Nucleic Acids Res, 1989 Aug 11, 17(15), 6299 - 317 DNA primase and the replication of the telomeres in Oxytricha nova; Zahler AM et al.; An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n . Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end . The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end . With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template . A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA . The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin . Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template . This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA . The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E . coli DNA polymerase I Klenow fragment . Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972). Nucleic Acids Res, 1989 Aug 11, 17(15), 6269 - 82 The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases; Bricteux-Gregoire S et al.; Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase) . For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease . We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction . We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results. Nature, 1989 Aug 10, 340(6233), 482 - 6 Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle; Bernstein HD et al.; Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP) . Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome . Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain . The properties of this latter domain suggest that it contains the signal sequence binding site . A previously uncharacterized Escherichia coli protein has strong homology to both domains . Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E . coli protein, ftsY, which resembles SR alpha . Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane . We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals. Biochemistry, 1989 Aug 8, 28(16), 6611 - 8 Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli; Adams J et al.; The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis . The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C . Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected . Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively) . Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L . The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L. J Biol Chem, 1989 Aug 5, 264(22), 13114 - 21 Isolation and molecular characterization of a cDNA encoding the 23-kDa subunit of human RNA polymerase II; Pati UK et al.; We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II . Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter . By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA . We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure . The cDNAs isolated both from oligoscreening and immunoscreening were identical . The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa) . The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II . The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors. J Biol Chem, 1989 Aug 5, 264(22), 12902 - 8 Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro; Elich TD et al.; Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T . D., and Lagarias, J . C . (1988) Plant Physiol . 88, 747-751) . Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T . D., McDonagh, A . F., Palma, L . A., and Lagarias, J . C . (1988) J . Biol . Chem . 264, 183-189) . Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein . The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies . The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein . The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide . A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin . ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation . When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced . These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment. J Mol Biol, 1989 Aug 5, 208(3), 509 - 10 First crystallization of a phosphoenolpyruvate carboxylase from Escherichia coli; Inoue M et al.; Two different forms of crystal for a phosphoenolpyruvate carboxylase from Escherichia coli were obtained by the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant . The hexagonal crystal in space group P6(2)22 (or P6(4)22) has cell dimensions of a = 131 A and c = 325 A, whereas the orthorhombic crystal in space group I222 has a = 119 A, b = 252 A and c = 83 A . A tetrameric molecule (396,244 Mr), a subunit of which contains 883 amino residues, has a crystallographic 2 symmetry in the hexagonal crystal or 222 symmetry in the orthorhombic crystal, respectively. J Mol Biol, 1989 Aug 5, 208(3), 501 - 5 Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization . Evidence for genetically distinct events at oriT; Kim K et al.; A segment of R1162 DNA containing genes for conjugative mobilization (Mob) and the origin of transfer (oriT) was integrated into the Escherichia coli chromosome . Bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector . The direction of transfer, together with the properties of mutated derivatives of oriT, indicate that initial cleavage of oriT, and subsequent religation after transfer, entail different mechanisms that can be distinguished genetically. J Biol Chem, 1989 Aug 5, 264(22), 13012 - 17 Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli; Vijgenboom E et al.; Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli . Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu . A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting . The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately . These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally. J Biol Chem, 1989 Aug 5, 264(22), 12752 - 3 Crystallization and preliminary x-ray characterization of thioredoxin reductase from Escherichia coli; Kuriyan J et al.; Single crystals of thioredoxin reductase, suitable for x-ray diffraction studies, have been obtained at room temperature by vapor diffusion of 10-20 mg/ml protein solution against 35% polyethylene glycol containing 200 mM ammonium sulfate . Good quality crystals appear spontaneously only from a protein solution that had been stored for more than a year at 4 degrees C, although large single crystals are reproducibly obtained from fresh protein solutions by micro-seeding . The space group is P6(3)22 (a = b = 123.8 A, c = 81.6 A), with one monomer of the enzyme (34.5 kDa) in the crystallographic asymmetric unit . The crystals are well ordered and diffract to beyond 2 A resolution. J Biol Chem, 1989 Aug 5, 264(22), 13321 - 8 Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase . The effect of defective cAMP binding on holoenzyme activation; Woodford TA et al.; The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses . The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites . In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated . All of the mutant RI subunits which bound {3H}cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit . Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional . The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed . Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM . The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites . Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit . The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites. J Biol Chem, 1989 Aug 5, 264(22), 13086 - 92 C-terminal truncation of p21H preserves crucial kinetic and structural properties; John J et al.; The human c-Ha-ras protooncogene product p21C was truncated at the C terminus by 23 amino acids . The resulting G-binding domain, p21 (1-166) = p21C', can be crystallized as a complex with the slowly hydrolyzing GTP analogues guanosin-5'-{beta,gamma-imido}triphosphate, guanosin-5'-{beta,gamma-methylene}triphosphate, and guanosin-5'-O-(3-thiotriphosphate) . We show here that this protein has biochemical properties very similar to those of the intact protein . Activating mutations in position 12 (Gly12----Val; Gly12----Arg) have the same effect on the properties of the truncated protein as on intact protein . Nuclear magnetic resonance (NMR) measurements show no apparent effect of the C-terminal deletion on the solution structure of p21 . This suggests that neither the structure of the G-binding domain nor any of its biochemical properties are markedly influenced by the truncation. J Mol Biol, 1989 Aug 5, 208(3), 429 - 43 Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition; Mellot P et al.; Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases . Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme . The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro . According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains . The former, the N-domain, contain a crevice that is believed to bind ATP . The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains . This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases . This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation . Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs. J Chromatogr, 1989 Aug 4, 476, 245 - 55 Isolation of recombinant hirudin by preparative high-performance liquid chromatography; Bischoff R et al.; The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described . rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation . Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium . The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases . Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants . Liquid secondary ion mass spectrometry was used to confirm its primary structure . The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active. Science, 1989 Aug 4, 245(4917), 522 - 4 Triggering of allostery in an enzyme by a point mutation: ornithine transcarbamoylase; Kuo LC et al.; The origin of allostery is an unanswered question in the evolution of complex regulatory proteins . Anabolic ornithine transcarbamoylase, a trimer of identical subunits, is not an allosteric enzyme per se . However, when the active-site residue arginine-106 of the Escherichia coli enzyme is replaced with a glycine through site-directed mutagenesis, the resultant mutant enzyme manifests substrate cooperativity that is absent in the wild-type enzyme . Both homotropic and heterotropic interactions occur in the mutant enzyme . The initial velocity saturation curves of the substrates, carbamoyl phosphate and L-ornithine, conform to the Hill equation . The observed cooperativity depends on substrate but not enzyme concentration . The finding underscores the possibility that a single mutation of the enzyme in the cell could turn transcarbamoylation into a regulatory junction in the biosynthesis of L-arginine and urea. J Chromatogr, 1989 Aug 4, 476, 147 - 58 Sodium dodecyl sulphate-protein complexes . Changes in size or shape below the critical micelle concentration, as monitored by high-performance agarose gel chromatography; Mascher E et al.; We have determined the sodium dodecyl sulphate (SDS) concentration needed to complete the formation of SDS-protein complexes . A Superose-6 column was equilibrated with SDS for 7 h . A sample of a native protein or an SDS-protein complex was applied, and the elution volume, Ve, was determined . Then the SDS concentration, CSDS, was changed, etc., i.e., Ve was determined as a function of CSDS . The critical micelle concentration of SDS (cmcSDS) was 1.8 mM in the eluent (ionic strength 0.10 M) . Native bovine carbonic anhydrase (BCA) formed an SDS complex above 0.2 mM SDS . As CSDS was increased, Ve decreased gradually in two main transitions, (TI) at 0.2-1.0 mM and (TII) at 1.2-2.0 mM SDS . These concentrations are corrected for a lag in the column equilibration with SDS . SDS-BCA, pre-equilibrated at 1.6 mM SDS, showed transitions similar to those observed with native BCA, except that transition TII included a minor transition at 2.0-2.2 mM SDS . The SDS complexes of reduced and carboxamidomethylated bovine serum albumin, of N-5'-phosphoribosylanthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli (PRAI-IGPS) and of two tryptic fragments of this enzyme behaved similarly . For SDS-PRAI-IGPS the major part of transition TII was completed at 1.6-1.7 mM SDS, as shown by analyses after 20-h column equilibrations with increasing as well as decreasing CSDS . The SDS complex of an integral membrane protein, the glucose transporter from human red cells, was smaller or less elongated than the SDS complexes of water-soluble proteins of the same polypeptide length . The formation of all five SDS-protein complexes investigated was practically completed at cmcSDS. Nature, 1989 Aug 3, 340(6232), 397 - 400 Complete mutagenesis of the HIV-1 protease; Loeb DD et al.; Retroviruses encode a protease which needs to be active for the production of infectious virions . A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease . Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein . Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation . These results show that random mutagenesis can be used to identify functionally important regions within a protein . Mutants with conditional phenotypes have also been identified within this collection. N Engl J Med, 1989 Aug 3, 321(5), 280 - 7 The cardiovascular response of normal humans to the administration of endotoxin; Suffredini AF et al.; Marked abnormalities in cardiovascular function accompany septic shock, and bacterial endotoxin is believed to be one of the principal mediators of these abnormalities . To evaluate the cardiovascular effects of endotoxemia in humans, we measured hemodynamic variables in nine normal subjects given an intravenous bolus dose of endotoxin (Escherichia coli, 4 ng per kilogram of body weight) and in six normal subjects given a bolus dose of saline, before and three hours after administration . All the subjects then underwent volume loading with normal saline (mean, 2217 ml) during the fourth and fifth hours after administration of the bolus, and the measurements were repeated . Three hours after the administration of endotoxin and before volume loading, the cardiac index had increased by 53 percent and the heart rate by 36 percent (both changes were significant; P less than or equal to 0.008), and the systemic vascular-resistance index had decreased by 46 percent (P = 0.004) . After volume loading (five hours after the administration of endotoxin), the left ventricular ejection fraction decreased by 1 percent of the base-line value in the subjects given endotoxin, but increased by 14 percent in the controls (P = 0.008) . The left ventricular end-diastolic and end-systolic volume indexes increased by 18 percent (P = 0.07) and 24 percent (P = 0.042), respectively . Left ventricular performance, as measured by the ratio of the peak systolic pressure to the end-systolic volume index, was depressed (a decrease of 0.90 in the subjects given endotoxin vs . an increase of 0.76 in the controls; P = 0.024) . We conclude that the administration of endotoxin to normal subjects causes a depression of left ventricular function that is independent of changes in left ventricular volume or vascular resistance . The changes in function are similar to those observed in septic shock and suggest that endotoxin is a major mediator of the cardiovascular dysfunction in this condition. Gene, 1989 Aug 1, 80(1), 171 - 6 A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment; Bej AK et al.; A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus . After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U . violacea transformants appeared on Hyg-agar at a frequency of 60-80 per microgram pUCH1 DNA . The Hyg phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions . Southern DNA-DNA hybridization revealed multiple integrations of the pUCH1 plasmid into the U . violacea nuclear DNA . In addition, Escherichia coli transformants appeared at a frequency of 12 per microgram nuclear fraction DNA from Hyg U . violacea transformants; these E . coli consistently contained a deleted pUCH1 plasmid . This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences. Hybridoma, 1989 Aug, 8(4), 435 - 48 Specific, high affinity colchicine binding monoclonal antibodies: development and characterization of the antibodies; Edmond Rouan SK et al.; Nine colchicine specific monoclonal antibodies have been developed by immunizing BALB/c mice with a colchicine-keyhole limpet hemocyanin (Col-KLH) conjugate prepared using a bishydroxysuccinimide coupling reagent . Of four immunization procedures examined, intraperitoneal injection of the antigen attached to acid treated E . coli resulted in the maximum antigen specific antibody titers . A colchicine bovine serum albumin (Col-BSA) conjugate, prepared using a water soluble carbodiimide coupling technique, formed the basis of an enzyme linked immunosorbent assay used for screening hybridomas for colchicine specific antibody secretion and for determining the relative affinity and specificity profile of the monoclonal antibodies . All antibodies demonstrated high affinity, saturable binding to colchicine and low cross-reactivity with a panel of compounds structurally related to colchicine . The IC50 for the highest affinity antibody, C44, was 3.6 +/- 0.84 nM colchicine in the competitive enzyme immunoassay . The affinity of this antibody determined from Scatchard analysis of antibody binding to tritiated colchicine was 0.66 +/- 0.11 nM . Antibody C44 has the level of specificity and affinity suitable for a sensitive and selective immunoassay of colchicine for monitoring therapeutic drug levels . In addition, this antibody provides a specific pharmacologic antagonist for studies of colchicine's therapeutic mechanism and has the potential to reverse colchicine toxicity. Zentralbl Hyg Umweltmed, 1989 Aug, 188(5), 421 - 38 {Nitrated polycyclic aromatic hydrocarbons (nitro-PAH) in the suspended substances of the atmosphere . 2 . Comparison of the mutagenicity of nitro-PAH and dust extracts of the air in the Ames, SOS repair induction and SCE test}; Schleibinger H et al.; Extracts of airborne suspended particulate matter were assayed in the Ames-test with strain TA 98, in the SOS chromotest (test on induction of the SOS repair) with strains E . coli PQ37 and S . typhimurium TA 1535 and in the sister chromatid exchange test . Samples from Berlin-Wedding were extracted by dichloromethane and tested up to the limits of solubility . All three test systems showed biologic activity . In the Ames- and the SOS chromotest these extracts revealed higher activity with S9-mix . 4 nitro-PAH, which are known to be present in ambient urban air, were assayed in these test systems, checking them additionally with strains TA 1538, TA 100 and TA 1535 in the Ames-test . In this test these compounds appeared to be direct acting frameshift mutagens and their activity is diminished by addition of S9-mix . On the contrary these compounds were either active at all or more active in the SOS chromotest in the presence of S9-mix . In the SCE-test only with exogenous activation a raise of the SCE frequency could be detected. J Gen Virol, 1989 Aug, 70 ( Pt 8), 2087 - 95 Protection of woodchucks from infection with woodchuck hepatitis virus by immunization with recombinant core protein; Roos S et al.; Woodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) core antigen (WHcAg) to investigate whether such immunization protects against WHV infection . The C gene was cloned into a pUC12 vector and expressed in Escherichia coli . Core particles purified by sucrose and CsCl gradient centrifugation had a buoyant density of 1.37 g/ml which corresponded to the density of WHcAg particles present in chronically infected liver . Two animals immunized with the recombinant antigen developed high antibody titres and were protected against infection after challenge with WHV . The surface antigen (WHsAg) and WHV DNA were not detected in the sera of immunized animals after challenge and these animals did not develop anti-WHs . Three control animals developed a typical WHV infection . The protection from WHV infection may depend not on the presence of antibodies against the core protein but on a