetric unit contains one dimer, and the solvent content of the crystals is 60% . The crystals are radiation sensitive. Biokhimiia, 1995 May, 60(5), 678 - 93 {Changes in biogenesis and secretion of periplasmic alkaline phosphatase, coded by the phoA gene included in the Escherichia coli DH1 plasmid}; Badiakina AO et al.; Augmented synthesis of periplasmic alkaline phosphatase from E . coli DH1 coded by the phoA gene within the composition of plasmid pHI-7 results in alterations in the enzyme biogenesis, such as accumulation of intermediate enzyme forms corresponding to various stages of its posttranslational modification, and alternative localization . The cell cytoplasm was found to contain a large proportion of alkaline phosphatase precursors in the form of insoluble aggregates . The mature enzyme was detected in both the periplasm and the cultural fluid in the form three methazymes with an increased (as compared with the original strain) content of methazymes I and II . The cell envelope of E . coli DHI transformed by plasmid pHI-7 with the phoA gene differed from that of the original strain by an increased acid phospholipid (cardiolipin/phosphatidylglycerol) ratio as well as by increased distribution density of intramembrane particles on the surface of the cytoplasmic membrane facing the cytoplasm . Changes in the biogenesis of the enzyme during its augmented synthesis are due to disturbances in the equilibrium between the synthesis of polypeptide chains, on the one hand, and their translocation and processing, on the other, apparently as a result of restricted secretion sites and lack of inhibition of translation of secreted proteins in bacteria. J Struct Biol, 1995 May-Jun, 114(3), 167 - 76 A structural model for the Escherichia coli DnaB helicase based on electron microscopy data; San Martin MC et al.; The DnaB protein is the major replicative DNA helicase in Escherichia coli . It hydrolyzes ATP to promote its translocation in the 5' to 3' direction on single-stranded DNA templates, facilitating the separation of strands of duplex DNA in its path . This places it on the lagging strands at replication forks during chromosomal DNA replication . Electron microscopic images of negatively stained DnaB protein have been studied and processed to produce a three-dimensional reconstruction of the protein oligomer at 2.7 nm resolution . While it is known that the native protein is a complex of six identical 52-kDa subunits, the specimen shows threefold rather than sixfold symmetry, with three outer stain-excluding regions surrounding another six, more massive, lobules . There is a channel through the particle that appears fully open on both sides . Based on these results, a structural model for the oligomer is presented, and functional implications are considered. Bioorg Khim, 1995 May, 21(5), 359 - 64 {Chemico-enzymatic synthesis, cloning and expression of a gene for an analog of human anaphylatoxin C5a}; Babkina IN et al.; Chemical-enzymatic synthesis and cloning of a gene for a human anaphylatoxin C5a analog were carried out . Recombinant plasmid pRC5a providing the expression of the synthetic gene in the Escherichia coli cells was obtained . The biological activity of the expression product was demonstrated by the chemotaxis activity test and by the release of myeloperoxidases from rat peritoneal cells that was induced by bacterial cell lysates containing the recombinant protein. Am J Vet Res, 1995 May, 56(5), 555 - 61 Cloning and expression of an antigenic domain of glycoprotein gE of pseudorabies virus in Escherichia coli and its use as antigen in diagnostic assays; Ro LH et al.; Use of a combination of an effective gE gene-deleted pseudorabies virus (PRV) vaccine with a companion diagnostic kit for PRV glycoprotein gE has proven successful in several pseudorabies-eradication programs . To produce a large quantity of functional gE protein for development of a PRV-gE diagnostic kit, an Escherichia coli expression system containing the distal region of the PRV-gE gene of a PRV strain CF was constructed . The expressed protein contained 134 amino acids of gE protein (amino acids 77-210) fused to a 19-amino acids tag containing 6 histidine residues . After induction, a truncated PRV-gE polypeptide of 18-kd was expressed to about 20% of the total E coli proteins . Results of immunoblot analysis indicated that this E coli-produced PRV-gE protein reacted specifically with serum from PRV-hyperimmunized pigs and from field PRV-infected pigs, but not with serum samples from specific-pathogen-free pigs or pigs inoculated with gE-deleted PRV vaccine . These data indicate