J Exp Med, 1992 May 1, 175(5), 1317 - 26 Membrane sorting during phagocytosis: selective exclusion of major histocompatibility complex molecules but not complement receptor CR3 during conventional and coiling phagocytosis; Clemens DL et al.; We have used immunocytochemical techniques and enzyme cytochemistry to examine the distribution of plasma membrane proteins during coiling phagocytosis of Legionella pneumophila and conventional phagocytosis of Escherichia coli . Whereas class I and class II major histocompatibility complex (MHC) molecules are relatively excluded from nascent phagosomes during conventional and coiling phagocytosis, the CR3 complement receptor persists in nascent phagosomes . The staining pattern for alkaline phosphatase activity resembles that of MHC molecules, with a marked exclusion of phosphatase activity from L . pneumophila coils and nascent phagosomes . The staining pattern for 5'-nucleotidase activity, on the other hand, resembles that of CR3 with intense staining in the inner layers of L . pneumophila coils . These results demonstrate that the cell has the ability to exclude selectively certain membrane proteins from the nascent phagosome during phagocytosis, thereby producing a phagosomal membrane markedly different from the plasma membrane from which it is derived. J Infect Dis, 1992 May, 165(5), 873 - 8 Inhibition of production of tumor necrosis factor by monoclonal antibodies to lipopolysaccharides; Vacheron F et al.; Murine monoclonal antibodies to lipopolysaccharides (LPS) from rough J5 mutant of Escherichia coli O111:B4 protected D-galactosamine-treated mice against lethal effects of LPS and provided protection in an experimental infection model with live E . coli . Three previously prepared anti-LPS antibodies were evaluated for ability to inhibit LPS-induced tumor necrosis factor (TNF) secretion . In vivo, TNF production was induced in mice treated with D-galactosamine and challenged with LPS . Two antibodies (D6B3 and D6B4) decreased serum TNF levels and prevented lethal effects of LPS . Nonprotective anti-LPS antibody (D9A2) and an unrelated antibody to neomycin did not reduce circulating TNF levels after LPS challenge . Pretreatment of mice with D6B3 and D6B4 protected mice from infection with E . coli and prevented serum TNF increases induced by E . coli infection . In nonprotected animals, high levels of TNF were detected in serum 3-6 h after infection; animals died within 24 h . In vitro, addition of protective antibodies to macrophage cultures at initiation of LPS stimulation inhibited TNF production . Nonprotective antibody D9A2 failed to block LPS-induced TNF production. J Bacteriol, 1992 May, 174(9), 3078 - 82 Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle; Smith DW et al.; A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication . Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle. J Bacteriol, 1992 May, 174(9), 3004 - 10 Site-specific proteolysis of mini-F plasmid replication protein RepE destroys initiator function and generates an incompatibility substance; Kline BC et al.; Plasmid F replication is controlled by a plasmid-specified Rep protein with both autorepressor and initiator functions . The mechanism by which these two functions of a Rep protein are balanced to achieve stable replication is unknown; however, we speculated in prior work that Rep protein modification could be involved . We report here that naturally proteolyzed F RepE protein has been detected and characterized . The processed molecule lost the first 17 N-terminal aminoacyl residues and initiator function but acquired increased specific DNA-binding affinity in the presence of Escherichia coli chromosomal DNA . When supplied in trans, the altered protein acts as an incompatibility substance and eliminates maintenance of F'lac . These findings indicate that protein processing has the potential to contribute to the overall control of DNA replication. J Bacteriol, 1992 May, 174(9), 2881 - 90 Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression; Barbier CS et al.; In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon . Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine . A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction . Determination of the ability of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP . CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced . Data obtained with cytR delta M149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein . Representative cytR mutations were combined with cytR delta M149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E . coli strain . Amino acid substitutions A209E and C289Y suppressed the transdominance of CytR delta M149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association . In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytR delta M149 . The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression. J Bacteriol, 1992 May, 174(9), 2843 - 50 Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Ruijter GJ et al.; Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity . Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc . Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme . Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr . Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled . Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc . These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate. J Bacteriol, 1992 May, 174(9), 2779 - 84 The lrp gene product regulates expression of lysU in Escherichia coli K-12; Lin R et al.; In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant . Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent . The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion . A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein . The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA. Cancer Res, 1992 May 1, 52(9), 2389 - 93 Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene; Owens JK et al.; We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents . In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs . The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein . Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein . The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested . The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein . This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine . On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels . We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene . Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme. Arch Biochem Biophys, 1992 May 1, 294(2), 434 - 9 Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179; Ma Q et al.; We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q . Ma, R . Wang, C . S . Yang, and A . Y . H . Lu, 1990, Arch . Biochem . Biophys . 283, 311-317) . Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis . Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction . The mutant enzymes were expressed in E . coli . The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis . The mutant proteins were purified to apparent homogeneity . The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type . The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled . Both mutant proteins contained two molecules of FAD per enzyme molecule . Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M . Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes . It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol . However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme. Virology, 1992 May, 188(1), 67 - 76 Genetic manipulation of African swine fever virus: construction of recombinant viruses expressing the beta-galactosidase gene; Rodriguez JM et al.; Homologous recombination is shown to be specifically induced in Vero cells by infection with African swine fever (ASF) virus . The frequency of recombination induced by ASF virus infection between cotransfecting plasmids is comparable to that found after infection with the prototype poxvirus, vaccinia virus . The induction of recombination is accompanied by replication of the plasmid templates in the ASF virus-infected cells . An ASF virus insertion/expression plasmid vector containing the Escherichia coli reporter gene beta-galactosidase (beta-gal) fused to a viral promoter sequence was constructed . Recombination between homologous sequences present in both the plasmid vector and the virus genome led to the generation of recombinant viruses expressing the beta-gal gene . Visual screening of beta-gal+ plaques allowed the isolation and plaque purification of recombinant ASF viruses . The characterization of a beta-gal+ virus isolate showed that the beta-gal gene had been stably inserted into the thymidine kinase locus of the virus genome, thus demonstrating that controlled genetic manipulation of ASF virus can be achieved by homologous recombination in infected cells. Infect Immun, 1992 May, 60(5), 2058 - 65 Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen; Haapasalo M et al.; Treponema denticola surface proteins were studied for their biochemical and biological characteristics . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins . Antiserum raised against whole cells of T . denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins . Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell . SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa . Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole-cell antiserum and anti-53-kDa-protein antibody . The aggregates dissociated into their subunits after heating to 70 degrees C . Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5 . The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested . A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T . denticola ATCC 35405 . The recombinant protein had exactly the same molecular mass as the major 53-kDa T . denticola surface protein and reacted with antisera raised against this protein . The role of T . denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay . Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T . denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed . Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA . Our results suggest that the 53-kDa major surface protein of T . denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species. J Virol, 1992 May, 66(5), 2717 - 23 The vaccinia virus B1R gene product is a serine/threonine protein kinase; Lin S et al.; The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases . To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase . Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1 . Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide . Casein and histone H1 were phosphorylated on serine and threonine residues . The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism . The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain . The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent . Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle . Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase. Eur J Biochem, 1992 May 1, 205(3), 1115 - 21 Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product; Wolber V et al.; The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter . The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents . Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31% . GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein . Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1) . An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1 . Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated . Correspondingly, our Nef preparation shows a low, but significant, ATPase activity . In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded. Blood, 1992 May 1, 79(9), 2196 - 200 Interleukin-1 receptor antagonist circulates in experimental inflammation and in human disease; Fischer E et al.; Interleukin-1 receptor antagonist (IL-1ra) is a 22-Kd protein that shares homology with IL-1 beta, binds to the IL-1 receptor, but has no known agonist properties . This inhibitor appears to be the first cytokine whose sole function is to block the actions of another cytokine . Exogenous IL-1ra administration has been shown to reduce mortality in experimental septic shock . We now report that IL-1ra is endogenously produced and circulates in experimental inflammation and in clinical disease . After experimental endotoxemia in human volunteers, IL-1ra concentrations increase from a baseline concentration of 460 +/- 200 pg mL-1 to 14,870 +/- 290 pg mL-1 at 3 hours (P less than .05) . IL-1ra is also detectable in all plasma samples from critically ill patients with a mean concentration of 8,680 +/- 2,060 pg mL-1 (range 320 to 55,370 pgs mL-1) . In nonhuman primates, Escherichia coli septic shock induces elevated plasma levels of IL-4ra (P less than .05) . However, in animals that eventually succumb to septic shock, Il-1ra appears in quantities presumed inadequate to block the pathologic sequelae associated with high IL-1 beta levels . The findings suggest that IL-1ra may play a role in modulating the systemic host responses to a variety of nonlethal disease states by altering the balance between cytokines and their antagonists. Photochem Photobiol, 1992 May, 55(5), 783 - 8 Effect of psoralen + ultraviolet-A on the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg; Esaki K et al.; It was shown that psoralen + UV-A inhibits the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg . This reaction required oxygen and there is a high possibility that the active oxygen species was in a singlet state . Oxygen did not act directly on C5a des Arg, but rather produced oxidized psoralen which inhibited C5a des Arg activity . The effect was dependent on the concentration and types of psoralen and on the UV-A dose . Among the psoralens, the inhibitory effect of 4,5',8-trimethylpsoralen was the strongest, followed by 8-methoxypsoralen and 3-carbethoxypsoralen and finally 4,4',6-trimethylangelicin . Psoralen + UV-A failed to inhibit the chemotactic activity towards chemotactants other than anaphylatoxin C5a such as casein, the cultured filtrate of E . coli, platelet-activating factor, leukotriene B4, 5-hydroxy-6,8,11,14-eicosatetraenoic acid and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid. Photochem Photobiol, 1992 May, 55(5), 723 - 7 Further characterization of monoclonal antibody indicates specificity for (6-4)-dipyrimidine photoproducts; Strickland PT et al.; Monoclonal antibody aUVssDNA-1 is produced by hybridoma cell line 25JF.C3B6 originally selected from cell fusions using spleen cells from mice immunized with UV-irradiated polydeoxynucleotides (Strickland and Boyle, Photochem . Photobiol . 34, 595-601, 1981) . Original and subsequent studies of the binding characteristics of aUVssDNA-1 indicated that it was specific for cyclobuta-dithymidine photoproducts . Those investigations examined action spectrum, short-wavelength photo-reversal, nucleotide sequence effects, and photoreactivation using E . coli photolyase and incandescent light . However, the more recent studies reported here examined acetophenone-UV-B photosensitization, UV-B photoisomerization, and photoreactivation using cloned E . coli photolyase and filtered incandescent light . The results indicate that aUVssDNA-1 recognizes photoproducts with characteristics of (6-4)-dipyrimidines . Thus, previous studies in which relatively rapid repair of cyclobuta-dithymidine photoproducts was inferred using this antibody, require re-interpretation in light of these new findings. J Periodontol, 1992 May, 63(5), 405 - 11 Effect of exogenously applied prostaglandin E2 on alveolar bone loss--histometric analysis; Miyauchi M et al.; The effect of prostaglandin E2 (PGE2) on alveolar bone resorption was examined in 8-week old Wistar rats by histometric analysis . One mg/ml PGE2 topically applied to gingival sulcus induced a marked increase in osteoclasts . The number of osteoclasts increased progressively and reached a maximum at 12 hours . Ultrastructurally, these osteoclasts were in active form with well developed ruffled borders and clear zones . The changes in numbers of osteoclasts after application of various concentrations of PGE2 were dose-dependent (0.001 to 1.0 mg/ml), but higher concentrations of PGE2 (2 mg/ml) were less effective . In addition, the number of osteoclasts in groups treated with both PGE2 and endotoxin was higher than those that received PGE2 only . These results indicate that bone resorption caused by PGE2 depends on activation and increase of osteoclasts, and suggests that endogenous PGE2 production by host cells stimulated by plaque-associated bacterial endotoxin may be an important pathogenetic factor in periodontal disease. J Periodontol, 1992 May, 63(5), 397 - 404 The effect of cyclosporin and lipopolysaccharide on fibroblasts: implications for cyclosporin-induced gingival overgrowth; Barber MT et al.; Drug-induced gingival overgrowth is an adverse side effect associated principally with 3 different types of drugs; specifically the antiepileptic phenytoin, the calcium channel antagonist nifedipine, and the immunosuppressant cyclosporin . The present study has analyzed the effect of cyclosporin and lipopolysaccharide on fibroblasts from 3 different sources: 1) normal healthy human gingiva (NHGF); 2) overgrown gingiva from 2 patients taking cyclosporin (CHGF); and 3) human fetal lung (WI-38) . Fibroblasts isolated from cyclosporin-associated gingival overgrowth were significantly less responsive to cyclosporin in terms of DNA, total protein, and proteoglycan synthesis . This finding supports the in vivo response where few fibroblasts are seen but marked overgrowth of fibrous tissue occurs . Lipopolysaccharide derived from Fusobacterium nucleatum and Escherichia coli was capable of inhibiting DNA synthesis significantly in all 3 fibroblast types . Total protein synthesis by CHGF cells was inhibited differentially by Fusobacterium nucleatum LPS and addition of cyclosporin to this system resulted in reversal of the inhibition . A synergistic effect was noted when the proteoglycan output of NHGF cells was assessed in response to co-incubation with cyclosporin and Escherichia coli LPS . The study shows that bacterial LPS may be an important co-factor in the pathogenesis of cyclosporin-induced gingival overgrowth. Mikrobiologiia, 1992 May-Jun, 61(3), 464 - 71 {Electrophysical analysis of Escherichia coli cell damage caused by silver ions}; Ivanov AIu et al.; The influence of Ag+ (0.5-10 microM) on Escherichia coli K-12 cells was studied by electrophoresis and electro-orientation spectroscopy methods . It was shown that the pH-dependency of the cell electrokinetic potential (phosphate-citrate buffer with ion strength 0.02) practically didn't changed after Ag+ treatment, but in low-conductive media electrophoretical mobility of intact and inactivated by heat (70 degrees, 15 min) cells gradually decreased as the Ag+ concentration increased . It was due to the Ag+ adsorption on the cell surface and could not be used for the definite characterization of the cell damage . The high-frequency decrease in the cell electro-orientation spectrum shifted to the region of lower frequencies, K+ was excreted by cells, slight raise of the medium pH occurred and significant changes of cell osmotic properties were observed as a result of Ag+ action . All these changes showed the disturbance of barrier properties of the cytoplasmic membrane . Besides the damaging action of Ag+ on cell membranes increased with the decrease of pH and decreased after the addition of Mg2+, Ca2+ and Sr2+ in low concentrations. Mikrobiologiia, 1992 May-Jun, 61(3), 455 - 63 {Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells}; Ivanov AI et al.; The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods . It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes . The degree and the character of these changes depend on cation valency and the initial value of cell EM . At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases . Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane . Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed . By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+ . Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface. Anal Biochem, 1992 May 1, 202(2), 268 - 74 Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate; Watanabe Y et al.; The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant . A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration . When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins . Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution . When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite . On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS . When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides . The mechanism of binding of the protein polypeptides was discussed on the basis of these results . The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite. New Biol, 1992 May, 4(5), 581 - 90 In situ lacZ retrovirus-marked lymphocytes define a B cell microenvironment in the lymph node medulla; Krall WJ et al.; A recombinant nonreplicating retroviral vector bearing the Escherichia coli lacZ indicator gene was used to mark a population of B cells in situ in murine lymphoid tissue . The retrovirus was surgically injected into popliteal lymph nodes during the primary immune response to DNP-CGG when B cell proliferation in the germinal centers was maximal . LacZ+ cells were initially detected in the perivascular medullary interstitium, where they expanded and persisted up to 2 weeks following retrovirus injection . Migrant lacZ+ B cells were detected in the spleen 3-18 weeks following immunization and resided in the red pulp or marginal zones . Two-color flow cytometric analysis using a fluorogenic substrate for beta-galactosidase revealed that lacZ+ cells bear kappa light chains and that at least 50% of these cells bound the hapten, DNP . Based on their location, life span, migratory capacity, antigenic specificity, and surface immunoglobulin density, lacZ+ cells define a distinct nonfollicular B cell population associated with other late developmental stages of B lymphocytes, including memory and plasma cells. New Biol, 1992 May, 4(5), 569 - 80 Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping; Kelley WL et al.; The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma . The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins . R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome . We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo . Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins . We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation . A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin . Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin . Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin . Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping. New Biol, 1992 May, 4(5), 520 - 6 Context rules of rightward overlapping reading; Peter K et al.; We have investigated the mechanism and sequence context rules governing ribosome frameshifting promoted by aminoacyl-tRNA limitation . In the case of one shifty sequence, frameshifting promoted by lysyl-tRNA limitation occurs at the sequence AAG C and is due to rightward movement of the ribosome so as to read the AGC triplet overlapping the hungry codon from the right . The frequency of this event is unaffected by sequence elements more than three bases to the left (upstream) or two bases to the right (downstream) of the hungry codon, and only slightly affected by the identity of the base two bases to the right . It is strongly affected by the base immediately to the right of the hungry codon, which becomes the wobble base of the shifted triplet; and by the third base of the hungry codon, even though the two synonyms (AAG and AAA) call for the same aminoacyl-tRNA; and by the identity of the base immediately to the left of the hungry codon . The latter result suggests that the aminoacyl-tRNA in the P site affects the maintenance of reading frame at the adjacent A site of the ribosome . However, the DNA sequence makes it seem unlikely that the P-site tRNA shifts to the right in concert with the A-site tRNA, a mechanism that can account for leftward frameshifting (in the opposite direction) in retroviral translation . The specificity of sequence determinants of leftwing versus rightwing frameshifting is discussed. AIDS Res Hum Retroviruses, 1992 May, 8(5), 537 - 43 In vivo derived HIV-1 nef gene products are heterogeneous and lack detectable nucleotide binding activity; Harris M et al.; Multiple HIV-1 nef genes were cloned from lymphocyte DNA of asymptomatic seropositive individuals by polymerase chain reaction (PCR) . Sequence analysis of these clones revealed a unique set of nef variants with premature terminations (PCRnef 1 and 6), mutations at sites of potential posttranslational modification (PCRnef 2 and 3) and deletions . In common with laboratory isolates of nef, strong sequence conservation was observed in the central domain of nef and in the myristylation target sequence, with variable domains toward the N- and C-termini of the molecule . The biochemical function of nef remains elusive however, as the products of these genes cloned into a bacterial expression system failed to reveal any nucleotide binding activity. Biotechniques, 1992 May, 12(5), 718 - 21 DH11S: an Escherichia coli strain for preparation of single-stranded DNA from phagemid vectors; Lin JJ et al.; A new E . coli strain DH11S {mcrA delta(mrr-hsdRMS-mcrBC) delta(lac-proAB) delta(rec1398) deoR rpsL srl- thi-/F'proAB+ lacIqZ delta M 15} has been constructed . Transformation of DH11S competent cells with any of several different phagemid vectors {pSPORT1, pBluescript II SK(+), pGEM11Zf(+)} results in the production of highly purified single-stranded DNAs upon the addition of M13KO7 helper phage . Contamination by double-stranded DNAs was observed with all the other studied strains (XL1-Blue, JM109, DH5 alpha F'IQ) . The optimal yield of single-stranded DNA production was obtained when glycerol stocks made from stationary phase cells or single colonies from overnight ampicillin plates of DH11S containing the phagemid vector were infected with M13KO7 helper phage using a wide range (1 to 100) of multiplicities of infection . Five different pSPORT1 clones containing cDNA inserts of various lengths (0.3 kb to 2.0 kb) were compared using these four different bacterial strains . The use of strain DH11S results in the best yields and quality of single-stranded DNA . Therefore, DH11S appears to be the best all-around host for various applications that require single-stranded DNA such as DNA sequencing, in vitro mutagenesis and construction of subtractive cDNA libraries. Plasmid, 1992 May, 27(3), 231 - 6 Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid; Tam JE et al.; The hypothetical origin of replication for the 7.5-kb plasmid common to Chlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region . To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar of C . trachomatis was examined by electron microscopy . The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2 . In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7-10 . The evidence presented suggests that C . trachomatis has a homologue to the Escherichia coli dnaA gene and that this homologue might be involved in replication of the C . trachomatis 7.5-kb plasmid. Antimicrob Agents Chemother, 1992 May, 36(5), 977 - 81 Pharmacologic modulation of interleukin-1 expression by amphotericin B-stimulated human mononuclear cells; Cleary JD et al.; Fever and chills occur frequently with amphotericin B (AB) administration, but the mechanism that causes these reactions has not been definitively established . A variety of proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor, have been shown to be important mediators of fever . In order to clarify the cellular and biochemical responses associated with AB-induced fever, the experiments described sought to (i) establish whether human mononuclear cells exposed to AB in vitro expressed IL-1 beta, (ii) evaluate whether clinically used premedications for fever prophylaxis in AB-treated patients were effective in down-regulating IL-1 beta expression in vitro, (iii) evaluate whether methylxanthine agents with immunomodulatory actions effected in vitro IL-1 beta expression, and (iv) define the dose and time dependency of the modulating effects . Peripheral blood mononuclear cells were isolated by density centrifugation and resuspended to 10(6) cells per ml in culture wells of Linbro plates . When cocultured for 2 h with human mononuclear cells, both Escherichia coli lipopolysaccharide and AB stimulated IL-1 beta expression in a dose-related fashion . AB-induced IL-1 beta expression was suppressed by hydrocortisone (HC), pentoxifylline, and an investigational theobromine, A81-3138, in a linear, dose-related manner . In contrast, indomethacin, meperidine, and diphenhydramine had no effect on IL-1 beta expression . Our in vitro data indicate that serum HC concentrations of greater than 1 to 2 micrograms/ml may be sufficient to modulate IL-1 beta expression . Pentoxifylline and A81-3138 may also be effective in modulating IL-1 beta expression by mononuclear cells at concentrations achievable in serum . These new agents may prove to be effective alternatives to HC or may be added with HC to suppress febrile reactions secondary to AB administration . Clinical studies with pentoxifylline as a premedication for AB seem warranted. Antimicrob Agents Chemother, 1992 May, 36(5), 1151 - 4 Genetics and regulation of outer membrane protein expression by quinolone resistance loci nfxB, nfxC, and cfxB; Hooper DC et al.; Quinolone resistance mutations (cfxB1, marA1, and soxQ1) that reduce porin outer membrane protein OmpF map near 34 min on the Escherichia coli chromosome . Another such mutation, nfxC1, was found in strain KF131 (nfxB, 19 min) . nfxC1 and cfxB1 mutants (selected with quinolones) differed slightly but reproducibly