Am J Pathol, 1989 Jan, 134(1), 7 - 10 Nick translation detection in situ of cellular DNA strand break induced by radiation; Maehara Y et al.; DNA strand break in HeLa cells induced by radiation was detected using the in situ nick translation method . The cells were exposed to radiation of 3, 6, 12, 18, and 24 Gy in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass . The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and {3H}-labeled dTTP . Autoradiographic observation was made of the level of break sites in the DNA . The DNA strand break appeared even with a 3 Gy exposure, increased 8.6 times at 24 Gy compared with the control cells, and this level correlated reciprocally to change in cell viability . This nick translation method provides a rapid in situ assay for determining radiation-induced DNA damage of cultured cells, in a semi-quantitative manner. J Med Microbiol, 1989 Jan, 28(1), 49 - 57 An endocytic process in HEp-2 cells induced by enteropathogenic Escherichia coli; Andrade JR et al.; Infection of HEp-2 cells by enteropathogenic Escherichia coli (EPEC) was examined by transmission and scanning electronmicroscopy . EPEC strains of serogroups O111:K58 and O55:K59 recently isolated from human patients did not exhibit enterotoxic activity, as judged by the Vero-cell and suckling-mouse assays, or invasive ability as judged by the Sereny test . These strains attached to and penetrated HEp-2 cells . Transmission electronmicroscopy showed bacteria in close contact with cell membranes 15 min after infection; later, intense swelling and budding of membranes and penetration of EPEC into the cell cytoplasm occurred . Intracellular bacteria were enclosed in membrane-bound vacuoles in the cell cytoplasm underlying localised adherence sites observed by light microscopy . Scanning electronmicroscopy showed morphologically altered membranes only at the sites of bacterial attachment . Bacteria inactivated by ultraviolet light were not internalised and cytochalasin B (greater than or equal to 10 mg/L) markedly inhibited uptake . These observations suggest that penetration of EPEC into HEp-2 cells occurs by an endocytic process in metabolically active bacteria. J Med Microbiol, 1989 Jan, 28(1), 43 - 7 The use of phage-sensitivity patterns for tracing heat labile toxin-positive (LT+) Escherichia coli; Monsur KA et al.; The heat-labile toxin (LT) positive Escherichia coli colonies from 785 stool specimens obtained during a cholera vaccine trial were examined for their phage-sensitivity pattern to 31 E . coli phages . These specimens originated from 105 index cases and their contacts . Isolates with common phage-sensitivity patterns were grouped and were studied further both serologically and for their plasmid profile . The largest group (42 isolates) belonged to serogroup O78 and the second largest group (19 isolates) belonged to serogroup O6 . There were 23 index cases which had E . coli with the same phage-sensitivity pattern as some of their contact strains . The identity of isolates from 16 index cases with strains from their respective contacts could be verified serologically . For the remaining seven index cases and their contacts, the isolates did not agglutinate with available antisera . However, subsequent studies demonstrated that, when the phage-sensitivity pattern among the strains matched, the plasmid profiles of these strains also matched . This further indicates the ability of phage-sensitivity patterns to serve as markers in tracing strains. J Am Acad Dermatol, 1989 Jan, 20(1), 87 - 8 Agranulocytosis caused by dapsone therapy for granuloma annulare; Potter MN et al.; Dapsone has been suggested as a useful drug in the treatment of granuloma annulare; however, adverse reactions include a potentially life-threatening agranulocytosis . We report the case of a 50-year-old woman in whom agranulocytosis and septicemia developed after 7 weeks of therapy with dapsone for granuloma annulare . Full recovery followed cessation of this drug, but caution is advised in prescribing dapsone for relatively benign skin conditions. Arch Biochem Biophys, 1989 Jan, 268(1), 74 - 80 Chemical modification of iron- and manganese-containing superoxide dismutases from Escherichia coli; Borders CL Jr et al.; The manganese-containing (MnSOD) and iron-containing (FeSOD) superoxide dismutases from Escherichia coli are extensively (greater than 95%) inactivated by treatment with phenylglyoxal . The relatively high concentrations of phenylglyoxal and high pH required for optimal inactivation suggest that inactivation may be due to modification of an arginine with a "normal" elevated pKa, i.e., one not in an active site cavity where the pKa is likely to be lowered because of lower solvent accessibility and decreased polarity of the local environment . Treatment of either enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-hydroxy-5-nitrobenzyl bromide, m-chloroperoxybenzoate, or tetranitromethane causes no inactivation, while 2,4,6-trinitrobenzenesulfonate, N-acetylimidazole, or diethyl pyrocarbonate cause 55-75% inactivation of each enzyme . Failure of hydroxylamine to reverse inactivation by the latter two suggests that in each instance loss of activity is due to lysine modification . The previously reported inactivation of FeSOD by H2O2 was further investigated, and no evidence was found for an affinity mechanism, i.e., a reversible binding of peroxide that precedes inactivation. Arch Biochem Biophys, 1989 Jan, 268(1), 67 - 73 Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase; Kojiro CL et al.; 31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia . Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+ . Na+ antagonized the K+ promoted appearance of the second resonance . These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group . An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca . 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase . The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C. Arch Biochem Biophys, 1989 Jan, 268(1), 379 - 87 Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli; Kashiwagi K et al.; A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E . coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine . In the absence of putrescine, doubling time of the mutant was 496 min . The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits . Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis . Under these conditions, we determined which proteins were preferentially synthesized . Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly . The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine . The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min . The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine . Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes . The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes . The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines. Arch Biochem Biophys, 1989 Jan, 268(1), 26 - 34 Interactions of oxaloacetate with Escherichia coli fumarate reductase; Ackrell BA et al.; Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA) . Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents . In these respects the enzyme behaves like cardiac succinate dehydrogenase . Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase . Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B . A . C . Ackrell, E . B . Kearney, and D . Edmondson (1975) J . Biol . Chem . 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme . However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase . The reason for the difference is not known . The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV). Proc Natl Acad Sci U S A, 1989 Jan, 86(2), 476 - 80 In vivo DNA loops in araCBAD: size limits and helical repeat; Lee DH et al.; Formation of a DNA loop by AraC proteins bound at the araI and araO2 sites, whose center-to-center distance is 211 base pairs, is necessary for repression of the araBAD promoter, PBAD, of Escherichia coli . To determine the upper and lower size limits of the loop, we constructed PBAD-reporter gene fusion plasmids with various spacings between araI and araO2 and measured their levels of expression . Spacings larger than about 500 base pairs resulted in elimination of detectable repression . No lower limit to spacing was found, suggesting that AraC protein itself possesses significant flexibility and its bending substantially aids formation of small loops . As the spacing between araI and araO2 varied, the activity of PBAD oscillated with an 11.1-base-pair periodicity, implying that the in vivo helical repeat of this DNA is 11.1 base pairs per turn. Proc Natl Acad Sci U S A, 1989 Jan, 86(2), 448 - 52 Retroregulation of the synthesis of ribosomal proteins L14 and L24 by feedback repressor S8 in Escherichia coli; Mattheakis L et al.; Previous studies on regulation of the spc operon containing genes for ribosomal proteins have shown that S8, encoded by the fifth gene of the operon in Escherichia coli, is a translational repressor and regulates the synthesis of the third gene product (L5) and distal gene products by acting at a site near the L5 mRNA translation initiation site . We have now shown that S8 also regulates the synthesis of the first and second gene products (L14 and L24) of the operon by acting at the same mRNA target site--that is, the site located distal to sites coding for L14 and L24--and that mRNA degradation is involved in this retroregulation . It was shown that single base substitutions in the target site, which abolish repression of the synthesis of L5 and L5-distal gene products by S8, also cause derepression of L14-L24 synthesis . Inhibition of L14-L24 synthesis by S8 was also shown by overproducing S8 in trans from a plasmid carrying the S8 gene under lac promoter/operator control . A strain carrying temperature-sensitive mutations in genes for polynucleotide phosphorylase and RNase II was found upon shift to nonpermissive temperature to show higher differential synthesis rates of L14-L24 (and L5) relative to those of several L5-distal spc operon gene products . We suggest that repression of distal ribosomal protein synthesis by S8 triggers nucleolytic cleavage of spc operon mRNA, followed by mRNA degradation by these 3'- to 5'- exonucleases, which is then responsible for inhibition of L14-L24 synthesis. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 85 - 9 Regulation of methionine synthesis in Escherichia coli: effect of the MetR protein on the expression of the metE and metR genes; Maxon ME et al.; A plasmid (pRSE562) containing the metE and metR genes of Escherichia coli was used to study the expression of these genes and the role of the MetR protein in regulating metE expression . DNA sequence analysis of the 236-base-pair region separating these genes showed the presence of seven putative met boxes . When this plasmid was used to transform either wild-type E . coli, metE mutant, or metR mutant, MetE enzyme activity increased 5- to 7-fold over wild-type levels . The metR gene was subcloned from pRSE562, and this plasmid, pMRIII, relieved the methionine auxotrophy of a metR mutant after transformation . The metR gene was also cloned into a vector containing the lambda PL promoter, and the MetR protein was overexpressed and purified to near homogeneity . This protein, when added to an in vitro DNA-dependent protein synthesis system in which the MetE and/or MetR proteins were synthesized, caused a large increase in the expression of the metE gene but a decrease in the expression of the metR gene . The in vitro expression of both genes was inhibited by the MetJ protein and S-adenosylmethionine in the presence or absence of MetR protein . These results provide evidence that the product of the metR gene is a trans-activator of the expression of the metE gene and that the expression of the metR gene is under autogenous regulation and is repressed by the MetJ protein. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 80 - 4 Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification; Kurose K et al.; Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+ . The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen . Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the bound oxygen . After light emission, aequorin may be regenerated by incubating the protein with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol . To understand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine . Six of the seven modified aequorins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin . Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type aequorin . The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 46 - 50 In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase; Wild JR et al.; The allosteric control of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli involves feedback inhibition by both CTP and UTP rather than just CTP alone . It has been known that CTP functions as a heterotropic inhibitor of catalysis; however, the inhibition by CTP alone is incomplete (50-70% at various aspartate concentrations) and there is only a partial occupancy of the allosteric binding sites by CTP at saturating concentrations . The logic of these allosteric characteristics can now be understood in that UTP is a synergistic inhibitor of ATCase in the presence of CTP even though UTP has no independent effect at pH 7.0 . When saturating concentrations of CTP are present, the concentration of substrate required for half-maximal activity (S0.5) of the native holoenzyme for aspartate increases from 5 to 11 mM . When CTP and UTP are both present, the aspartate requirement increases further (S0.5 = 17 mM) . At aspartate concentrations less than 5 mM, the heterotropic inhibition of ATCase is 90-95% in the presence of both pyrimidine nucleotides . UTP does enhance the binding of CTP to the holoenzyme but the number of tight binding sites does not change (n = 3) . The binding of UTP is stabilized in the presence of CTP although its binding characteristics are not as strong as those of CTP . The recent crystallographic studies of Kim et al . {Kim, H.K., Pan, Z., Honzatko, R.B., Ke, H.M . & Lipscomb, W.N . (1987) J . Mol . Biol . 196, 853-875} have described a structural asymmetry across the molecular two-fold axis that is consistent with these CTP/UTP interactions . The synergistic inhibition of ATCase by both CTP and UTP provides a satisfying logic for ensuring a balance of endogenous pyrimidine nucleotide pools. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 343 - 6 Cloned diphtheria toxin within the periplasm of Escherichia coli causes lethal membrane damage at low pH; O'Keefe D et al.; Acidic pH within endosomal vesicles of sensitive animal cells triggers a conformational change in diphtheria toxin (DT) that is believed to cause the B chain to insert into the vesicular membrane and the enzymic A chain to be released into the cytosol . In artificial lipid bilayers, DT forms ion-conductive channels under mildly acidic conditions (pH approximately 5) . Here we report a related phenomenon in Escherichia coli strains that secrete certain cloned DT-related proteins into their periplasm: the cells are rapidly killed at pH 5 but remain unharmed at pH 7 . Expression of full-length DT (an active-site mutant, to comply with the National Institutes of Health recombinant DNA guidelines) causes acid-sensitivity, whereas expression of the A chain alone does not . The killed cells are not lysed, but inner-membrane functions are impaired (membrane potential, active transport, and ion impermeability) . We propose that acidification of DT within the periplasm induces its insertion into the inner membrane, lethally damaging the permeability barrier . This discovery provides a potentially important selection procedure for mutations affecting the membrane insertion function of DT . Similar approaches may be useful in studying other proteins that undergo condition-dependent interaction with membranes. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 302 - 6 Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells; Goodwin RG et al.; A cDNA encoding biologically active human interleukin 7 was isolated by hybridization with the homologous murine clone . Nucleotide sequence analysis indicated that this cDNA was capable of encoding a protein of 177 amino acids with a signal sequence of 25 amino acids and a calculated mass of 17.4 kDa for the mature protein . Recombinant human interleukin 7 stimulated the proliferation of murine pre-B cells and was active on cells harvested from human bone marrow that are enriched for B-lineage progenitor cells . Analysis of RNA by blot hybridization demonstrated the presence of two size classes of interleukin 7 mRNA in human splenic and thymic tissue. Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 292 - 6 Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis; Mollison KW et al.; C5a is an inflammatory mediator potentially involved in a number of diseases . To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis . Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity . Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor . Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR . Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15 . Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency. Protein Seq Data Anal, 1989, 2(1), 9 - 16 Amino acid sequence analysis of the glutamate synthase enzyme from Escherichia coli K-12; Gosset G et al.; The amino acid sequence for the two subunits of the glutamate synthase of Escherichia coli K-12 was compared to the protein sequences compiled in the National Biomedical Research Foundation databank . Similarities were detected between the small glutamate synthase subunit and three members of the flavin-containing pyridine nucleotide-disulphide oxidoreductase superfamily, and also with three members of a lactate dehydrogenase family . Two segments in this glutamate synthase subunit showed similarity to regions previously proposed as part of dinucleotide-binding sites in some members of these two families . Similarity can be extended if the predicted secondary structure is considered . Based on these data, residues 148-260 and 289-409 in the small GOGAT subunit are proposed as dinucleotide-binding regions . Comparison of the amino acid sequence of the large glutamate synthase subunit with the glutamine phosphoribosylamine:pyrophosphate phosphoribosyltransferases of B . subtilis and E . coli revealed a significant similarity between the amino termini of these three enzymes . In these last two amidotransferases, the glutamine-binding site has been located in their amino-terminal region . The comparison with a second group of glutamine amidotransferases did not show any significant global similarity with the large glutamate synthase subunit . However, this polypeptide contains a small segment that shares similarity with a 13-amino acid segment proposed as part of the glutamine-binding site in this second group of amidotransferases. J Comput Assist Tomogr, 1989 Jan-Feb, 13(1), 71 - 4 Computed tomography in the diagnosis and treatment of solitary splenic abscesses; van der Laan RT et al.; Solitary splenic abscess is a rare entity and difficult to diagnose . Late recognition results in a high mortality . Recently percutaneous drainage has proved to be beneficial . The CT and ultrasound findings of two patients with splenic abscesses are reported . In one patient the solitary splenic abscess was drained percutaneously . In the other patient, a HTLV-III-positive man, a splenectomy was performed. J Cell Physiol, 1989 Jan, 138(1), 165 - 74 In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells; Meyrick B et al.; A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability . Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels . In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin . Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin . We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium . Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species . Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis . In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis . Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel . To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study. Blood, 1989 Jan, 73(1), 166 - 71 Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis; Lord ST et al.; The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen . Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment . By selective modification of residues in this region, we probed the features that participate in thrombin interactions . We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli . Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000 . The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain . When cell lysates were incubated with thrombin, FPA was released . By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions . Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost . Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition . Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed. Mutat Res, 1989 Jan, 210(1), 15 - 22 Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli; Janion C et al.; In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E . coli dam- cells are able to mutate following MMS treatment . Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E . coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains . The mutagenicity of MMS was also tested in several of these strains . The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system . The role of strand breaks in repair of mismatches induced by alkylating agents is discussed. Mutat Res, 1989 Jan, 210(1), 1 - 8 Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats; Jain AK et al.; The effects of tea extracts and their ingredients, catechins and L-ascorbic acid (AsA), on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in vitro and in the stomachs of rats using E . coli WP2 and S . typhimurium TA100 . The extracts of green tea and black tea leaves decreased the mutagenic activity of MNNG to E . coli WP2 in vitro in a desmutagenic manner . Catechins such as (-)-epigallocatechin from green tea leaves and the low-molecular-weight tannin fraction isolated from black tea extract with HP-20 resin also exhibited inhibitory effects against the mutagenic activity of MNNG . A desmutagenic effect of AsA on MNNG-induced mutagenicity was observed depending on the dose, though it was complicated . The effects were also demonstrated in the stomachs of rats by assaying the bacterial mutagenic in vitro; the tea extracts previously given orally to rats reduced the mutagenic activity of MNNG remarkably, though simultaneous administration showed less effect . The effectiveness of tea extracts for the decrease of MNNG-induced mutagenesis in vitro and in vivo suggests that the habitual drinking of tea may reduce the tumor-initiating potency of MNNG-type nitrosoureido compounds if they are formed in the stomach. Infect Immun, 1989 Jan, 57(1), 283 - 8 Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG; Yamaguchi R et al.; The gene for immunogenic protein MPB64 found in culture filtrates of only Mycobacterium tuberculosis and some strains of Mycobacterium bovis BCG was cloned by using a single-probe method and was