cellular immune response to WHcAg. Virology, 1989 Aug, 171(2), 579 - 87 Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein; Kotwal GJ et al.; The objective of this study was to identify and characterize the gene encoding a protein of approximately 12 kDa that is secreted from cells infected with the vaccinia virus . The absence of this protein from the medium of cells infected with a spontaneous deletion mutant (6/2) suggested that the open reading frame (ORF) was located within a 12,800-base pair segment near the left end of the genome (G . Kotwal and B . Moss, Nature (London) 335, 176-178, 1988) . Antibody to the 12-kDa protein immunoprecipitated an appropriate size in vitro translation product of mRNA that hybridized to a DNA segment containing an ORF (N1L) that could encode a 13.8-kDa polypeptide . The similarity in the sizes of the in vitro translation product and the secreted protein was consistent with the absence of processing . Transcriptional analysis revealed major and minor early RNA start sites preceding the N1L ORF as well as a late RNA start site with an atypical TAAAAT sequence . The N1L gene was interrupted by replacing a segment of the ORF with the Escherichia coli beta-galactosidase gene . When two-dimensional polyacrylamide gel electrophoretic patterns of {35S}methionine-labeled proteins secreted from cells infected with parental and recombinant viruses were compared, a spot missing from the latter corresponded in molecular weigh and isoelectric point with that predicted from the N1L ORF . The latter analysis revealed the presence of other secreted proteins of similar molecular weight but different isoelectric points that also appear to map within the left end of the vaccinia genome . The recombinant virus was attenuated as judged by the increased intracranial LD50 for mice but nevertheless induced antibody and cytotoxic responses after intradermal and intraperitoneal injections . Relative to the parental virus, the recombinant was also more attenuated for immunodeficient nude mice, based on their survival time after infection. Arch Biochem Biophys, 1989 Aug 1, 272(2), 281 - 9 Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications; Berthiaume L et al.; Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle . Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture . The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively . Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence . Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases . Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases . Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme . Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology . The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed. Ryumachi, 1989 Aug, 29(4), 259 - 67 {Pathological changes in cartilage of the knees in rabbits immunized with Escherichia coli 0:14}; Sakai N; Pathological changes in the articular cartilage were found in the knees of rabbits immunized with heat-killed Escherichia coli for 8-10 months at significantly higher rate (33.3%, 12 of 36 knees, p less than 0.05) than in those immunized for 1-4 months (17.6%, 6 of 34) . Cartilage degeneration showed two types which were pannus invasion at the cartilage-synovial junction, and degeneration at superficial layer of the articular cartilage as seen in rheumatoid arthritis of human . Depositions of immunoglobulin were found in 10 out of 16 knees (62.5%) with the pannus invaded lesion and 5 out of 8 knees (75%) with the degenerated superficial layer of articular cartilage. Acta Chir Scand, 1989 Aug, 155(8), 409 - 12 Psoas abscess complicating Crohn's disease; Shokouh-Amiri MH et al.; Six cases of psoas abscess complicating Crohn's disease are presented . The most common manifestations were weight loss, pain, fever and a palpable mass in the flank or iliac fossa . CT-scan confirmed the diagnosis . The treatment of choice in this condition is either laparotomy with drainage and primary resection of affected bowel or initial ultrasound-guided percutaneous drainage followed by early resection of the affected bowel. Biochem Cell Biol, 1989 Aug, 67(8), 468 - 72 Production and interconversion of 1,2-propanediol and hydroxyacetone by Escherichia coli; Commodari F et al.; The anaerobic metabolism of marginally lethal levels of {13C}formaldehyde by Escherichia coli (K12, MU352, CRB, and CR63) was followed in vivo by 13C NMR . The products include 1,2-propanediol . Under aeration, the 1,2-propanediol is converted to hydroxyacetone . The hydroxyacetone is reconverted to 1,2-propanediol when aeration is stopped . The process can be cycled by varying the rate of aeration. Biochem Cell Biol, 1989 Aug, 67(8), 404 - 10 Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli; Brisson A et al.; The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli . To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase . Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme . Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures . The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E . coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli. Bioorg Khim, 1989 Aug, 15(8), 1078 - 90 {Synthesis of human proinsulin in Escherichia coli cells}; Efimov VA et al.; Expression of the synthetic gene for human proinsulin in E . coli has been investigated . The proinsulin gene has been expressed directly under the control of a synthetic promoter of phage fd DNA and a promoter of tryptophan operon, or using fusions with fragments of some bacterial proteins . These fusions gave insoluble polypeptide products amounting to 20-30% of total cellular protein . The scheme for isolating proinsulin from bacterial cells was developed . Proinsulin was cleaved from leader polypeptides by treatment with cyanogen bromide and converted into human insulin. Bioorg Khim, 1989 Aug, 15(8), 1070 - 7 {Construction of a family of artificial genes of human insulin}; Efimov VA et al.; Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier . The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized . The constructions obtained were cloned in plasmid vectors. Bioessays, 1989 Aug-Sep, 11(2-3), 62 - 7 Invertebrate cytokines: the phylogenetic emergence of interleukin-1; Beck G et al.; Cytokines are polypeptides released by activated vertebrate blood cells which have profound effects on other blood cells and which have hormone-like properties affecting other organ systems as well . In recent years a wide variety of these mediators has been isolated and characterized . Many of these molecules have subsequently been cloned and expressed in E . coli . The tremendous importance of these proteins to host immune and non-specific defense systems along with the striking similarities of their properties among different species suggested to us that cytokines may have been proteins that have been conserved through evolution . Investigations of the evolution of cytokines will help us decipher the complex cellular, humoral and molecular interactions that regulate host defenses . Studies of the invertebrates will shed light on the phylogenetic emergence of these molecules as well. Genetika, 1989 Aug, 25(8), 1384 - 90 {Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells}; Kaplan AM et al.; Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied . Stable maintenance of RP4::D3112 plasmid in E . coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression . The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid . Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration . The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology. FEMS Microbiol Lett, 1989 Aug, 51(3), 267 - 71 Partial complementation of pyruvate dehydrogenase deficiency by independently expressed lipoyl and catalytic domains of the dihydrolipoamide acetyltransferase component; Russell GC et al.; Two compatible plasmids encoding a hybrid lipoyl domain and a defective pyruvate dehydrogenase (PDH) complex which lacks lipoyl domains, were co-expressed in a strain of Escherichia coli deleted for the PDH complex genes . In vivo complementation between the mutant complexes and the independent lipoyl domains was observed using growth tests in liquid and solid media . However, no PDH complex activity could be detected in the corresponding cell-free extracts . This suggests that untethered lipoyl domains can interact productively with the three types of active site in the multienzyme complex, but this association is disrupted in cell-free extracts. Can J Microbiol, 1989 Aug, 35(8), 779 - 85 Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress; Deutch CE et al.; L-Proline, which is accumulated by Escherichia coli during growth in media of high osmolality, also induces the synthesis of the enzyme degrading it to glutamate . To determine if proline catabolism is inhibited during osmotic stress, proline utilization and the formation of proline dehydrogenase were examined in varying concentrations of NaCl and sucrose . Although the specific growth rate of E . coli with proline as the sole nitrogen source diminished as the solute osmolality increased, a comparable reduction in growth rate occurred with ammonium as the primary nitrogen source . Proline catabolism, as measured in whole cells by the conversion of {14C}proline to {14C}glutamate, was only slightly inhibited by solute osmolalities up to 1.0 osmol/kg; more than 50% of the initial activity was still found at 2.0 osmol/kg . By contrast, the specific activity of proline dehydrogenase in bacteria grown in the presence of added solutes decreased to less than 20% of the control level . This reduction was related to a lower rate of synthesis, but was independent of genes currently known to be involved in osmoregulation or proline metabolism . The specific activities of tryptophanase, beta-galactosidase, and histidinol dehydrogenase were also reduced under similar growth conditions . These results indicate that while proline catabolism is not directly inhibited by high solute concentrations, prolonged exposure to osmotic stress leads to its reduction