from marA1 (selected with tetracycline) and soxQ1 (selected with menadione) mutants in quinolone resistance and linkage to zdd2208::Tn10kan (33.7 min) . For nfxB nfxC1 and cfxB1 mutants, as previously shown for marA mutants, resistance and reduced OmpF required the micF locus encoding an antisense RNA complementary to ompF mRNA and were associated with increased micF expression. Nippon Seikeigeka Gakkai Zasshi, 1992 May, 66(5), 525 - 38 {Immunohistochemical localization of interleukin-1 and lipopolysaccharide (LPS) of experimental arthritis in the ankles of rats immunized with LPS extracted from Escherichia coli}; Noyori K; Sprague Dawley (SD) rats were immunized by subcutaneous injections with heat-killed E . coli 0:14 and lipopolysaccharide (LPS) extracted from E . coli for 15, 29 and 39 weeks which induced arthritis in the ankle . Localization of interleukin-1 (IL-1) and LPS in the ankle joints were investigated immunohistochemically . Serum IgM rheumatoid factor-like substance (RFLS) and anti-LPS IgM were detected by enzyme-linked immunosorbent assay (ELISA) . Rats immunized with LPS for 39 weeks developed synovial lining cell hyperplasia in 25 of 40 ankles and lymphoid cell infiltration in 25 and pannus formation in 23, the rates of which were significantly higher than those of control and rats immunized with LPS for 15 and 29 weeks . The induction rate of arthritis in rats immunized with LPS was the same as that in rats immunized with E . coli . LPS and IL-1 were located in synovial cells and pannus in arthritic joints . Changes of RFLS level in rats immunized with LPS were elevated more gradually than those in rats immunized with E . coli . These findings suggest that LPS could stimulate IL-1 and RFLS production and may induce arthritis in rats resembling rheumatoid arthritis. Indian J Med Res, 1992 May, 95, 121 - 4 Nonradioactive polynucleotide gene probe assay for the detection of three virulence toxin genes in diarrhoeal stools; Bright BD et al.; The present study describes a nonisotopic DNA-DNA hybridization assay for the detection of enterotoxigenic Escherichia coli (ETEC) in which the probes were labelled with the hapten molecule digoxigenin and after hybridization, DNA hybrids were detected by antidigoxigenin alkaline phosphatase conjugate . A blinded study carried out on a battery of enterotoxigenic and nonenterotoxigenic Esch . coli by dig-probe hybridization assay were compared with the results of a radiolabelled toxin gene probe hybridization assay (performed earlier) . The three digoxigenin labelled probes gave a 100 per cent specificity and sensitivities of 95.45, 100 and 100 per cent for LT, STh and STp respectively . These results were comparable to those with the radioactive probes. Microb Pathog, 1992 May, 12(5), 383 - 9 Expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli using a two cistron system; Guzman CA et al.; Expression of Bordetella pertussis filamentous hemagglutinin (FHA) has been achieved in Escherichia coli K-12 . This involved the construction of a two cistron system where the first cistron was provided by the NH2-terminus (first 98 amino acids) of MS2 polymerase . When the FHA gene sequences were fused to the first cistron, higher levels of expression were obtained and the fusion protein aggregated in inclusion bodies . FHA expressed by the two cistron system, however, appeared to be diffusely dispersed in the cytoplasm. Vet Parasitol, 1992 May, 42(3-4), 189 - 98 A specific DNA probe which identifies Babesia bovis in whole blood; Petchpoo W et al.; A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli . Several recombinants which hybridized strongly to radioactively labeled B . bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B . bovis DNA . It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B . bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia . pMU-B1 contained a 6.0 kb B . bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA . In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B . bovis geographic isolates, Mexican strain M and Thai isolate TS4 . Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B . bovis strains. Biull Eksp Biol Med, 1992 May, 113(5), 514 - 6 {Role of T-lymphocytes in the mechanisms of hemopoiesis-inducing microenvironment activation in inflammation}; Dygai AM et al.; On the model of acute infectious peritonitis in mice, the inflammation was shown to accompany pronounced activation of bone marrow haemopoiesis . The increased number of committed precursor cells of erythro- and granulomonocytopoiesis, morphologically differentiated elements and enhancing colony-stimulating and erythropoietic activities of GIM cells origin were shown as well . Thy 1.2+ cells, migrating to bone marrow, were shown to play an important role in hemopoiesis stimulation inflammation . These stimulate the processes of myeloid precursor proliferation directly (by means of lymphokines) and in cooperation with monocytes-macrophages of GIM. Bioorg Khim, 1992 May, 18(5), 646 - 59 {Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor}; Korobko VG et al.; Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out . Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein . In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide . In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro . Expression of the hybrid genes in E . coli and properties of the recombinant proteins were studied . The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells . Procedures for the isolation of both hybrid proteins were developed . The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active . Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures. Mol Biol (Mosk), 1992 May-Jun, 26(3), 596 - 600 {Cloning, primary structure determination and expression of preproinsulin cDNA from human insulinoma in Escherichia coli}; Chekhranova MK et al.; A human insulinoma cDNA library was constructed in expression plasmid vector pUEX1 . Clone pUEX1Ins12 was selected from human insulinoma cDNA library by means of hybridization with the insulin probe and a nucleotide sequence of the insertion was determined . It codes for full size amino acid sequence preproinsulin and furthermore, contains the entire 3'-end of noncoding mRNA region and 44 nucleotides from the 5'-untranslated region . The bacterial strain pUEX3Ins8 producing preproinsulin as beta-galactosidase fusion protein was constructed. Indian J Pediatr, 1992 May-Jun, 59(3), 305 - 7 Failure to isolate verotoxin producing Escherichia coli from patients of haemolytic uremic syndrome; Kishore K et al.; Haemolytic Uremic Syndrome (HUS) been defined as the simultaneous occurrence of acute renal failure in children with haemolytic anemia and thrombocytopenia . This clinical condition that has been recognized is an important cause of acute renal failure in children. Chem Pharm Bull (Tokyo), 1992 May, 40(5), 1266 - 7 Homeostasis as regulated by activated macrophage . VI . Protective effect of LPSw (a lipopolysaccharide from wheat flour) against acute infection by Toxoplasma gondii in mice; Suzuki Y et al.; An oral administration of partially purified LPSw, a lipopolysaccharide (LPS) from wheat flour, at a concentration of 20 ng/ml in drinking water beginning 1d after infection significantly decreased mouse mortality and prevented animal weight loss in acute infection with Toxoplasma gondii . Whereas 71% (5/7) of mice in a control group that did not receive LPSw died of toxoplasmosis, only 14% (1/7) of mice treated with LPSw died (p less than 0.05) . The administration of LPS purified from Bordetella pertussis also significantly decreased the mortality of infected mice . LPS from Escherichia coli and synthetic lipid A (LA-15-PP(506)), however, did not show a significant decrease in mortality. Farmaco, 1992 May, 47(5), 551 - 65 4-(1,2-Benzisothiazol-3-yl)alkanoic and phenylalkanoic acids: synthesis and anti-inflammatory, analgesic and antipyretic activities; Bordi F et al.; Continuing their studies on benzisothiazolyl derivatives, Authors refer to the preparation and pharmacological properties of 4-(1,2-benzisothiazol-3-yl) alkanoic and phenylalkanoic acids . All substances were tested for anti-inflammatory, analgesic and antipyretic properties . As reference compounds, 1,2-benzisothiazolin-3-one and 4-(3-oxo-1,2-benzisothiazolin-3-yl) phenylacetic acid, as prototypes of benzisothiazolinonic derivation . Ibuprofen, as a prototype of substituted arylalkanoic acids, and Phenylbutazone were used . Analysis of the data leaded to the following conclusions . Introduction of the aryl moiety, passing from benzisothiazolylalkanoic to benzisothiazolyl-phenylalkanoic structures, produced a remarkable increase of activity . 2-{4-(1,2-benzisothiazol-3-yl)phenyl} propionic and 2-{4-(1,2-benzisothiazol-3-yl)phenyl}butyiric acids showed anti-inflammatory, analgesic and antipyretic properties comparable to those of Ibuprofen . Substantial differences in variations in activities were observed comparing the properties of benzisothiazolylphenylalcanoic acids with those of the benzisothiazolinonic series, object of preceding studies. Biotechniques, 1992 May, 12(5), 630 - 1 Rapid analysis of lambda gt11 fusion proteins without subcloning or lysogen induction; Runge SW; Current methods of analyzing fusion proteins from lambda gt11 clones involve either subcloning of the insert DNA into a plasmid expression vector or production of lambda gt11 lysogens that are subsequently induced . Both of these methods can be quite time-consuming . The present communication describes a novel strategy for induction of the fusion protein that is both simple and rapid . Liquid cultures of E . coli Y1090R- infected with the lambda gt11 clone were induced directly to produce the fusion protein . Following the preparation of a crude bacterial cell lysate, fusion products were subjected to Western blot analysis. Anal Biochem, 1992 May 1, 202(2), 375 - 83 Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping; Clogston CL et al.; Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method . In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor . Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended . However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization . By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor . In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method . The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis . Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms. Appl Biochem Biotechnol, 1992 May, 33(2), 117 - 38 Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli; Grunfeld H et al.; Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent . The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein . The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two . The yield of the process is also shown to be particularly dependent on the recombinant protein concentration . At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained . Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme . This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column . The product was composed of roughly equal amounts of one-chain and two-chain t-PA . The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated. Mol Microbiol, 1992 May, 6(10), 1259 - 62 A metal-binding motif implicated in RNA recognition by an aminoacyl-tRNA synthetase and by a retroviral gene product; Miller WT et al.; A randomly generated mutation in Escherichia coli alanine tRNA synthetase compensates for a mutation in its cognate tRNA . The enzyme's mutation occurs next to a Cys-X2-Cys-X6-His-X2-His metal-binding motif that is distinct from the zinc finger motif found in some DNA-binding proteins . Instead, the synthetase's metal binding domain resembles the Cys-X2-Cys-X4-His-X4-Cys metal-binding domain of the gag gene product of retroviruses . For Ala-tRNA synthetase, the metal bound at the Cys-His motif is important specifically for the tRNA-dependent step of catalysis, and the enzyme-tRNA interaction is dependent on the geometry of metal co-ordination to the enzyme . These data, and the demonstrated sensitivity of RNA packaging to mutations in the metal-binding domain of the gag gene product of retroviruses, suggest that an aminoacyl-tRNA synthetase and retroviruses have adopted a related metal-binding motif for RNA recognition. J Biochem (Tokyo), 1992 May, 111(5), 633 - 7 Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques; Frorath B et al.; Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease . A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach . The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients . All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO) . Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants . A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO. Biochem Int, 1992 May, 26(6), 1115 - 24 10Sa RNA: processing by and inhibition of RNase III; Makarov EM et al.; Characterization of the maturation of precursor 10Sa RNA revealed that RNase III processed p10Sa RNA to two intermediate molecules . We showed that the intermediates are not conformers and both are larger than the mature 10Sa RNA . Cell extracts further process the RNase III products to an RNA molecule which has a different conformation than 10Sa RNA but is approximately the same size as 10Sa RNA . An inhibitor of p10Sa RNA processing by RNase III was identified . It is a protein, with a molecular mass of approximately 17 kDa. Rheum Dis Clin North Am, 1992 May, 18(2), 377 - 90 Antiribosomal antibodies in systemic lupus erythematosus; Elkon KB et al.; ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease . Anti-P antibodies appear to be a highly specific diagnostic marker for SLE because they are rarely detected in other multisystem autoimmune disorders . ARA are most frequently directed against the P proteins, and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested . Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances . This may be particularly true of patients with affective disorders . The clinical utility of serologic tests for anti-P in central nervous system lupus must await large, prospective studies . Other ARA antibodies have been detected in patients with SLE . These antibodies include anti-28S rRNA, anti-S10, and anti-L12 . In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P . The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome . The L12 protein is the mammalian homologue of the E . coli and yeast proteins known to bind to the 28S rRNA epitope . These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome . This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production . Despite these findings, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful . It therefore seems likely that the ribosomal components must be altered to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production. Mol Gen Genet, 1992 May, 233(1-2), 269 - 77 Structural and functional analysis of the Bz2 locus of Zea mays: characterization of overlapping transcripts; Schmitz G et al.; Analysis of the transcription pattern of the Bz2 locus revealed that overlapping transcripts are derived from opposite DNA strands . The most abundant transcript (sense transcript) has an open reading frame coding for a protein of 241 amino acids, whilst in the antisense orientation no open reading frame has been detected; the antisense transcripts are detected only in those tissues that show high levels of sense transcript . Particle gun experiments indicate that the sense transcript is sufficient to provide the Bz2 function . The promoter driving the sense transcript contains the elements usually found in front of eukaryotic genes . In addition an element with similarity to the C1 and R binding sites identified in the Bz1 promoter is found . Further upstream in the promoter region a transposon-like insertion has been identified . This element has features similar to members of the Ac/Ds transposable element family . The putative Bz2 protein shows similarity to various other plant proteins and to an Escherichia coli protein . All related proteins have in common the fact that they are involved in stress responses. Mol Gen Genet, 1992 May, 233(1-2), 122 - 8 Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase; Klein B et al.; A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata . This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa . The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach . Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed . An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity . Hybridization studies revealed that in C . lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds. Int J Biochem, 1992 May, 24(5), 737 - 49 Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates; Srivastava RA et al.; 1 . A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E . coli cells . 2 . Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E . coli cells . 3 . Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+ . Other divalent cations could not replace Mn2+ . 4 . Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal . 5 . The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C . 6 . Further characterization of this activity indicated that it is RNase III . 7 . To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E . coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C) . 8 . This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E . coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III. Am J Physiol, 1992 May, 262(5 Pt 2), R786 - 93 Kinetics of endotoxin-induced acute-phase protein gene expression and its modulation by TNF-alpha monoclonal antibody; Sharma RJ et al.; The kinetics of cytokine release and acute-phase protein gene expression in liver were investigated in rats receiving a single intraperitoneal bolus dose of Escherichia coli lipopolysaccharide (LPS) . Transient elevation of plasma tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were detected . Hepatic messenger RNAs for two acute-phase proteins, alpha 1-acid glycoprotein and alpha 2-macroglobulin, were measured by Northern blotting and were found to increase to a maximum at 24 h, returning to normal by 72 h; plasma concentrations showed a slower but more sustained rise . For albumin, hepatic mRNA was reduced, being minimum at 24 h with a similar but more prolonged fall in plasma concentration . Pretreatment of rats with TNF-alpha monoclonal antibody 4 h before LPS ameliorated weight loss and anorexia, partially suppressed the rise in IL-6 and reduced the increase in hepatic mRNA and plasma concentrations of alpha 1-acid glycoprotein and alpha 2-macroglobulin . For albumin, however, such pretreatment had no effect on the fall in either hepatic mRNA or plasma concentration . Thus we have defined an in vivo role of TNF-alpha in the control of endotoxin-induced acute-phase protein generation. Mol Microbiol, 1992 May, 6(9), 1219 - 29 Translation initiation in Escherichia coli: sequences within the ribosome-binding site; Ringquist S et al.; The translational roles of the Shine-Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied . The Shine-Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence . Each Shine-Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long . Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG . Translation was also affected by residues 3' to the initiation codon . The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon . The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation. Gene, 1992 May 1, 114(1), 109 - 14 Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth; Kenri T et al.; The Escherichia coli metY gene, encoding tRNA(f2Met), was split by the kanamycin-resistance-encoding gene . The resulting mutant exhibited the same growth rate as the wild type, indicating that tRNA(f2Met) is not indispensable as is the case with the metZ gene encoding tRNA(f1Met) {Kenri et al., Gene 103 (191) 31-36} . beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or a trpA'::lac'Z fusion gene, in the metY mutant . The lac repressor from the lacI gene and the chimeric protein from a hupB'::lac'Z fusion gene, whose start codons are GUG, were also synthesized efficiently in the insertion mutant . These results provide evidence that tRNA(f2Met) is not essential for growth of E . coli and that the start codons, AUG and GUG, are both recognized by tRNA(f1Met), a major N-formyl methionine-specific tRNA, in the tRNA(f2Met)-depleted cells . We were unable to construct mutants deficient in both tRNA(f1Met) and tRNA(f2Met) by P1 phage-mediated transduction with the metY and metZ mutations . Moreover, the ampicillin-resistance marker of the pUC9 plasmid carrying metZ+ was not cured at 42 degrees C in host cells with the polAts and metY-metZ double mutations . These results indicate that either tRNA(f1Met) or tRNA(f2Met) is required for the growth of E . coli. Mutat Res, 1992 May, 273(3), 263 - 9 Molecular cloning and DNA sequencing of the radC gene of Escherichia coli K-12; Felzenszwalb I et al.; The radC102 mutation that sensitizes E . coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome . E . coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR . The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector . A library of EcoRI/KpnI DNA fragments of E . coli strain BH20 was inserted into pUC19 . One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10 . The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment . The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced . The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500 . The order of the genes in this region of the E . coli chromosome is: fpg--rpmBG--radC--pyrE. J Neurosci, 1992 May, 12(5), 1628 - 39 Analysis of cis-regulatory elements in the 5' flanking region of the Drosophila melanogaster choline acetyltransferase gene; Kitamoto T et al.; We have analyzed the cis-regulatory elements in the 5' flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6) . DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation . A 7.4 kb 5' flanking sequence directed beta-galactosidase expression in the adult optic lobes and other well-defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein . In contrast, the proximal 3.3 kb and 1.2 kb of 5' flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct . Our results indicate that both qualitative and quantitative regulatory elements are present in the 5' flanking DNA and that these elements distinguish various subsets of cholinergic neurons . We have also fused the same 5' flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies . Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature-dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild-type ChAT expression to overcome these mutant phenotypes. J Bacteriol, 1992 May, 174(10), 3370 - 6 Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase; Monticello RA et al.; To evaluate whether expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase is sufficient to cause membrane proton permeability, plasmids carrying different combinations of the uncB, E, and F genes, encoding the a, c, and b subunits of the F0 sector, cloned behind the inducible lac promoter in pUC9 or pUC18, were constructed . The effects of inducing F0 synthesis in an unc deletion strain were monitored by measuring cell growth rate, quantitating F0 subunits by immunoblotting, and measuring the ability of membranes to maintain a respiration-induced proton gradient and to bind F1 and carry out energy-coupling reactions . The levels of functional reconstitutable F0 in membranes could be increased four- to sixfold with no change in cellular growth rate or membrane proton permeability (assayed by fluorescence quenching) . These results were obtained in uninduced cultures, so the F0 genes were presumably being transcribed from some promoter besides lac . Induction of transcription of all three F0 genes produced increased amounts of F0 subunits in membranes as determined by immunoblot and F1-binding assays, but, when reconstituted with F1, the F0 in membranes isolated from induced cultures was significantly less functional than the F0 in membranes isolated from uninduced cultures . Such induction did result in growth inhibition, but there was no correlation between growth inhibition and either increased membrane proton permeability or the presence of functional, reconstitutable F0. J Bacteriol, 1992 May, 174(10), 3204 - 11 Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46; Laine PS et al.; Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1 . A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113 . It has very little or no effect on ssb-1 or ssb-3 . All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels . The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda . The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411 . The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain. Mutat Res, 1992 May, 282(1), 39 - 42 A recA-ada hybrid gene inducible by DNA damage; Kleibl K et al.; A damage-inducible expression vector was constructed in which the original recA structural gene was replaced by the protein-coding region of the ada gene . The O6-alkylguanine-DNA alkyltransferase encoded by the ada gene can be measured by a rapid and highly sensitive assay . The introduction of this construct into an appropriate host cell provides an effective bacterial assay for genotoxins. Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3995 - 8 The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli; Persson BC et al.; One of the most abundant modified nucleosides in tRNA is 5-methyluridine (m5U or rT, ribothymidine) . The enzyme tRNA(m5U54)methyltransferase {S-adenosyl-L-methionine:tRNA (uracil-5-)-methyltransferase, EC 2.1.1.35} (the trmA gene product) catalyzes S-adenosylmethionine-dependent methylation of the uracil in position 54 (T psi C loop) in all Escherichia coli tRNAs to form m5U . Hitherto no modified nucleoside in tRNA has been shown to be essential for growth, although their importance in fine tuning the function of tRNA is well established . In this paper, we show that the structural gene trmA is essential for viability, although the known catalytic activity of the tRNA(m5U54)methyltransferase is not. Mutat Res, 1992 May, 267(1), 77 - 88 Multivariate statistical analysis of mutational spectra of alkylating agents; Benigni R et al.; A series of multivariate statistical methods were used to explore the current knowledge on the mutational spectra of alkylating agents (AA) in bacterial and mammalian cells . The data relative to lac I and gpt genes of Escherichia coli were considered . The analysis focused on the distribution of GC to AT transitions, which account for the majority of AA-induced mutations . The statistical analysis of 15 different mutational spectra obtained by various laboratories pointed to a number of biological factors involved in the mutational process . First of all, factor and cluster analyses demonstrated that the mutational profiles obtained in mammalian cells form a homogeneous cluster different from the cluster formed by the bacterial cell mutational spectra . SN1-type AAs give rise to classes of mutational spectra statistically different from the spectra induced by the SN2-type AAs . The analysis of the mutated sequences of both genes pointed to a correlation between mutation induction by SN1 AAs, which react through a positively charged alkylating intermediate, and the occurrence of mutations at guanines preceded 5' by a purine . Moreover, our statistical analysis showed that the distribution of AA-induced mutations is not affected by the transcriptional activity of the target gene, but is strongly determined by the sequence specificity of AA-induced mutagenesis and by the structure of the target proteins . The agreement of our results with the findings of previous studies indicates that the multivariate data analysis methods are a sensitive and reliable tool for exploring the mechanisms underlying complex biological processes . The novelty of the present results lies in their quantitative character, and in the clarity of the graphical displays . We propose the use of this methodological approach to explore the large bulk of information available on mutational spectra. Mutat Res, 1992 May, 267(1), 67 - 76 Properties of R-plasmid pEB017, which confers both enhanced UV-radiation resistance and mutability to wild-type, recA and umuC strains of Escherichia coli K12; Obaseiki-Ebor EE et al.; The R-plasmid, pEB017, restored recombination ability to recA56 and conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability to wild-type, recA56 and umuC36 strains of Escherichia coli K12 . Comparatively, pEB017 enhanced UV-radiation mutability in a umuC strain, and also enhanced UV-radiation and nitrofuran mutability in a wild-type strain several-fold more than did another R-plasmid, pKM101 . Plasmid pEB017 also mediated about a 3-fold enhancement of the SOS induction of beta-galactosidase synthesis in a recA strain, compared with the normal recA+ gene of E . coli . A BamHI fragment of pEB017 DNA was cloned into plasmid vector pBR322 to yield pEB021 . The BamHI fragment in pEB021 (3.5 kb) is about 170 bp longer between the BamHI and PstI sites on the left end of the recA-like fragment, compared with published data on a similarly cloned recA gene from E . coli . Plasmid pEB021 conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability in wild-type and recA strains, and restored recombination ability in a recA strain . The introduction of pEB021 into a umuC strain made the cells slightly more resistant to killing by UV-irradiation, and promoted a small amount of UV-mutability in an otherwise nonmutable strain . These results suggest that R-plasmid pEB017 has a recA-like gene that mediates the enhanced resistance to UV-radiation and enhanced UV-radiation mutability, but which seems different in several important aspects from the normal recA gene in E . coli. Mutat Res, 1992 May, 267(1), 1 - 17 Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: role of DNA breaks; DeMarini DM et al.; Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay . The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response . In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here . This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction . Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage . Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA . Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage . These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage . Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks . The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase. J Bacteriol, 1992 May, 174(9), 2858 - 64 Acquisition of apparently intact and unmodified lipopolysaccharides from Escherichia coli by Bdellovibrio bacteriovorus; Stein MA et al.; The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS) . Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios . The relocalized trimers were found associated with the same LPS originally bound to them in the E . coli . The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios . This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey . One or two other bands were identical in migration to the LPS of prey-independent mutants of B . bacteriovorus and represented bdellovibrio-synthesized LPS . The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS . The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS . Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS. Virology, 1992 May, 188(1), 47 - 56 Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase; McCormack SJ et al.; A molecular cDNA clone of the human RNA-dependent P1/eIF-2 alpha protein kinase was expressed in Escherichia coli . Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe . Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA . Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids . Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (greater than 54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins. Virology, 1992 May, 188(1), 33 - 46 Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells; Thomis DC et al.; A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase . The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da . Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN) . Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified . Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb . Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma . Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli . In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment . The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction . The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis . The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases . The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L. J Virol, 1992 May, 66(5), 2952 - 65 Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1; Johnson PA et al.; Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells . Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes . This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer . However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons . In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation . Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5 . Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE . We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible . After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time . It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells. Biosci Biotechnol Biochem, 1992 May, 56(5), 746 - 50 Expression and purification of recombinant 3C proteinase of Coxsackievirus B3; Miyashita K et al.; We have cloned various lengths of coxsackievirus B3 cDNA encompassing the region encoding the 3C proteinase, which is essential to the viral replication cycle . Such viral cDNAs were fused in frame to the 5'terminal portion of the lacZ' gene carried on the vector pUC118 to express mature 3C proteinase in Escherichia coli . In the E . coli cells containing pCXB108 or pCXB117, constructed for this study, a large amount of 23-kDa protein was synthesized in the presence of IPTG . This protein was purified and was shown to be intact 3C proteinase . These data suggest that 3C proteinase, expressed as a part of a fusion protein, was active in E . coli and released itself from the precursor fusion protein by autocatalytic cleavage. Biosci Biotechnol Biochem, 1992 May, 56(5), 716 - 9 Cloning and sequence analysis of cDNA encoding Rhizopus niveus lipase; Kugimiya W et al.; Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R . niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme . A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme . The lipase gene was expressed in Escherichia coli as a lacZ fusion protein . The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa . The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved. Indian J Med Res, 1992 May, 95, 115 - 20 Occurrence of colonization factor antigens I & II in enterotoxigenic Escherichia coli associated diarrhoea in Iran & correlation with severity of disease; Katouli M et al.; The occurrence of colonization factor antigens I and II (CFA/I and II) and type 1 somatic pili was investigated in 197 enterotoxigenic Esch . coli (ETEC) isolated from 197 patients of diarrhoea (aged under 3 yr) during February 1985 to March 1986 in Tehran, Iran . Among ETEC strains, 154 strains were heat-stable enterotoxin (ST) producers, 27 strains were heat-labile enterotoxin (LT) producers, and 16 strains produced both toxins . Sixty five (33%) strains showed mannose-resistant haemagglutination (MRHA) of human and/or bovine erythrocytes; of these, 51 (86%) strains were positive for CFA/I and II . Seventy one (36%) strains also exhibited type 1 somatic pili . CFA/I was found in 4 (15%) LT producing, 24 (16%) ST producing, and 2 (13%) LT/ST producing strains . In contrast, CFA/II was only found in ST producing strains (17 strains) and those producing both toxins (4 strains) . Patients having CFAs-positive ETEC strains had a significantly (P less than 0.001) higher number of stool evacuation per day and a longer duration of diarrhoea than those having CFAs-negative strains . Fifty nine patients had mixed infections of ETEC strains and other enteropathogens . CFA/I or II (CFAs)-positive and CFAs-negative ETEC strains were found in 17 and 42 patients with mixed infections respectively . The mean number of stool evacuations per day was much higher in patients with ETEC and rotavirus than those with only ETEC infection (P less than 0.001) . However, severity of the disease was not affected by the presence or absence of CFA/I or II in ETEC strains found in these patients. Microb Pathog, 1992 May, 12(5), 367 - 75 Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors; Willemsen PT et al.; The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli . The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E . coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa . Depending on the K88 serotype tested, differences in binding to these proteins were observed . In particular, E . coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins . The influence of age of the pig on the presence of K88 receptors was also investigated . One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs . The presence of receptors in the brush border fraction was shown to be independent of age . The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure. Biochem J, 1992 May 1, 283 ( Pt 3), 727 - 35 Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation; Katwa LC et al.; Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes . ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM . The strongest inhibitors were adenosine 5'-{beta gamma-imido}triphosphate (App{NH}p) (36%) and adenosine 5'-{beta-thio}diphosphate (ADP{S}) (41%) . Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS) . Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP{S} (41%) . While App{NH}p and MgGDP{S} appeared to be acting at different sites, they also interfered with each other . These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase . Inhibition by low concentrations of MgApp{NH}p and MgATP was weaker above 0.1 mM, while MgGDP{S} and magnesium guanosine 5'-{gamma-thio}triphosphate (MgGTP{S}) inhibited in a single phase . Inhibition by MgApp{NH}p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP{S} and MgGTP{S} . Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP . This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP . These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation. J Trauma, 1992 May, 32(5), 600 - 6; discussion 606-7 Adaptive regulation in skeletal muscle glutamine metabolism in endotoxin-treated rats; Austgen TR et al.; The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases . In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values . Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats . As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment . Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion . The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis . The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool. J Bacteriol, 1992 May, 174(10), 3133 - 9 Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants; Donnelly CE et al.; The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins . Our previous work has shown that the GroE proteins of E . coli are required for UV mutagenesis . This process requires the umuDC genes which are regulated by the SOS regulon . As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD' . In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes . We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations . However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus . Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains . In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC. Int J Gynaecol Obstet, 1992 May, 38(1), 41 - 3 Amoxycillin/clavulanic acid (augmentin) compared with triple drug therapy for pelvic inflammatory disease; Uri FI et al.; Sixty Jordanian women with pelvic inflammatory disease (PID) were studied . Of these, 31 were given oral amoxycillin/clavulanic acid (augmentin) for a mean duration of 8.4 days and 29 were given a standard triple drug regimen of oral ampicillin, intramuscular gentamicin and metronidazole tablets/pessaries for a mean duration of 7.2 days . Bacterial culture (cervical and high vaginal swabs) was positive in every case, most often E . coli but sometimes more than one pathogen was isolated . No gonococci were isolated and tests for chlamydia in 16 patients (8 in each group) were negative, suggesting a dissociation between the etiology of PID and sexually transmitted disease in this Jordanian study . After 3 days of treatment, more patients in group I (augmentin) showed diminution of symptoms of pain and discharge (P less than or equal to 0.05) compared to group II . At the end of treatment, complete cure or satisfactory improvement was recorded in 93.1% and 92.9% of cases in the two groups, corresponding to in vitro bacterial efficacy of 90.4% and 96.5%, respectively . No serious side effects were noted in either regimen . The results of this comparative study suggest that oral amoxycillin/clavulanic acid (augmentin) may be a convenient alternative to the triple drug regimen usually administered for the treatment of pelvic inflammatory disease. Infect Immun, 1992 May, 60(5), 2083 - 91 Ability of enteroaggregative Escherichia coli strains to adhere in vitro to human intestinal mucosa; Knutton S et al.; A collection of 44 enteroaggregative Escherichia coli (EAggEC) strains isolated from infants with diarrhea in India and the United Kingdom were examined for their ability to adhere in vitro to human intestinal mucosa and by electron microscopy for production of putative adherence factors . None of the strains adhered to human duodenal mucosa, and six strains tested did not adhere to ileal mucosa; all 44 strains, however, adhered to human colonic mucosa in localized aggregates . Electron microscopy of infected colonic mucosa indicated fimbrially mediated adhesion of the EAggEC strains . Four morphologically distinct kinds of fimbriae, including a new morphological type of E . coli fimbriae consisting of bundles of fine filaments, were identified among the EAggEC strains; this new type of fimbria was observed in 43 of the 44 EAggEC strains . Forty-three of the 44 EAggEC strains were positive with a DNA probe developed to identify EAggEC, and most of the strains belonged to serotypes unrelated to the other major classes of diarrheic E . coli . These results suggest that EAggEC may be a large-bowel pathogen and colonize the colon by a fimbrially mediated adhesion mechanism. Infect Immun, 1992 May, 60(5), 1963 - 71 Characterization of F107 fimbriae of Escherichia coli 107/86, which causes edema disease in pigs, and nucleotide sequence of the F107 major fimbrial subunit gene, fedA; Imberechts H et al.; F107 fimbriae were isolated and purified from edema disease strain 107/86 of Escherichia coli . Plasmid pIH120 was constructed, which contains the gene cluster that codes for adhesive F107 fimbriae . The major fimbrial subunit gene, fedA, was sequenced . An open reading frame that codes for a protein with 170 amino acids, including a 21-amino-acid signal peptide, was found . The protein without the signal sequence has a calculated molecular mass of 15,099 Da . Construction of a nonsense mutation in the open reading frame of fedA abolished both fimbrial expression and the capacity to adhere to isolated porcine intestinal villi . In a screening of 28 reference edema disease strains and isolates from clinically ill piglets, fedA was detected in 24 cases (85.7%) . In 20 (83.3%) of these 24 strains, fedA was found in association with Shiga-like toxin II variant genes, coding for the toxin that is characteristic for edema disease strains of E . coli . The fimbrial subunit gene was not detected in enterotoxigenic E . coli strains . Because of the capacity of E . coli HB101(pIH120) transformants to adhere to isolated porcine intestinal villi, the high prevalence of fedA in edema disease strains, and the high correlation with the Shiga-like toxin II variant toxin-encoding genes, we suggest that F107 fimbriae are an important virulence factor in edema disease strains of E . coli. J Comp Pathol, 1992 May, 106(4), 341 - 50 The effect of mixed live vaccines of Newcastle disease and infectious bronchitis on the chicken respiratory tract; Nakamura K et al.; The effect of mixed live vaccines of Newcastle disease (ND) and infectious bronchitis (IB) on specific pathogen-free chickens aged 7 days was investigated . The chickens were inoculated intranasally with mixed live vaccines with and without Escherichia coli, or with E . coli alone . "Vaccine 1" consisted of ND virus strain B1 and IB virus strain ON; "vaccine 2" consisted of ND virus strain B1 and IB virus strain H120 . The tracheas of chickens inoculated with the vaccines and E . coli and with the vaccines alone showed multiplication of E . coli and histological lesions (loss of cilia, degeneration and hyperplasia in epithelial cells and cellular infiltration of subepithelial tissues) . In the tracheas of chickens inoculated with vaccines and E . coli, multiplication of E . coli was greater than in chickens given vaccine alone . There were no histological lesions, and only mild, transient multiplication of E . coli in chickens inoculated with E . coli alone . The results suggest that these mixed live vaccines, especially vaccine 2, play a role in inducing or enhancing colibacillosis in the chicken. J Biochem (Tokyo), 1992 May, 111(5), 589 - 93 Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers; Akiyama H et al.; An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described . By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion . The digests could be analyzed with commercial automated DNA sequencers . Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning . Nor is the synthesis of oligonucleotide primers needed . This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods. Anticancer Res, 1992 May-Jun, 12(3), 873 - 80 Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase; Baratte B et al.; A universal intracellular factor, the "M phase-promoting factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms . In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13 . In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25 . We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds . The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts of Escherichia coli and easily purified by affinity chromatography on glutathione-agarose . Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined . Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity . However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor . The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery. Curr Genet, 1992 May, 21(6), 499 - 505 The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning, physical mapping and gene location; Moulinier T et al.; The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation . A representative library was constructed in E . coli by molecular cloning at the HindIII restriction site of pBR322 . Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule . Its size was assessed at about 80,500 bp . Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes . The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E . coli rrnB operon . A comparison of A . aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed. Kidney Int, 1992 May, 41(5), 1170 - 4 Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli; He CJ et al.; Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation . TM antigen was previously found by immunohistochemical methods in rabbit glomeruli . We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli . Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry . One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM . TM activity correlated with the protein content of the glomerular extracts (r = 0.94) . These extracts prolonged rat plasma activated partial thromboplastin time . Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E . coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner . Incubation with TNF suppressed their anticoagulant activity . In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody . These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases. Mol Endocrinol, 1992 May, 6(5), 805 - 14 The unique C-termini of the thyroid hormone receptor variant, c-erbA alpha 2, and thyroid hormone receptor alpha 1 mediate different DNA-binding and heterodimerization properties; Katz D et al.; Thyroid hormone receptors (TRs) mediate the regulation of gene transcription by thyroid hormone (T3) by binding to T3-responsive elements (TREs) in target genes . c-erbA alpha 2 is a C-terminal TR variant which does not bind T3 and is a dominant inhibitor of T3 action . When synthesized in Escherichia Coli, alpha 2 formed two TRE-binding complexes similar to the monomeric and homodimeric forms of TR alpha 1 . However, alpha 2 did not bind nearly as well as TR alpha 1 . Furthermore, alpha 2 failed to bind DNA with proteins that heterodimerized with TR alpha 1 . TR alpha 1 and alpha 2 also did not bind DNA as heterodimers with one another . The differences between TR alpha 1 and alpha 2 were further analyzed by studying a variety of C-terminal mutants synthesized in reticulocyte lysates . Deletion of the last 20 of the 122 unique amino acids (aa) of alpha 2 increased its DNA binding to approximately the level of TR alpha 1, indicating that the C-terminus of alpha 2 is an inhibitory domain . This alpha 2 mutant (alpha 2 delta C) was still unable to heterodimerize with nuclear proteins, as were C-terminal deletion mutants of TR alpha 1 . We hypothesized that fusion of TR alpha 1-specific sequences to the C-terminus of alpha 2 delta C would transfer the property of heterodimerization . Indeed, although alpha 2/alpha 1 chimeras containin