sequenced . The gene analysis revealed that the structural gene for MPB64 consisted of 618 base pairs, and its deduced molecular weight was 22,400 . Twenty-two amino acids for a putative signal peptide and 205 amino acids for the MPB64 protein were observed . In the coding region, the third letter of the codon showed a biased codon and a high G+C content (78.5%) . The gene was expressed in Escherichia coli by using an E . coli expression vector . The product showed migration similar to that of the authentic MPB64 protein by electrophoresis and reacted with the polyclonal and the monoclonal antibodies raised against the MPB64 protein . The strict specificity of MPB64 could be applied to immunodiagnosis of tuberculosis. Infect Immun, 1989 Jan, 57(1), 196 - 203 Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum; Hsu PL et al.; The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp . pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T . pallidum subsp . pallidum-derived) antigen . An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701 . Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T . pallidum subsp . pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E . coli . Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E . coli . The 47-kDa antigen sequence did not reveal a typical leader sequence . The overall G+C content for the DNA corresponding to the structural gene was 53% . Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope . No repetitive primary sequence epitopes were found . The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease. Obstet Gynecol, 1989 Jan, 73(1), 31 - 4 Human decidua: a source of interleukin-1; Romero R et al.; These studies were conducted to determine whether human decidua produces interleukin-1 in response to bacterial endotoxin . Explants of human decidua were incubated with and without Escherichia coli endotoxin for 20 hours . When tested for interleukin-1-like activity with the D10.G4.1 T-cell bioassay, supernatants from endotoxin-stimulated decidua contained significantly more interleukin-1 activity than did supernatants from unstimulated decidua . This activity could not be attributed to interleukin-2, as determined in the CTLL/2 assay for interleukin-2 . Interleukin-1-like activity was due to interleukin-1, as demonstrated by the blockade of this bioactivity with antibodies against interleukin-1: interleukin-1 alpha and interleukin-1 beta . Antibodies against interleukin-1 alpha blocked the activity in five of six cases . In one instance, the bioactivity could be attributed to a mixture of interleukin-1 alpha and interleukin-1 beta . These data demonstrate that human decidua can produce interleukin-1 in response to bacterial endotoxin. J Virol, 1989 Jan, 63(1), 111 - 21 Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases; Loeb DD et al.; Processing of the retroviral gag and pol gene products is mediated by a viral protease . Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor . Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol . Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases . Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites . The inclusion of a charged amino acid at the processing site blocks cleavage . A subset of these substitutions also inhibits processing at the nonmutated sites. Symp Soc Exp Biol, 1989, 43, 249 - 58 Goblet cell mucin in human foetal colon, its composition and susceptibility to enzyme degradation: a histochemical study; Filipe MI et al.; This work is part of an investigation into G . I . mucin susceptibility to enzyme degradation in normal and disease states . Formalin-fixed/paraffin embedded foetal (14-23 weeks) and neonatal colonic tissue was stained for mucins (neutral, N- and O-acylated sialomucins and sulphomucins) and PNA, UEA1, and Limax flavus . Enzymes tested: neuraminidases, alpha- and beta-galactosidase (E . coli and B . testis), beta-N-acetyl-glucosaminidase, alpha-fucosidase, single or in sequence, with and without prior neuraminidase treatment and followed by the stains . Acid mucins predominate throughout foetal life, sulphation occurs at 14 weeks and O-acylated sialomucins at 23 weeks . PNA and UEA1 are seen in traces or not detected . The mucin profile at birth is similar to the adult . Colonic mucins are susceptible to neuraminidase which abolishes Limax staining . The glycosidases effect on PNA is seen only with prior neuraminidase treatment and is particularly marked with beta-Gal(BT) in Neu----beta-Gal----beta-N-AcetylGlc than with beta-Gal (EC) . Fucosidase with prior neuraminidase treatment has effect on UEA1 (decreases) and PNA (increases) affinities . Neuraminidase is essential as a first step in the process and by using beta-galactosidases EC and BT it was possible to show different PNA binding affinities . Preliminary data demonstrate the feasibility of this histochemical approach to the study of colonic mucins and forms the basis for further studies in the adult. Yi Chuan Xue Bao, 1989, 16(6), 442 - 7 {The features of the 3' terminal sequences of the 18S rRNA gene from silkworm Attacus ricini}; Ling MH et al.; The 3' terminal sequence of the gene for 18S rRNA of silkworm Attacus ricini have been sequenced . Comparison of this sequence with the 18S rDNA of silkworm Bombyx mori, Drosophila melanogaster, rat and the 16S rDNA of E . coli has shown that there is a remarkable homology between them . Moreover, the stem and loop formation of 3' regions of these rDNAs are very similar . There is a conservative EcoR1 site in the 3' region of these rDNAs . These results may contribute to the understanding of the functions of the 3' end of 18S rDNA in protein synthesis, in proceeding of rRNA precursor, and to the understanding of the evolutionary relation of rDNAs. Acta Med Hung, 1989, 46(4), 253 - 61 Studies on the monocyte interleukin-1 and