as part of a more general metabolic response. Biokhimiia, 1989 Aug, 54(8), 1247 - 53 {The effect of mutations in ribosomal proteins S4, S12 and L7/L12 on EF-Tu-dependent expenditure of GTP in the process of codon-specific elongation and misreading of poly(U)}; Kakhniashvili DG et al.; The effect of mutations in ribosomal proteins S4 (rpsD12), S12 (rpsL282) and L7/L12 (rplL265) of Escherichia coli K12 on the EF-Tu-dependent expenditure of GTP during codon-specific elongation (poly(Phe) synthesis on poly(U} and misreading (poly(Leu) synthesis on poly(U}, was studied . Under the conditions used the mutations in proteins S4 and L7/L12 did not practically affect the EF-Tu-dependent expenditure of GTR during the poly(Phe) synthesis on poly(U): the GTP/Phe ratio was about 1, as in the case of the wild strain . Under the same conditions, the ribosomes with a mutant S12 protein tended to discard some amount of Phe-tRNA, as a result of which the GTP/Phe ratio increased to about 3 . The marked inhibition of misreading by ribosomes with a mutant S12 protein was accompanied by a significant increase of GTP expenditure at the stage of EF-Tu-dependent non-cognate aminoacyl-tRNA binding . In mutant S 12 proteins the GTP/Leu ratio was about 30-40, whereas in the wild type it was about 12 . In contrast, stimulation of misreading by ribosomes with mutant S4 and L7/L12 proteins was accompanied by a decrease of the EF-Tu-dependent expenditure of GTP by 2-3 GTP molecules per one Leu residue included into the peptide. J Biomol Struct Dyn, 1989 Aug, 7(1), 181 - 6 Additional Watson-Crick interactions suggest a structural core in large subunit ribosomal RNA; Haselman T et al.; Two new Watson-Crick type interactions in 23S-like ribosomal RNA have been identified by comparative sequence analysis . These interactions, A1269/U2011 and C1270/G2010 (E . coli numbering) along with the previously proposed A1262/U2017 suggest an anti-parallel helical arrangement characteristic of secondary structure in the 1265/2015 region of 23S rRNA . Nested within these three interactions are three universal juxtapositions which in principle allow the formation of an irregular helix containing two additional A-G interactions and a universal A-U pair . Whether or not this extended helix is biologically significant is uncertain . The proponderance of interactions in the 1265/2015 region and its location relative to the known structural domains of 23S rRNA suggest that this region may be part of a central structural core similar to that already known in 16S rRNA. Protein Eng, 1989 Aug, 2(8), 597 - 604 Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices; Henneke CM et al.; The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids . The alignment studies have been performed as the first step in a project aimed at homology modelling E . coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes . The effects of several important variables in producing realistic alignments have been investigated . The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E . coli proteins was explored . The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated . The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed. Mol Gen Mikrobiol Virusol, 1989 Aug, (8), 17 - 20 {Cloning of chloroplast psbA and rbcL-genes from cotton Gossipium hirsutum}; Ul'masov TN et al.; The fragments of cotton Gossipium hirsutum c.v . 108-f chloroplast genome were cloned in Escherichia coli cells . The cloned psbA and rbcL genes have been selected using the heterologous probes from spinach . The preliminary attempts to clone the complete psbA gene in pUC19 vector failed, probably, due to the toxicity of its product to Escherichia coli cells, and its 5'- and 3'-ends were cloned separately . Reconstruction experiments revealed that while the complete psbA gene was unable to be stably inherited by Escherichia coli cells, its structural part lacking the promoter region could be readily cloned in the bacterial cells. Yakugaku Zasshi, 1989 Aug, 109(8), 574 - 81 {Biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence}; Fujii T et al.; It was found that a plasmid which had a foreign deoxyribonucleic acid (DNA) between two repeated sequences did not multiply in E . coli recBCsbcB, even if it multiplied in wild-type E . coli, E . coli recBC or E . coli recBCsbcBrecF when the insert was longer than 351 base pair . The multiplication of these plasmids were, however, inhibited when a plasmid expressing recF gene was introduced into E . coli recBCsbcBrecF . The inviability of the plasmid carrying the repeated sequence in E . coli recBCsbcB was discussed by the mechanism of recombination, and the functions of recF, recBC and sbcB were speculated . When E . coli recBC was transformed with pDR1 which was a derivative of pBR322 carrying a directly repeated sequence between which a DNA fragment derived from plasmid R6K with its origin was inserted, the intramolecular recombinant appeared . The recombinant recovered was, however, only the plasmid which had the replication origin of pBR322 . The result suggests that pBR322 is compatible with pDR1 but R6K is not . The replication origin of R6K seems to be preferrentially used by pDR1. Acta Med Okayama, 1989 Aug, 43(4), 197 - 202 Non-radioactive hybridization probes prepared using M13 phage vector and the universal sequencing primer; Ikeda S et al.; Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer . The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E . coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label . Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA . The long tag sequence derived from the M13 vector may increase the sensitivity of the detection . The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates . As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development. Vet Microbiol, 1989 Aug, 20(4), 357 - 68 In vitro adhesion of K88ac+ Escherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs; Valpotic I et al.; Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells . The cells and brush borders were tested for their ability to bind K88-piliated enterotoxigenic E . coli Strain M1823B (K88ac) and E . coli Strain 1476 (K-12, K88ac) . Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac+ enterotoxigenic E . coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation . In vitro adhesion of K88ac-bearing E . coli was always negative with buccal epithelial cells . K88ac strains varied in their ability to adhere to lymphocytes and rectal epithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders . The non-piliated control E . coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes . It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88+ enterotoxigenic E . coli in the porcine small intestine. Gene, 1989 Aug 1, 80(1), 109 - 18 Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone; Redmond TM et al.; We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector . The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined . These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP . This cDNA sequence validates the intron: exon boundaries predicted from the gene . A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1 . Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination . This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots . This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP . Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein. Genes Dev, 1989 Aug, 3(8), 1226 - 32 Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation; Parsell DA et al.; To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escherichia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of lambda repressor . These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response . In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response. EMBO J, 1989 Aug, 8(8), 2417 - 24 tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli; Springer M et al.; Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site . The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species . We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way . We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression . The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity . The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity . The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged . The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way. EMBO J, 1989 Aug, 8(8), 2411 - 5 In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X; Amitsur M et al.; During phage T4 infection of Escherichia coli strains containing the prr locus the host tRNALys undergoes cleavage-ligation in reactions catalyzed by anticodon nuclease, polynucleotide kinase and RNA ligase . Known genetic determinants of anticodon nuclease are prr, which restricts T4 mutants lacking polynucleotide kinase or RNA ligase, and stp, the T4 suppressor of prr encoded restriction . The present communication describes an in vitro anticodon nuclease assay in which the specific cleavage of tRNALys is driven by an extract from E . coli prrr (restrictive) cells infected by phage T4 . The in vitro anticodon nuclease reaction requires factor(s) encoded by prr, is stimulated by a synthetic Stp polypeptide and appears to require additional T4 induced factor(s) distinct from Stp. Berl Munch Tierarztl Wochenschr, 1989 Aug 1, 102(8), 266 - 72 {The administration of homeopathic drugs for the treatment of acute mastitis in cattle}; Merck CC et al.; The general principles of homeopathic therapy are described together with a number of homeopathic drugs used for the treatment of acute bovine mastitis . Fifty cows with acute mastitis were used in the study . The initial treatment comprised aconitum D 4, phytolacca D 1 and bryonia D 4 . In subsequent treatments phytolacca D 1, bryonia D 4 and lachesis D 8 either singly or in combination were used; mercurius solubilis D 4 was also used . Encouraging results, especially in the treatment of cases of E.coli mastitis, were achieved. Am J Vet Res, 1989 Aug, 50(8), 1294 - 6 Cytotoxin activity on Vero cells among Escherichia coli strains associated with diarrhea in cats; Abaas S et al.; The role of Escherichia coli as a causative agent of diarrhea in cats was investigated . Isolates of E coli from healthy and diarrheic cats were serotyped and investigated for their biochemical characters, production of cytotoxin activity on Vero cells, heat-labile enterotoxin, heat-stable enterotoxin, and hemagglutination of erythrocytes from other animal species . None of 48 investigated strains produced heat-labile enterotoxin or heat-stable enterotoxin, nor did they agglutinate erythrocytes . Most strains were hemolytic and belonged to O-serotypes 2 and 6 . Cytotoxin activity on Vero cells was significantly more common and produced in greater amounts among E coli strains isolated from diarrheic cats, and was neutralized by anti-Shiga-like toxin I serum. Mol Gen Genet, 1989 Aug, 218(2), 284 - 8 Ars region TL-DNA on octopine type Ti plasmids; Suzuki Y et al.; In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae . T-DNA: YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10(3)-10(4) transformants per microgram plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable . The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication . The ars region was mapped on the noncoding region between the coding regions corresponding to no . 