that, although the recombinant gE protein is produced in E coli, it still retains the antigenicity of the viral gE glycoprotein . Comparison between the recombinant gE protein, using immunoblot analysis with a commercial gE ELISA containing natural PRV-gE protein, revealed comparable test performance . This finding indicated that recombinant gE protein produced by E coli can be used for development of a companion serologic assay for a PRV-gE gene-deleted vaccine. Virus Res, 1995 May, 36(2-3), 299 - 309 Complete nucleotide sequence of a cytopathic hepatitis A virus strain isolated in Italy; Beneduce F et al.; The molecular basis of the cytopathic effect induced in cell culture by some hepatitis A virus (HAV) strains and variants has not been determined . In order to assess the molecular mechanism(s) underlying this particular phenotype the genome of an Italian cytopathic isolate (strain FG) was sequenced from cDNAs obtained by RT-PCR . Sequence analysis revealed the presence of mutations common to either adapted or cytopathic variants of HAV . In particular, amino acid deletions in proteins VP1 and 3A were detected . Expression of protein 3A in E . coli showed that the N-terminal deletion renders this protein toxic to bacteria. Virus Res, 1995 May, 36(2-3), 269 - 78 Identification and characterization of a Marek's disease virus gene encoding DNA polymerase; Sui D et al.; DNA sequence analysis revealed a gene encoding the Marek's disease virus (MDV) DNA polymerase (pol) within the BamHI-E fragment of the long unique region of the virus genome . Identification is based on an extensive amino acid homology between the MDV open reading frame and the DNA pol (UL30) of the herpes simplex virus . We describe here a 3540-base-pair fragment of the MDV DNA encoding 1180 amino acids with a M(r) of 133,920 daltons as the viral DNA pol gene, with the analysis of transcription and translation . In Northern blot hybridization, a transcript of 4.0 kb was detected in GA-MDV-infected duck embryo fibroblast (DEF) cells . An antiserum was generated in rabbit using TryE-pol fusion protein expressed in E . coli . This antiserum specifically immunoprecipitated a protein of 135 kD from lysates of MDV-GA-infected DEF cells . MDV DNA pol showed extensive homology to five distantly related herpesviruses: equine herpesvirus (EHV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV) . Comparison of amino acid sequences among the herpesviruses highlights nine highly conserved regions . Three of the conserved regions are in the N-terminus in the 3'-5' exonuclease domains and the remaining six are in the C-terminus in the catalytic domains . The predicted structural characters are in good agreement with the published data on a number of human herpesvirus DNA pol . The identification of MDV DNA pol gene may lead to a better understanding of MDV replication. Shock, 1995 May, 3(5), 369 - 75 Maintenance of dynamic microvascular function and structure in a rat model of endotoxic shock by blockade of the interleukin-1 receptor; Norman JG et al.; The microvascular and macrovascular effects of IL-1 receptor antagonist (IL-1ra) were examined in rat cremaster muscle A1, A2, and A3 arterioles by videomicroscopy to better define its protective effects during endotoxemia . Mean arterial pressure (MAP), arteriolar diameters, and responses to norepinephrine (NE) and acetylcholine (ACh) were examined hourly after the administration of Escherichia coli endotoxin (6 mg/kg intravenously) . Animals received saline (Control) or IL-1ra (.2 mg/kg/min intravenously) beginning 1 h prior to endotoxin . Serum tumor necrosis factor-alpha (TNF-alpha) and nitrate/nitrite (NO) were determined terminally . Aortic endothelium was examined by electron microscopy (EM) . All Control animals, but no IL-1ra animals, died within 6 h (p < .01).IL-1ra significantly attenuated endotoxin-induced vasoconstriction of A1 and A2 arterioles (p < .01), while MAP and NE threshold remained at baseline (p < .01 vs . Control) . Serum TNF and NO were elevated following endotoxin (p < .001), but only TNF was decreased (p < .005) in animals receiving IL-1ra . Aortic endothelium was damaged in all Control animals but was spared with IL-1 antagonism . IL-1ra increases survival during endotoxic shock and attenuates production of TNF but not NO . IL-1ra maintains MAP, arteriolar diameters, reactivity of arterioles to NE and ACh, and the integrity of the aortic endothelium. Nat Genet, 1995 May, 10(1), 111 - 3 A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase; Frosst P et al.; Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease . Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism . 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine . Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease . We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes . The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR . Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels . This mutation in MTHFR may represent an important genetic risk factor in vascular disease. J Chir (Paris), 1995 May, 132(5), 264 - 6 {Abscess of the rectovaginal wall in women . Apropos of a case}; Andze G et al.; Recto-vaginal septum abscess is a rare entity in women . The authors report a rare clinical observation of a recto-vaginal septum abscess in a 40 year old woman, hospitalized and treated in the Surgical Unit of the Yaounde General Hospital . Medical literature was reviewed and surgical indications discussed. Prikl Biokhim Mikrobiol, 1995 May-Jun, 31(3), 308 - 10 {Use of barium salts of pentoso-1-phosphoric acids in the enzymatic synthesis of ribothymidine and bromvinyldeoxyuridine}; Bokut' SB et al.; The use of barium salts of ribose 1-phosphate and 2-deoxyribose-1-phosphate as the donors of the carbohydrate part of the required nucleosides in the reaction of enzymatic glycosylation of heterocyclic bases was studied . It was shown that application of indicated substrates in the synthesis of modified nucleosides-ribothymidine and bromovinyldeoxyuridine, catalyzed by the nucleoside phosphorylases from Escherichia coli BM-11, leads to the 96-98% yield of required products even at an equimolar ratio of original pentose 1-phosphates and heterocyclic bases in the reaction mixtures. J Virol Methods, 1995 May, 53(1), 75 - 90 Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli; Kelsall SR et al.; Major capsid proteins (MCPs) of various papillomaviruses have recently been expressed in heterologous cells as soluble and functional polypeptides . The host cells for producing these proteins have so far been of eukaryotic origin; however, E . coli has potential utility a host, with advantages over eukaryotic cells such as relatively simple culture requirements and greater ease of mutation of expressed sequences . We studied the expression by E . coli of the MCP of human papillomavirus type 16 (HPV16) using the gene derived from the 'prototype' HPV16 genome . Using expression vector pTrc99A, the protein was produced in full-length unfused form at levels of 3-4% of cell protein . Soluble polypeptide was detected, albeit at low levels . The level of solubility was not increased by growing cells at low temperature and slowing the rate of protein synthesis . The soluble protein was degraded at its carboxy terminus by an outer membrane protease of E . coli, OmpT, giving rise to two slightly shortened protein species of 52K and 56K in addition to the full-length 57K polypeptide . Since the MCP of prototype HPV16 is known to be prone to excessive aggregation compared with other papillomaviral MCPs, the recovery of soluble polypeptide indicates that E . coli is worth consideration as an alternative host to eukaryotes for producing these proteins. J Virol Methods, 1995 May, 53(1), 63 - 73 Assay of HIV-1 protease activity by use of crude preparations of enzyme and biotinylated substrate; Yu SL et al.; An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV) . The protease and its substrate, the gag precursor, were generated separately in Escherichia coli . The HIV-1 protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system . Pin17/24 consists of an N-terminal peptide, which is biotinylated in E . coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments . Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody . When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased . The binding was specific and the reaction was inhibited by a known HIV protease inhibitor . Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay . The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors. Genetics, 1995 May, 140(1), 411 - 4 Inversions with deletions and duplications; Gordon AJ et al.; Complex mutational events, including de novo inversion with deletion and duplication of sequence, have been observed but are difficult to model . We propose that nascent leading-strand misalignment upon the lagging-strand template during DNA replication can result in the inversion of sequence . The positioning of this misalignment and of the realignment of the leading strand back into the leading-strand template will determine if the inversion is accompanied by deletion and duplication of sequence . We suggest that such strand misalignment-realignment events may occur at the replication fork during concurrent DNA replication. Genetics, 1995 May, 140(1), 27 - 45 Suppression of recJ exonuclease mutants of Escherichia coli by alterations in DNA helicases II (uvrD) and IV (helD); Lovett ST et al.; The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination . We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated . Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes . A recessive amber mutation, "uvrD517am," at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially . The uvrD517am mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517am as a suppressor . However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517am derivative was greatly elevated, equal to that in a uvrD insertion mutant . The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD . The identity of the third cosuppressor "srjD" is not known . Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences . To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ. Synapse, 1995 May, 20(1), 75 - 84 Immunochemical localization of calcium/calmodulin-dependent protein kinase I; Picciotto MR et al.; Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was originally identified in rat brain based on its ability to phosphorylate site 1 of synapsin I . Recently a cDNA for the rat brain enzyme has been cloned and the primary structure elucidated {Picciotto et al . (1993), J . Biol . Chem., 268:26512-26521} . The rat cDNA encoded a protein of 374 amino acids with a calculated M(r) of 41,636 . Antibodies have now been raised against the recombinant kinase expressed in E . coli as a glutathione-S-transferase fusion protein . Immunoblot analysis of rat cortex lysates revealed two major immunoreactive bands of approximately M(r) 38,000 and 42,000 . Minor immunoreactive species of slightly lower M(r) were also detected . Two distinct CaM kinase I activities were partially purified from rat brain and shown to correspond to the two major immunoreactive species . A variety of immunoreactive species of M(r) 35-43,000 were detected in "brain" tissue from cow, zebra finch, goldfish, Xenopus, lamprey, and Drosophila . In rat brain, immunocytochemistry revealed strong staining in cortex, hippocampus, amygdala, hypothalamus, brain stem, and choroid plexus . The labelling was mainly observed in neuropil but clusters of intensely labelled neuronal cell bodies were also detected all along the neuraxis . Neuronal nuclei and glial cells did not appear to be stained . Subcellular fractionation studies confirmed the cytosolic localization of the kinase in the brain . In various rat non-neuronal tissues and in a number of cell lines, immunoreactive species of approximately M(r) 38,000 and approximately 42,000 were detected at lower levels than that detected in brain . The M(r) 38,000 and 42,000 species were also found in different ratios and at different levels in the non-neuronal tissues . These results support a role for CaM kinase I in the regulation of multiple neuronal processes . Furthermore, the widespread cell and tissue distribution suggests that CaM kinase I may function as a ubiquitous multi-functional protein kinase . Finally, the multiple immunoreactive species may represent isoforms of CaM kinase I. Immunol Invest, 1995 May, 24(4), 643 - 52 Suppression of immune parameters in animal models of morphine dependence; Portoles JM et al.; Implantation of pellets containing 75 mg of morphine induced short term (4 day) morphine dependence and markedly reduced total number of spleen cells of BALB/c mice, without affecting total body or liver weight . Polyclonal responses induced by anti-CD3 antibodies, Concanavalin A or Escherichia coli lipopolysaccharide in the remaining spleen cells of morphine-treated mice were also inhibited . Cytofluorimetric analysis indicated that the proportion of major functional lymphocyte populations (Ig+, CD3+, CD4+ and CD8+ lymphocytes) were not significantly changed in the spleen from morphine-dependent mice . Furthermore, expression levels of surface Ig, CD3, CD4, and CD8, were similar in spleen cells from control or morphine-treated mice . So, morphine dependence in BALB/c mice under these controlled conditions results in a specific defect in lymphoid cell number and function, with no incidence on body weight or particular lymphocyte subsets. Genetika, 1995 May, 31(5), 617 - 21 {2,4,6-trinitrotoluene and 2,4-diamino-6-nitrotoluene: the absence of recA-dependent mutagenesis?