tumour necrosis factor-alpha production in patients with alcoholic liver cirrhosis; Muzes G et al.; Interleukin-1 and tumour necrosis factor-alpha activities by E . coli lipopolysaccharide-triggered monocytes were studied in patients with chronic alcoholic liver disease . Monocytes from cirrhotic patients were shown to have significantly reduced IL-1 and TNF-a activities, compared with that from age and sex matched healthy controls . These findings indicate further immunoregulatory disturbances concerning alcoholic liver cirrhosis. Acta Med Hung, 1989, 46(4), 245 - 52 Defective production of interleukin-1 and tumour necrosis factor-alpha by stimulated monocytes from patients with systemic lupus erythematosus; Muzes G et al.; Interleukin-1 and tumour necrosis factor-alpha activity by E . coli lipopolysaccharide-triggered monocytes was studied in patients with systemic lupus erythematosus in various stages of activity . Monocytes from both groups of SLE patients produced significantly less tumour necrosis factor-alpha activity than those of age and sex matched healthy controls . However, interleukin-1 activity was only significantly reduced in patients with active stage of the disease . These findings indicate further immunoregulatory disturbances in monocyte function concerning SLE. Yi Chuan Xue Bao, 1989, 16(2), 89 - 96 {Primary structure of the leading sequence of 16S rRNA gene in Brassica napus chloroplast}; Gao JG et al.; The 840bp leading sequence of 16S rRNA gene (including 140bp 16S rRNA gene) was sequenced . Screening for structure elements common to tRNA reveals a gene coding for tRNA(Val) . An open reading frame, and three E . coli RNA polymerase binding sites were found . In front of the open reading frame, the tRNA(Val) gene, and the 16S rRNA gene, a stable stem-loop structure can be formed . We suggested that these stem-loop structures may have some effect on the gene transcription which are located on the inverted repeat sequence in chloroplast. Mol Reprod Dev, 1989, 1(3), 201 - 7 Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation; West JD et al.; In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored . This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised . Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA . These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific . In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa. J Steroid Biochem, 1989, 34(1-6), 369 - 74 A highly charged sequence of chick hsp90: a good candidate for interaction with steroid receptors; Binart N et al.; The sequence of the entire chick 90 kDa heat shock protein (hsp90), the non hormone binding component of the heterooligomeric form of steroid receptors, is reported . A comparison of the amino acid sequence of the chick hsp90 to that of the homologous hsp90 from yeast to man, reveals 64-96% identity respectively, and even with E . coli hsp90 an identity of 44% is observed . Analysis of the sequence and a secondary structure prediction of chick hsp90 suggest that two hydrophilic regions A and B, predicted in alpha-helix may play a role in the interaction of hsp90 with other proteins such as steroid hormone receptors . While there are regions of the sequences completely conserved in all hsps90, the most negatively charged hydrophilic region (A) is absent in the E . coli protein. Proteins, 1989, 6(3), 259 - 66 A hydrophobic cluster forms early in the folding of dihydrofolate reductase; Garvey EP et al.; The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site-directed mutagenesis and by physicochemical means . The absence of this fluorescence-detected phase in the refolding of the Trp-74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp-74 . In addition, water-soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and native-like conformers . These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp-74 from solvent. Int Arch Allergy Appl Immunol, 1989, 90(4), 364 - 7 Relation between splenic antibody formation and serum antibody levels to sheep erythrocytes as studied at the level of individual mice; Hol C et al.; The relation between splenic antibody formation and serum antibody levels after intraperitoneal immunization of mice with sheep erythrocytes was studied in individual animals . A strong correlation between plaque-forming cell numbers in the spleen and haemolysin and haemagglutinin titres in the serum was observed . These results are at variance with recent data on the development of splenic auto-haemolysin-forming cells and corresponding serum levels after injection of mice with Escherichia coli endotoxin . The background behind the discrepancy between the antigen-driven immune response and the more polyclonal stimulation of B cells with endotoxin is being discussed. Genome, 1989, 31(1), 45 - 52 The human recombination strand exchange process; Moore SP et al.; A mechanism for the initiation of general recombination that involves the formation of left-handed Z-DNA heteroduplex segments adjacent to right-handed B-DNA heteroduplex segments is discussed . The paranemic nature of this initiation structure allows for homology recognition in the absence of strand cleavage . This model suggests that proteins catalyzing recombination initiation via the formation of paranemic joint should in some capacity recognize Z-DNA . Other studies have shown that both the RecA protein of Escherichia coli and the Rec1 protein of Ustilago maydis have a greater affinity for Z-DNA than B-DNA . Here we have used Z-DNA affinity chromatography to purify a peptide of approximately 120 kilodaltons from a human tumor cell line that catalyzes a simple recombination strand-transfer reaction similar to one developed for the characterization of the RecA and Rec1 proteins . We report details of the characterization of the human strand-transfer activity and identified a