5 and no . 7 mRNA, and its minimal length determined in this experiment was about 150 bp. Mol Gen Genet, 1989 Aug, 218(2), 249 - 56 Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer; Blasco F et al.; The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed . It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively . The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide . In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide . This suggested that the latter is an electron transfer unit of the nitrate reductase complex . Such a view is opposite to the current description of the nitrate reductase . The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction . The NarG polypeptide was found to display a high degree of homology with numerous E . coli molybdoproteins . Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs. Mol Gen Genet, 1989 Aug, 218(2), 190 - 8 Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86; Maas R et al.; Many plasmids belonging to the F incompatibility groups contain more than one basic replicon . The chimeric plasmid pCG86 is an example of such a multireplicon plasmid . The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied . The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication . In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility . This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated . We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon. Mol Gen Genet, 1989 Aug, 218(2), 183 - 9 Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene; Ezaki B et al.; The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42 degrees C . We cloned the wild-type E . coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein . Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR--thrA--seg-2--leuB, consistent with the locus of the dnaK gene . Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution . These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene . The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30 degrees C in contrast to plasmids pBR322, pACYC184 and pSC101, which did . The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30 degrees and 42 degrees C. Epidemiol Infect, 1989 Aug, 103(1), 211 - 5 Necrobacillosis and immunity in mice; Smith GR et al.; The study arose from the recent finding that sub-lethal numbers of certain bacterial species greatly enhanced the infectivity of Fusobacterium necrophorum . A severe F . necrophorum infection in mice, cured with metronidazole, produced significant though slight resistance, which was demonstrable by challenge with a minute dose of F . necrophorum (less than 20 organisms) suspended in a sub-lethal dose of Escherichia coli (300 x 10(6) organisms) to enhance fusobacterial infectivity . In an earlier comparable experiment, challenge with F . necrophorum alone, in necessarily large doses (greater than or equal to 3 x 10(6) organisms), failed to demonstrate that a single cured fusobacterial infection gave rise to resistance; such an infection neither protected against the fatal necrobacillosis produced by challenge nor prolonged survival . A sub-lethal E . coli infection was also shown by challenge with a minute dose of F . necrophorum (less than 10 organisms), suspended in a sub-lethal dose of E . coli (152 x 10(6) organisms), to produce significant though slight protection against necrobacillosis . The degrees of resistance demonstrated were too slight to give any encouragement to the prospect of an effective necrobacillosis vaccine. Circ Shock, 1989 Aug, 28(4), 385 - 94 Temporal response of lipoprotein lipase-suppressing mediator and tumor necrosis factor in lipopolysaccharide -injected and -infused rats; Bagby GJ et al.; This study was initiated to compare the temporal response of serum lipoprotein lipase-suppressing mediator (LSM) and tumor necrosis factor (TNF) in lipopolysaccharide (LPS) -infused or -injected rats . Serial blood samples were obtained over a 5-day period from rats implanted with vascular catheters . Control rats infused with saline exhibited no detectable LSM activity during the 5-day observation period . LPS administered either by injection or infusion (6 h or 5 d) resulted in detection of serum LSM and TNF activities during the early period of observation with the LSM temporal response (8 h) outlasting the TNF response (3 h) . The LSM response lasted a little longer in LPS-infused rats than it did in injected rats, but in each case LSM activity was not detected in serum samples collected at or after 12 h post-LPS . Neither the duration nor the magnitude of the TNF response differed between LPS-infused vs . -injected rats . Despite similarities in the LSM and TNF pattern in all rats receiving LPS, lethality was greater in LPS-infused animals than it was in LPS-injected rats . The results indicate that differences in lethality between LPS-injected and -infused rats cannot be explained solely by a differential response of serum TNF. Biotechnol Appl Biochem, 1989 Aug, 11(4), 401 - 12 Expression of repetitive human calcitonin genes in Escherichia coli; Gigova L et al.; In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E . coli under the control of a constitutive synthetic phage promoter . The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc . The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene) . This effect held true as well for the proteins coded by the corresponding repetitive hCT genes . The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats . The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E . coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics) . One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene . The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E . coli strains in order to find the optimal host for production of rhCT . The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1. Pediatr Res, 1989 Aug, 26(2), 158 - 61 Direct long-term effects of L-asparaginase on rat and human pancreatic islets; Clausen N et al.; L-Asparaginase, an effective agent in the treatment of acute lymphoblastic leukemia, may induce a diabetic state . The pathogenesis of the diabetogenic effect was studied in cultured pancreatic islets . Mean serum concentrations in three children with acute lymphoblastic leukemia were 2.4 U/mL (range 1.4-4.5) before and 31.5 U/mL (range 18.6-51.8) immediately after an intravenous injection of 1000 U/kg L-asparaginase . Glucose-induced insulin release from pancreatic islets of rat and man was measured after 3 and 7 days of culture in media with or without clinically relevant concentrations of Escherichia coli L-asparaginase (0.01-100 U/mL) . After culture, the remaining insulin, glucagon, and DNA in the islets were determined . After 7 days of culture of adult rat or human islets, both the accumulation of insulin in the medium and the content of insulin and glucagon in the islets were significantly reduced in the presence of 100 U/mL L-asparaginase compared with controls . Addition of 10(-6) M hydrocortisone to the culture medium enhanced this effect . In newborn rat islets a significant reduction in insulin release and content was observed already in the presence of 0.1 U/mL asparaginase, whereas the glucagon content was unchanged . Removal of the drug resulted in partial recovery of the insulin secretion . To elucidate the mechanisms of of action of the drug, insulin biosynthesis was studied in islets cultured in asparagine-free medium with or without asparaginase . No difference in biosynthesis was seen between media with or without asparagine, whereas 0.1 U/mL asparaginase caused about a 50% reduction under both conditions.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Virol, 1989 Aug, 70 ( Pt 8), 1961 - 74 Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene; Hammond J et al.; The sequence of 1015 nucleotides from the 3' poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones . This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region . The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV . The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of Mr 30910 . The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions . The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses . The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5'-proximal one-fifth of the genes . Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion . A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins . The BYMV 3'-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract. Arthritis Rheum, 1989 Aug, 32(8), 1041 - 4 An adsorption procedure to increase the specificity of enzyme-linked immunosorbent assays for Lyme disease without decreasing sensitivity; Fawcett PT et al.; Previous efforts to use adsorption techniques to enhance the specificity of enzyme-linked immunosorbent assays for antibodies to the spirochete that causes Lyme disease resulted in unacceptable reductions in assay sensitivity . We present here evidence that preadsorption of serum with Escherichia coli can enhance test specificity without significantly reducing test sensitivity. Anesth Analg, 1989 Aug, 69(2), 163 - 8 Alterations in host defense associated with inhalation anesthesia and blood transfusion; Waymack JP et al.; Anesthesia and blood transfusions have been demonstrated to impair immune function . The present study evaluated whether these impairments led to increased susceptibility to infectious complications in two animal models . Both transfusions and anesthesia (methoxyflurane) were found to increase susceptibility to peritoneal Escherichia coli infections . This susceptibility increased with time after the transfusions but decreased with time after anesthesia . Neither transfusions nor anesthesia altered susceptibility to an intravenous Escherichia coli challenge in rats. Surgery, 1989 Aug, 106(2), 439 - 43 Failure of tumor necrosis factor to produce hypotensive shock in the absence of endotoxin; Neilson IR et al.; Tumor necrosis factor (TNF) is reported to cause a shock syndrome similar to that produced by endotoxin (LPS) . The purpose of this study was to determine the relationship between TNF and LPS in causing shock . Eighty rats received infusions of either TNF, LPS, or TNF plus LPS, as compared with saline solution . Temperature, blood, and tissue specimens were obtained at 2 hours . Blood pressure was measured over 4 hours in a separate group of awake rats . Mortality was assessed over 24 hours . Neither TNF (1 mg/kg) nor LPS (1 mg/kg) altered hematocrit, blood gases, temperature, or caused hypotension or mortality . If the same dose of TNF was combined with LPS, however, there was significant (p less than 0.05) hemoconcentration and metabolic acidosis associated with hypotension and 100% mortality by 4 hours . Pathologic changes were restricted to the small intestine and occurred in this group only . It was concluded that TNF does not cause hypotension or shock in the rat . TNF will cause lethal shock, however, if combined with a sublethal dose of endotoxin . This suggests that synergy between TNF and endotoxin is important in septic shock. Surgery, 1989 Aug, 106(2), 216 - 22; discussion 222-3 Protein kinase C: a potential pathway of endothelial cell activation by endotoxin, tumor necrosis factor, and interleukin-1; Magnuson DK et al.; Human endothelial cells exposed to lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin-1 (IL-1) in vitro acquire a cell surface property that promotes the adherence of neutrophils (PMNs) . The common mechanism by which endothelial cells are activated by these agents is unknown . We examined adherence of PMNs to cultured human umbilical vein endothelium (HUVE) pretreated with LPS (100 ng/ml), TNF (100 U/ml), and IL-1 (1 U/ml) in medium alone or medium containing protein kinase inhibitors H-7 or HA-1004 . Both compounds inhibit a similar spectrum of protein kinases, but H-7 is an effective inhibitor of protein kinase C, whereas HA-1004 is not . We found that H-7 (25 mumol/L) reduced the adherence of PMNs to LPS-, TNF-, and IL-1-stimulated HUVE monolayers to 16.7% +/- 3.0%, 12.1% +/- 2.5%, and 18.3% +/- 2.9% of control, respectively (mean plus or minus standard error of three experiments); HA-1004 (25 mumol/L) did not inhibit endothelial adhesiveness . Cytotoxicity of H-7 was less than 10% in LPS-, TNF-, and IL-1-treated HUVE . Protein synthesis, as measured by the incorporation of tritiated amino acids, was not significantly impaired in LPS-treated HUVE concurrently exposed to H-7 . We conclude that protein kinase C appears to be a necessary common mediator of endothelial cell activation by LPS, TNF, and IL-1. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6087 - 91 Characterization and functional reconstitution of a soluble form of the hydrophobic membrane protein lac permease from Escherichia coli; Roepe PD et al.; Lac permease, a polytopic membrane protein from Escherichia coli, has been purified in soluble form by overexpressing the lacY gene by means of the T7 RNA polymerase system . Soluble permease is dissociated from membranes with urea or other chaotropes and appears after the membrane is saturated with newly synthesized permease . Remarkably, this form of the permease appears to remain soluble in phosphate buffer at neutral pH after removal of urea, although it aggregates in a time- and concentration-dependent manner . Importantly, soluble permease behaves as a monomer during size-exclusion chromatography with or without urea, contains less than 3 mol of organic phosphate per mol of protein, and is largely helical . Soluble permease binds p-nitrophenyl alpha-D-galactopyranoside approximately 40% as well as permease in the native environment of the membrane and can be reconstituted into phospholipid vesicles that catalyze lactose counterflow or active transport in response to a membrane potential (interior negative) . The results suggest that lac permease can assume a nondenatured conformation in aqueous solution. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6052 - 6 Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli; Forst S et al.; EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals . We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by {gamma-32P}-ATP . We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR . The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate . The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA . Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR . Furthermore, in a delta envZ strain of E . coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid . Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6023 - 7 Homology of aspartyl- and lysyl-tRNA synthetases; Gampel A et al.; The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced . The identity of the gene is confirmed by the following evidence . (i) The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase . (ii) In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis . (iii) Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA . The primary structures of the cytoplasmic and mitochondrial aspartyl-tRNA synthetases are similar to the yeast cytoplasmic lysyl-tRNA synthetase, suggesting that the two types of synthetases may have a common evolutionary origin . Searches of the current protein banks also have revealed a high degree of sequence similarity of the lysyl-tRNA synthetase to the product of the Escherichia coli herC gene and to the partial sequence of a protein encoded by an unidentified reading frame located adjacent to the E . coli frdA gene . Based on the sequence similarities and the map positions of the herC and frdA loci, we propose herC to be the structural gene of the constitutively expressed lysyl-tRNA synthetase of E . coli and the unidentified reading frame to be the structural gene of the heat-inducible lysyl-tRNA synthetase. Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5898 - 902 Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences; Traver CN et al.; A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA . A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells . A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate . This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters . Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence . An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene. Mutat Res, 1989 Aug, 213(2), 165 - 73 RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli; Shwartz H et al.; Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme . Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA . Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition . Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication . These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens. Metabolism, 1989 Aug, 38(8 Suppl 1), 6 - 13 Muscle glutamine concentration and protein turnover in vivo in malnutrition and in endotoxemia; Millward DJ et al.; A comparison of the changes in the concentration of glutamine {Gln} in skeletal muscle in a variety of catabolic states with the attendant changes in rates of protein synthesis and degradation indicates a number of substantial correlations which provide insight into both the way in which {Gln} is regulated in muscle and possible regulatory influences of {Gln} on protein balance . There is a striking direct correlation between {Gln} and the rate of protein synthesis in the whole data set . Further examination of this relationship in protein deficiency shows that the changes in {Gln} correlate mainly with the reductions in ribosomal concentration (RNA/protein) and with the decrease in the rate of protein degradation . Because the fall in {Gln} in protein deficiency is also correlated with the decrease in free T3 concentrations, it is suggested that in this case the correlations of {Gln} with rates of protein turnover may be incidental, reflecting thyroidal influences on both protein turnover and glutamine transport . In contrast, in endotoxemia the changes in {Gln} were highly correlated with the ribosomal activity, kRNA, and in this case {Gln} was inversely correlated with the rate of protein degradation . Similar correlated changes occur in starvation and in response to glucocorticoids, and it is suggested that the reductions in {Gln} in endotoxemia could be causally related to the development of insulin resistance and the inhibition of the translational phase of protein synthesis which occurs in these circumstances . The mechanism of the reduction in {Gln} and any linked inhibition of protein synthesis is unknown, but it is shown to be independent of prostaglandin production.