}; Karamova NS et al.; The genotoxicity of 2,4,6-trinitrotoluene (2,4,6-TNT) and its amino derivative, 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT), was studied using the Escherichia coli tester strain PQ37 in the SOS chromotest . The compound 2,4,6-TNT, without metabolic activation, virtually failed to induce an SOS effect in cells of the tester bacteria . Consequently, mutagenic activity of 2,4,6-TNT, which was shown earlier in the Ames test, does not depend on SOS mutagenesis . It was demonstrated that metabolic activation with the microsomal S9 human placenta fraction results in a threefold increase in the induction factor of the SOS effect caused by 2,4,6-TNT . The absence of the SOS-inducing activity of 2,4-DA-6-NT, regardless of the presence of a microsomal activating mixture, is shown . Thus, 2,4-DA-6-NT does not belong to metabolites of 2,4,6-TNT, responsible for the genotoxicity of this compound. Abdom Imaging, 1995 May-Jun, 20(3), 219 - 21 Delayed peritoneal and retroperitoneal abscesses caused by spilled gallstones: a complication following laparoscopic cholecystectomy; Rioux M et al.; Many complications following laparoscopic cholecystectomy have been reported . We report a case of delayed peritoneal and retroperitoneal abscesses caused by spilled gallstones from a laparoscopic cholecystectomy performed 1 year earlier . This diagnosis was suggested only at sonography because the aggressive behavior of the lesions containing nonopaque gallstones suggested, by computed-tomography scan, peritoneal metastatic disease. Abdom Imaging, 1995 May-Jun, 20(3), 217 - 8 Abscess formation as a late complication of dropped gallstones; Maldjian C et al.; We describe a case of abscess formation 1.5 years postoperatively in a patient with dropped gallstones from laparoscopic cholecystectomy . The entity was initially recognized on computed tomography (CT) and the diagnosis was confirmed with ultrasound . Although this is a rare complication of laparoscopic cholecystectomy, it should be recognized as a potential source of abscess formation even in a patient presenting months after the procedure. Biotechniques, 1995 May, 18(5), 832, 835 - 8, 840-2 Variable region sequence modulates periplasmic export of a single-chain Fv antibody fragment in Escherichia coli; Ayala M et al.; Using PCR, we have cloned antibody heavy and light chain variable region (VH and VL) coding sequences specific for a recombinant hepatitis B virus surface antigen (HBsAg) and assembled these for expression as single-chain Fv (scFv) fragments in Escherichia coli periplasm using the ompA signal peptide . The vectors also encoded N- or C-terminal His6 extensions to allow for the purification of the expressed proteins using immobilized metal affinity chromatography (IMAC) . We found that the VH-linker-VL configuration of the scFv was not exported to the periplasm but remained associated with cellular insoluble material, from which it could be extracted, renatured to its active form by gentle dialysis and purified using IMAC . The molecular size of the scFv suggests that the ompA signal peptide was not processed . Based on previous reports, we hypothesized that the arginine in framework 1 (FR1) of the VH might interfere with translocation to the periplasm by means of the signal peptide . Because no arginines are present in FR1 of VL, we reversed the order of the V-regions in the scFv and observed efficient export of the active scFv to the periplasm . Furthermore, when the arginine in FR1 of VH was mutated to glycine in the original VH-linker-VL construct, active scFv was also exported to the periplasm . Thus, exposed positive charges near the signal peptide may account for at least some of the often-encountered difficulties in bacterial scFv expression. Biotechnol Prog, 1995 May-Jun, 11(3), 288 - 93 Fnr, a global transcriptional regulator of Escherichia coli, activates the Vitreoscilla hemoglobin (VHb) promoter and intracellular VHb expression increases cytochrome d promoter activity; Tsai PS et al.; The oxygen-regulated promoter (Pvhb) of the Vitreoscilla hemoglobin gene has been applied to direct high-level expression of several cloned proteins, including Vitreoscilla hemoglobin (VHb), which improves productivity of many aerobic processes . In an effort to gain a better understanding of the regulation of Pvhb, and to guide further optimization of this technology, we investigated whether the Escherichia coli global regulatory molecules Fnr and the Arc system (ArcA and ArcB), which control the expression of various genes under either aerobic or anaerobic conditions, also regulate Pvhb activity in E . coli . The activity of Pvhb and the expression of VHb in E . coli were activated by Fnr, but were relatively unaffected by the Arc system under microaerobic conditions (DO less