potential human recombination complex. Symp Soc Exp Biol, 1989, 43, 423 - 8 Binding of pig small intestinal mucin glycopeptides to fimbriated enterotoxigenic Escherichia coli; Lindahl M et al.; Fimbriated enterotoxigenic Escherichia coli were found to specifically bind mucin glycopeptides from pig small intestine . Bacteria with N-acetyl galactosamine-specific fimbriae (F41) were found to have two populations of binding sites for mucin glycopeptides . High affinity sites (5 X 10(2) per cell) with a dissociation constant of 1 X 10(-7) M . Bacteria with sialic acid specific fimbriae (K99) were estimated to possess 3 X 10(3) binding sites per cell with a dissociation constant of 6 X 10(-7) M. Cancer Commun, 1989, 1(4), 243 - 51 Cloning of the cDNA and sequence of the human proliferating-cell nucleolar protein P120; Fonagy A et al.; The 120 kDa proliferating-cell nucleolar antigen described by Freeman et al . (Cancer Res . 48:1244; 1988) is the most cancer specific of the proliferation-associated nucleolar proteins identified thus far . It is localized in a novel nucleolar microfibrillar structure recently described by Ochs et al . (Cancer Res . 48:6523; 1988) . The amino acid sequence has been determined by a combination of cDNA and genomic DNA sequences . This molecule contains, consecutively, four major domains: a basic domain, an acidic domain, a hydrophobic and methionine-rich domain, and a domain rich in cysteine and proline residues . The isolated cDNA was shown to code for the HeLa P120 protein as shown by a similarity in immunoreactivity, mobility on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and patterns of partial digestion of the Escherichia coli-expressed P120 and the HeLa nucleolar P120 protein . This protein is of special interest because it is expressed in early G1 and, in studies to date, it has not been detected in benign tumors and most normal resting tissues. Revis Biol Celular, 1989, 21, 347 - 60 Proteolysis induced by metal-catalyzed oxidation; Levine RL; Many enzymes are now known to be subject to site-specific, covalent modification mediated by activated oxygen species . Oxidatively modified enzymes generally lose catalytic activity, gain carbonyl groups in their side chains, and become susceptible to proteolytic degradation . Thus, oxidative modification is one of the covalent alterations which marks proteins for degradation . This degradation is mediated by specific intracellular proteinases which degrade only the modified proteins . One can then view the turnover as occurring in two distinct steps: 1) Metal-catalyzed oxidative modification marks the protein for degradation . 2) The marked protein is degraded by a specific proteinase . Utilizing a model metal-catalyzed oxidation system (ascorbate/iron/oxygen) studies on bacterial glutamine synthetase revealed several functional and structural changes . Analysis of the time courses of these changes established correlations between specific structural alterations and increased susceptibility to proteolytic degradation. Am J Physiol Imaging, 1989, 4(4), 151 - 4 Left ventricular volume and function changes during beta-blockade and accompanying atrial pacing; Dormehl IC et al.; Radionuclide ventriculography was performed at rest and during transoesophageal pacing on nine anaesthetized baboons (Papio ursinus), alternatively with and without administration of a beta-blocker . Left ventricular volumes, cardiac output, and ejection fraction were determined at each heart rate . A gradual decrease in end-diastolic, end-systolic, and stroke volume followed with increasing heart rate, significantly more pronounced under beta-blockade . Cardiac output and ejection fraction showed no significant changes, although tendencies to increase were observed without beta-blockade . Comparisons of these parameter changes were drawn to those previously observed during the first 5 h of septic shock (E . coli) . We conclude that the tachycardia present during this phase of shock is not the only contributory factor to cardiac function changes but that other depressant influences prevail as the added effect of the beta-blocker seems to suggest. Am J Physiol Imaging, 1989, 4(4), 136 - 42 Use of indium-111 oxine to study pulmonary and hepatic leukocyte sequestration in endotoxin shock and effects of the beta-2 receptor agonist terbutaline; Sigurdsson GH et al.; The dynamic behavior of indium-111 oxine-labeled leukocytes was simultaneously recorded in multiple organs during endotoxin shock in sheep . Also, the effects of the beta-2 receptor agonist terbutaline were studied . An experimental protocol was designed to mimic a clinical condition in an intensive care setting as far as possible . The animals were ventilated with 50% oxygen to avoid hypoxemia and were given large amounts of intravenous fluids to reduce adverse effects of hypovolemia . A moderate dose of E . coli endotoxin (10 micrograms/kg bwt) was given by intravenous infusion to 14 adult sheep, seven of them receiving continuous intravenous infusion of terbutaline (20 micrograms/kg/hr) during 4 hr, starting 30 min after endotoxin, when signs of lung injury had developed . The other seven acted as controls . A marked pulmonary and hepatic leukocyte sequestration together with a sharp drop in leukocyte counts in peripheral blood occurred within minutes after start of the endotoxin infusion in both groups . However, no changes were observed in the kidneys or the gut . After 60 min and until the end of the experiment, there was a significantly lower activity in the lungs and in the liver of the animals treated with terbutaline than in the controls (P less than .01) . Furthermore, less marked hemodynamic and respiratory alterations occurred in the terbutaline group compared with the controls . This study confirms the results of other investigators showing that significant leukocyte sequestration occurs in the lungs during endotoxemia, but it also demonstrates that leukocytes sequestrate in the liver, although slightly less than in the lungs.