(ABSTRACT TRUNCATED AT 250 WORDS) J Trauma, 1989 Aug, 29(8), 1076 - 84; discussion 1084-5 Complement depletion with Naje haje cobra venom factor limits prostaglandin release and improves visceral perfusion in porcine endotoxic shock; Fink MP et al.; We tested the hypothesis that complement (C')-dependent release of prostaglandin (PG) I2 is an important factor contributing to the development of hypotension and low systemic vascular resistance index (SVRI) in endotoxic shock . Two groups (n = 7) of pentobarbital-anesthetized pigs (12-15 kg) were infused over 40 min with Escherichia coli lipopolysaccharide (LPS; 200 micrograms/kg) and continuously resuscitated with normal saline (1 ml/kg min): LPS-Control (no pretreatment) and LPS-Decomplemented (pretreatment 18 hr before study with 500-1,500 units of Naje haje cobra venom factor, CVF) . Prior treatment with CVF: i) decreased the mean titer of total hemolytic C' to 15.9% of pretreatment levels; ii) significantly decreased post-LPS plasma concentrations of immunoreactive TxB2 (TxA2 metabolite) and 6-keto-PGF1 alpha (PGI2 metabolite); iii) abrogated the early transient decrease in cardiac index observed in the LPS-Control group; iv) tended to improve post-LPS visceral perfusion assessed using radioactive microspheres; and v) had no discernible effect on the late sustained decrease in SVRI observed following infusion of LPS . We conclude that C' activation is a major determinant of LPS-induced prostanoid release in vivo, although our results do not support the view that C'-dependent release of PGI2 is an important factor contributing to low SVRI in resuscitated endotoxic shock. J Infect Dis, 1989 Aug, 160(2), 243 - 7 Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center; Bower JR et al.; Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization . Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact . Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters . All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein . Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II . Heat-labile and heat-stable enterotoxins were not present by gene probe analysis . Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis . The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism. Eur J Biochem, 1989 Aug 1, 183(2), 371 - 9 Iron metabolism of Escherichia coli studied by Mössbauer spectroscopy and biochemical methods; Matzanke BF et al.; To date it has barely been recognized that the nature of about 75% of the Escherichia coli iron pool is unknown . Here we report the isolation of two iron species representing major components of iron metabolism in various growth states of E . coli . In vivo Mossbauer spectroscopy was applied to obtain information on the intracellular distribution pattern of iron in E . coli K12 W3110 . Only two types of iron could be detected in the cell spectra: hexacoordinated Fe2+ and Fe3+ high-spin complexes . Other iron-requiring compounds are at least one order of magnitude less abundant in E . coli . The Mossbauer parameters of these complexes fit neither cytochromes nor iron-sulfur proteins nor ferric holo-bacterioferritin . They are sensitive to metabolic changes and inhibitors . The ratio of Fe/subunit, Fe2+/Fe3+ interconversion, chromatographic and electrophoretic data exclude bacterioferritin as the main iron metabolite in E . coli . Bacterioferritin can be observed only at very high ferric ion concentrations in the medium . The 55Fe fluorograms of both cytoplasmic and membrane fractions exhibit two exclusive bands with apparent molecular masses of 17 and 15 kDa, respectively . The two bands comprised 70% of the applied radioactivity . In gel filtration the main iron peak elutes at 155 kDa yielding two bands with apparent molecular masses of 17 and 15 kDa on SDS/PAGE . We therefore conclude that the iron species form a protein with an apparent molecular mass of 155 kDa containing 17-kDa and 15-kDa subunits . The iron content of the protein is 44 micrograms Fe/mg protein which corresponds to approximately 13 iron ions/subunit . No iron protein exhibiting the observed features has been described so far . Additional Mossbauer experiments suggest that these novel iron proteins are not restricted to E . coli but that similar components are detectable in several bacterial and fungal systems, thus pointing to a general occurrence. Eur J Biochem, 1989 Aug 1, 183(2), 339 - 45 Modulation of arginine decarboxylase activity from Mycobacterium smegmatis . Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme; Balasundaram D et al.; Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity . The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa . The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation . The enzyme is specific for arginine (Km = 1.6 mM) . The holoenzyme is completely resolved in dialysis against hydroxylamine . Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature . The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths . A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations . The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme . Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function . Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively . These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme. Eur J Biochem, 1989 Aug 1, 183(2), 311 - 6 Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene; Harford S et al.; In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity . One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity . A serine to phenylalanine mutation appears to be responsible for the loss of toxicity. Eur J Biochem, 1989 Aug 1, 183(2), 281 - 4 The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes; Remme J et al.; The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in {32P}orthophosphoric acid for about four generations . The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids . This procedure preferentially labels growing peptidyl chains . Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis . The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site . The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model. Ann Surg, 1989 Aug, 210(2), 239 - 45 Peripheral blood leukocyte kinetics following in vivo lipopolysaccharide (LPS) administration to normal human subjects . Influence of elicited hormones and cytokines; Richardson RP et al.; Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol . This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects . A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers . Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol . Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion . A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion . A profound monocytopenia (p less than 0.01) was noted one hour after infusion . Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion . During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01) . There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations . A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage . Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes . The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration . This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets. J Bacteriol, 1989 Aug, 171(8), 4494 - 7 Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli; Damagnez V et al.; The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III . The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired . We show that the mismatch repair system of mutD5 strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication . This recovery depends on de novo protein synthesis. J Bacteriol, 1989 Aug, 171(8), 4479 - 85 Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I; Weglenski P et al.; Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I (NRI), the product of glnG, were obtained by two different selection procedures . The mutant proteins were purified and characterized . The concentrations of mutant proteins needed to activate transcription at the glnAp2 promoter were three to four times lower than that of the wild-type NRI . The rate of phosphorylation of these proteins and the stability of mutant NRI phosphate were found to be similar to those of the wild-type NRI . In one of the mutants, the site of the mutation was localized in the DNA region specifying the central domain of NRI. J Bacteriol, 1989 Aug, 171(8), 4457 - 65 Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon; Xie QW et al.; Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli . We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes . We have now studied the upstream promoter and the downstream terminator regions of this operon more completely . We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene . Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t . An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t . By comparing our DNA fragments with those prepared from this region of the E . coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E . coli chromosome . We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E . coli chromosomal map. J Bacteriol, 1989 Aug, 171(8), 4349 - 54 Sequence and overexpression of the menD gene from Escherichia coli; Popp JL; The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase . DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein . Possible promoter and ribosome binding sites are present . Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of {35S}methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector . This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes. J Bacteriol, 1989 Aug, 171(8), 4315 - 9 Isolation and mapping of Escherichia coli mutations conferring resistance to division inhibition protein DicB; Labie C et al.; Temperature-sensitive dicA mutants of Escherichia coli, dicA1(Ts), are blocked for cell division, owing to derepressed expression of a division inhibition gene, dicB . We isolated mutants which survived a high temperature in the dicA1 background and which survived induced expression of dicB carried by a high-copy-number plasmid . Most of the mutations conferred very slow growth on the cells . Two were mapped to the 90-min cluster of genes involved in translation and transcription, in or very close to gene rpoB . The majority of the other mutations were found to cause variable degrees of minicell formation and to map within or very close to the minB locus . Contrary to these mutations, the canonical min-1 mutation did not confer resistance to DicB. J Bacteriol, 1989 Aug, 171(8), 4303 - 14 Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA; Mulder E et al.; The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants . In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random . We conclude that constriction positioning depends in an unknown way on nucleoid replication activity. J Bacteriol, 1989 Aug, 171(8), 4241 - 7 Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358; Nucifora G et al.; The structural basis for induction of the mercury resistance operon with inorganic mercury and with the organomercurial compound phenylmercuric acetate was addressed by DNA sequencing analysis and by lac fusion transcription experiments regulated by merR in trans from broad-spectrum-resistance plasmid pDU1358 (Hg2+ and phenylmercury responding) . The lac fusion results were compared with those from a narrow-spectrum-resistance (Hg2+ responding but not phenylmercuric responding) operon and the pDU1358 merR deleted at the 3' end . The nucleotide sequence of the beginning region of the broad-spectrum mer operon of plasmid pDU1358 was determined, including that of the merR gene, the operator-promoter region, the merT and merP genes, and the first 60% of the merA gene . Comparison of this sequence with DNA sequences of narrow-spectrum mer operons from transposon Tn501 and plasmid R100 showed that a major difference occurred in the 3' 29 base pairs of the merR gene, resulting in unrelated C-terminal 10 amino acids . A hybrid mer operon consisting of the merR gene from pDU1358, a hybrid merA gene (determining mercuric reductase enzyme), and lacking the merB gene (determining phenylmercury lyase activity) was inducible by both phenylmercury and inorganic Hg2+ . This shows that organomercurial lyase is not needed for induction by organomercurial compounds . A mutant form of pDU1358 merR missing the C-terminal 17 amino acids responded to inorganic Hg2+ but not to phenylmercury . Thus, the C-terminal region of the MerR protein of the pDU1358 mer operon is involved in the recognition of phenylmercury. J Bacteriol, 1989 Aug, 171(8), 4207 - 16 Transcription mapping of the Escherichia coli chromosome by electron microscopy; French SL et al.; The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli . On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E . coli . The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E . coli chromosome; over 600 kilobase pairs in the 84- to 90-min and 72-min regions can now be recognized . Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities . As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rrnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs . The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters. J Bacteriol, 1989 Aug, 171(8), 4170 - 7 Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis; Couto LB et al.; Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea . Following fixation of mutations in E . coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E . coli 6451 (recA Strr) . Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host . Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent . Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis . The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells . When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels . In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction . This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase . These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced . The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs. J Bacteriol, 1989 Aug, 171(8), 4112 - 20 Stabilization of the 3' one-third of Escherichia coli ribosomal protein S20 mRNA in mutants lacking polynucleotide phosphorylase; Mackie GA; Mutations which largely inactivate polynucleotide phosphorylase and which render RNase II thermolabile exert two effects on the metabolism of the two nested mRNAs which encode ribosomal protein S20 . (i) The lifetime of both mRNA species is extended 2.5-fold at 38 degrees C in a strain harboring both mutations . (ii) A relatively stable truncated fragment of these mRNAs accumulates to significant levels in strains lacking polynucleotide phosphorylase . The truncated RNA (Po RNA) is 147 to 148 residues long and is coterminal with the 3' ends of intact S20 mRNAs . Its 5' end appears to be generated by endonucleolytic cleavage to the 5' side of a G residue in the sequence AACCGAUC . The data are consistent with the hypothesis that S20 mRNAs can be degraded by alternative pathways . The normal pathway depends on functional polynucleotide phosphorylase and is concerted, since S20 mRNAs disappear without accumulation of detectable intermediates in the decay process . The slower alternative pathway is followed when polynucleotide phosphorylase is inactivated by mutation . This pathway is distinguished by segmental rather than concerted degradation of S20 mRNAs and involves at least one endonucleolytic cleavage . The 5' two-thirds of S20 mRNAs decays significantly more quickly than the 3' third in this latter mode of mRNA turnover. Endocrinology, 1989 Aug, 125(2), 1097 - 9 Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies; Chang CS et al.; Complementary DNA segments that encode different domains of human and rat androgen receptors were fused to the Escherichia coli trpE gene using pATH expression vectors . Fusion proteins expressed by the bacteria were used to immunize rats and rabbits to obtain polyclonal antibodies to androgen receptors . Spleen cells of immunized rats were fused with myeloma cells to obtain stable hybridomas that produced monoclonal antibodies . Gradient centrifugation and immuno-precipitation assays indicated that the antibodies interacted with androgen receptors specifically. Circ Res, 1989 Aug, 65(2), 502 - 14 Effects of recombinant human tumor necrosis factor alpha, lymphotoxin, and Escherichia coli lipopolysaccharide on hemodynamics, lung microvascular permeability, and eicosanoid synthesis in anesthetized sheep; Kreil EA et al.; We infused recombinant human tumor necrosis factor alpha (rhTNF alpha), lymphotoxin (rhLT), and Escherichia coli 0111:B4 lipopolysaccharide (LPS) into anesthetized sheep with a lung lymph fistula to compare their effects on systemic and pulmonary hemodynamics, lung lymph dynamics, and eicosanoid release . rhTNF alpha (25-150 micrograms/kg, n = 6 sheep), but not rhLT (25 micrograms/kg, n = 3), rapidly increased lung lymph and plasma levels of 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and caused profound systemic vasodilation and hypotension . Meclofenamate pretreatment (10 mg/kg) of three other sheep given 25 micrograms/kg rhTNF alpha prevented the increase of lymph and plasma 6-k-PGF1 alpha levels, systemic vasodilation, and the early (less than 2 hrs) but not the late (4-6 hours) hypotension caused by rhTNF alpha . LPS (1 micrograms/kg, n = 11) induced a briefer increase of lymph 6-k-PGF1 alpha levels than did rhTNF alpha while plasma 6-k-PGF1 alpha levels did not increase . LPS induced more gradual hypotension than did rhTNF alpha but did not cause systemic vasodilation . LPS and rhTNF alpha, but not rhLT, increased lymph thromboxane B2 (TXB2) levels during the first hour of study, whereas only LPS acutely increased plasma TXB2 levels . LPS caused acute pulmonary vasoconstriction and greater acute pulmonary artery hypertension than did either rhTNF alpha or rhLT . Whereas LPS-treated sheep required less fluid transfusion than rhTNF alpha-treated sheep to maintain mean systemic arterial pressure greater than 50 mm Hg, LPS infusion caused a greater increase of lung lymph protein clearance . rhTNF alpha caused minimal alterations of lung microvascular permeability . We conclude that eicosanoid mediators contribute importantly to differences of systemic and pulmonary hemodynamics caused by these agents in sheep . rhTNF alpha cannot account for all of the LPS-induced hemodynamic, lung lymph, and eicosanoid responses in sheep. Cell Immunol, 1989 Aug, 122(1), 262 - 73 Molecular mimicry between uveitopathogenic site of retinal S-antigen and Escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction; Singh VK et al.; Experimental autoimmune uveitis (EAU) is caused by the immunization of microgram amounts of a soluble retinal protein, known as S-antigen, in susceptible animal strains including primates . The disease serves as an animal model of ocular inflammation . We induced EAU and pinealitis in Lewis rats with small synthetic peptides, corresponding to the amino acid sequence in Escherichia coli protein, which contains six consecutive amino acids identical to a uveitopathogenic site in human S-antigen (peptide M) . EAU and pinealitis induced in rats by synthetic peptide derived from E . coli was indistinguishable from those induced by native S-antigen or other uveitopathogenic synthetic peptides corresponding to the amino acid sequence of S-antigen . Furthermore, lymph node cells from animals immunized with either peptide M or peptide derived from E . coli protein showed significant proliferation in the presence of either peptide when tested in vitro for lymphocyte mitogenesis using {3H}thymidine . Thus, molecular mimicry, a process by which an immune response directed against a nonself protein cross-reacts with a normal host protein, may play a role in autoimmunity. Infect Immun, 1989 Aug, 57(8), 2553 - 8 The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity; Pinkney M et al.; Site-directed mutagenesis of the TGC codon in a cloned streptolysin O (SLO) gene exchanged the single Cys residue in SLO for either Ala or Ser . The parent wild-type SLO (SLO.Cys-530) and the SLO.Ala-530 and SLO.Ser-530 mutant toxins, expressed in Escherichia coli, were purified and analyzed . Wild-typ