than 2% air saturation) . We also examined the possibility of VHb affecting cytochrome d promoter activity during microaerobiosis . The presence of VHb increased the activity of beta-galactosidase from a cytochrome d promoter-lacZ fusion by 1.5-fold . This indicates that VHb affects oxygen-regulated transcription of E . coli genes and may contribute to the modified physiology observed in VHb-expressing E . coli. Biotechnol Prog, 1995 May-Jun, 11(3), 265 - 9 Purification by immobilized metal affinity chromatography of human atrial natriuretic peptide expressed in a novel thioredoxin fusion protein; Wilkinson DL et al.; A fusion protein that contains human atrial natriuretic peptide (ANP) at its carboxy terminus has been genetically engineered with the objective of being able to produce the peptide in a process with a relatively simple purification procedure . The fusion protein also includes a (His)6 metal affinity binding site at the amino terminus, followed by Escherichia coli thioredoxin, a factor Xa protease recognition site, and ANP . With induction of the tac promoter at 30 degrees C, the expression level of the fusion protein was high (10% of total cell protein as measured by densitometry) and it was almost completely (92%) expressed as a soluble protein in the cytoplasm . A step gradient elution with imidazole of a column of Ni2+ chelated to iminodiacetic acid-agarose saturated with proteins in crude cell extract gave a very nearly pure fusion protein . After digestion of the purified fusion protein with factor Xa protease, ANP of exactly the correct size (to within 2 Da) was observed by coupled HPLC/mass spectrometry. Vet Immunol Immunopathol, 1995 May, 46(1-2), 83 - 92 Serological diagnosis of feline immunodeficiency virus infection using recombinant transmembrane glycoprotein; Calzolari M et al.; We developed an antibody detection enzyme-linked immunosorbent assay (ELISA) using recombinant surface (SU), transmembrane (TM) and capsid (CA) antigens of feline immunodeficiency virus (FIV) expressed in Escherichia coli . The three antigens were tested with sera collected from experimentally infected cats in order to follow the course of seroconversion and of the antibody levels throughout the infection . An early and marked increase of TM antibodies was observed . Antibodies to TM were demonstrated at high levels throughout the observation period . The immune response to SU and to CA was less pronounced and in some cats the level of antibodies to SU and CA tended to decline 6 months after infection . In addition, 413 FIV negative and positive cat sera were tested in order to define for each antigen the diagnostic sensitivity, specificity and efficiency . TM showed the highest diagnostic sensitivity (98%) while its specificity was 97% . Its diagnostic efficiency of 97% was better than that of SU and CA and exceeded that of tests utilizing conventionally grown and gradient purified FIV . Therefore, recombinant TM can be considered a very important antigen for FIV ELISA testing . An interesting perspective is offered in the combination of TM with other recombinant antigens in a dot assay form. Vet Immunol Immunopathol, 1995 May, 46(1-2), 115 - 25 FIV vaccine studies . II . Clinical findings, hematological changes and kinetics of blood lymphocyte subsets; Hofmann-Lehmann R et al.; Five groups of cats were vaccinated with different recombinant feline immunodeficiency virus (FIV) SU vaccines expressed either in Escherichia coli or in the Baculovirus system . In Part I of this series, we described the humoral immune response and outcome of intraperitoneal FIV challenge exposure . Additionally, all cats were monitored for clinical and hematological changes and the course of blood lymphocyte subsets . These results are described in this present paper . A great increase of antibodies was found after vaccination with different recombinant FIV antigens, which did not protect the cats from intraperitoneal FIV challenge infection . This observation was paralleled by an increase of eosinophils during vaccination which was even more pronounced after challenge infection . After FIV challenge, infection lymphadenopathy, gingivitis, pharyngitis, changes in total leukocytes and neutrophils and a decrease in the CD4+:CD8+ ratio were found in cats of all groups and were considered as a sign of the FIV infection taking place, independent of vaccination . The following observations suggest that in these cats a TH2-like immune response was elicited: the high counts of eosinophils, the nature of antigen and adjuvant (aluminium hydroxide) and the high amounts of antigens used for immunization . Clearly, this type of immune response did not protect the animals from intraperitoneal FIV challenge infection. Vet Immunol Immunopathol, 1995 May, 46(1-2), 103 - 13 FIV vaccine studies . I . Immune response to recombinant FIV env gene products and outcome after challenge infection; Lutz H et al.; We have vaccinated five groups of cats (n = 25) four times with five preparations of recombinant feline immunodeficiency virus (FIV) env gene products; one group (n = 7) served as control . The vaccine formulations were as follows: (1) envelope glycoprotein of FIV Zurich 2 (FIV Z2) expressed in a Baculovirus system and isolated by gel electroelution (denatured form); (2) insect cells expressing FIV Z2 glycoprotein; (3) envelope glycoprotein of a Boston strain (FIV Bangston) expressed in insect cells and isolated by gel electroelution (denatured form); (4) glycosylated Bangston envelope protein made in insect cells and isolated in a native form; (5) non-glycosylated Bangston envelope protein made in Escherichia coli . All cats were challenged with 20 50% cat infective doses (CID50) of FIV Z2 previously titrated in cats . All vaccinated cats developed high enzyme-linked immunosorbent assay (ELISA) antibodies to the homologous antigen; crossreactivity to heterologous antigens was seen at a lower level . Virus neutralizing antibodies (tested with Petaluma virus) reached titers up to 32 . After challenge, all cats seroconverted (as judged by anti gag antibodies in Western blot) and became infected (as judged by virus isolation and/or polymerase chain reaction) between 4 and 11 weeks with the exception of one cat . It is concluded that it is relatively easy to induce high ELISA antibody titers using recombinant env gene products, ELISA antibody titers do not correlate with virus neutralization or with protection. J Med Entomol, 1995 May, 32(3), 338 - 45 Effects of Dermacentor andersoni (Acari: Ixodidae) salivary gland extracts on Bos indicus and B . taurus lymphocytes and macrophages: in vitro cytokine elaboration and lymphocyte blastogenesis; Ramachandra RN et al.; Cattle and laboratory animal species-acquired resistance to tick infestation has an immunological basis involving antigen presenting cells, B-lymphocytes, T-lymphocytes, and cytokines . Tick infestation has been shown to impair guinea pig antibody responses to a thymic-dependent antigen and in vitro responsiveness of lymphocytes to T-cell mitogens . Tick salivary gland extracts inhibited in vitro proliferative responses of normal murine lymphocytes to the T-cell mitogen concanavalin A (Con A) and enhanced reactivity of normal B-lymphocytes to the mitogen E . coli lipopolysaccharide (LPS) . Salivary gland extracts collected daily during engorgement were shown to inhibit normal murine macrophage elaboration of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF) as well as murine T-lymphocyte production of interleukin-2 (IL-2) and interferon-gamma (IFN-G) . Peripheral blood mononuclear cells collected from purebred Bos indicus and B . taurus were significantly inhibited in their in vitro responses to Con A by salivary gland extracts prepared daily from female Dermacentor andersoni stiles during the course of engorgement . Percentage of suppression of Con A responsiveness was similar for both B . indicus and B . taurus cells . The overall responsiveness of B . indicus derived T cells is significantly greater than that of similar cells from B . taurus, when mean counts per minute of methyl-tritiated-thymidine incorporation were compared for both groups . Cells of B . indicus origin were 34.5% more reactive . In vitro responsiveness of the same cell populations to LPS were significantly enhanced by the presence of tick salivary gland extracts . B . indicus lymphocyte reactivity to LPS was significantly greater (42.9%) than that of similar B . taurus cells in the absence of salivary gland extracts . B . indicus and B . taurus macrophage elaboration of IL-1 were suppressed in a similar manner by tick salivary gland extracts prepared on days 5-9 of engorgement . B . indicus macrophages produced more IL-1 than similar cells of B . taurus origin either in the presence (45.6%) or absence (43.0%) of LPS . Macrophages derived from both genetic backgrounds were significantly suppressed in their LPS induced production of TNF in the presence of tick salivary gland extracts collected on days 0-9 of engorgement . B . indicus might be able to develop more vigorous immune responses to foreign immunogens presented to the animal during tick feeding. J Clin Microbiol, 1995 May, 33(5), 1375 - 7 Identification of enteropathogenic