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Biochem, 1989, 21(11), 1211 - 5 Regulation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics; Carr S et al.; 1 . Escherichia coli heat-stable enterotoxin (ST) induces a secretory diarrhea by binding to receptors on brush borders of intestinal villus cells, activating particulate guanylate cyclase and increasing intracellular concentrations of guanosine 3',5'-cyclic monophosphate (cyclic GMP) . 2 . However, little is known concerning coupling of receptor-ligand interaction to enzyme activation . 3 . This study compares the kinetics of toxin-receptor binding and enzyme activation to better understand this transmembrane signal cascade . 4 . Toxin receptor binding was linear and saturable with 50% of maximum displacement of {125I}ST by unlabeled toxin observed at 1.1 x 10(-7) M . ST increased the maximum velocity (Vmax) of guanylate cyclase with magnesium or manganese as the cation substrate without altering the affinity of the enzyme for its substrate or its positive cooperativity . 5 . The concentration of toxin yielding half-maximum stimulation of guanylate cyclase was 1.2 x 10(-6) M, 10-fold higher than the affinity of the ligand for its receptor . 6 . These data are consistent with the suggestion that ST-receptor interaction is coupled to activation of particulate guanylate cyclase . 7 . However, the discrepancy between the affinity of ST for its receptor and its efficacy in activating the enzyme suggests that this coupling is complex . 8 . Possible mechanisms underlying this coupling are discussed. Eur Biophys J, 1989, 17(4), 201 - 10 A theoretical study of glucosamine synthase . Part I . Molecular mechanics calculations on substrate binding; Tempczyk A et al.; Glucosamine synthase transfers the gamma-amino group of glutamine to fructose, producing 1-glucosamine which is the key constituent of bacterial and fungal cell walls . In this study, model calculations were performed on substrate binding to the enzyme active site . Two models of the active site of glucosamine synthase were proposed, which assume two different sequences of aminoacids, Cys-Gly-Ile and Cys-Ala-Cys, the first one being the N-terminal sequence of the Escherichia coli enzyme . Several initial geometries were assumed for these tripeptides, the energy was then optimized by means of molecular mechanics . It has been found that the structure which is both energy optimal and satisfies the assumed cysteine sulphur arrangement consists of combinations of C7eq and C7ax conformations of single residues . Molecular mechanics calculations were then performed on glutamine and D-fructose-6-phosphate, which are the substrates of the enzymatic catalysis, and on their complex with the enzyme glutamine-binding site . The spatial configuration of the compounds under study, which is optimal as far as the reaction path is concerned, also turned out to be an energy minimum. Comp Biochem Physiol A, 1989, 94(2), 323 - 31 Effect of Escherichia coli infection on growth and protein metabolism in broiler chicks (Gallus domesticus); Tian S et al.; 1 . A controlled experimental Escherichia coli infection was developed in broiler chicks . 2 . Infection with E . coli significantly reduced feed intake, altered growth of the whole body, eviscerated carcass, skeletal muscles, heart and liver . Organ weight and/or the proportions of organs within the body were affected . 3 . Protein accumulation in the eviscerated carcass, extensor digitorum communis and sartorius muscles was severely inhibited by infection, and to a greater extent than body weight . 4 . Failure of muscle tissue to accumulate protein was associated with a significant decline in protein synthesis, when measured in vitro (-48%; P less than 0.05) and in vivo (-42%; P less than 0.001) . Protein degradation also declined (-28.7%), but to a smaller extent than protein synthesis . 5 . Although the infected chicks showed no viable bacteria at day 12 after infection, chicks did not reach the same body weight as controls by day 30 after infection. Microbiol Immunol, 1989, 33(4), 265 - 75 Purification and characterization of the CS2 pili of colonization factor antigen II produced by human enterotoxigenic Escherichia coli; Honda T et al.; A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied . Analysis revealed that CS2-possessing ETEC was predominant among isolates from traveller's diarrhea at Osaka, Japan . TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl-Sepharose CL-4B . It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili . This suggests that TH61 pili purified in this study are CS2 pili . Subunit (pilin) of the purified pili has a molecular weight of about 16,000 . Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili . These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells. Life Sci, 1989, 45(6), 533 - 41 Involvement of platelet-activating factor (PAF) in endotoxin-induced intestinal motor disturbances in rats; Pons L et al.; Intestinal myoelectrical activity was investigated in conscious fasted rats chronically implanted with Nichrome electrodes in the duodeno-jejunum . Motility of the small intestine was characterized by the presence of migrating myoelectric complex (MMC) occurring regularly at 16.2 +/- 5.8 minute intervals . Intravenous administration of endotoxin (E . coli S.0111:B4) at a dose of 50 micrograms/kg increased the interval between MMC to 112.6 +/- 26.8 min, the duration of these effects being dose-related between 10 to 100 micrograms/kg . Such a typical myoelectrical alteration, corresponding to rapidly propagated groups of spike bursts, was mimicked by the IP administration of PAF at doses of 10 to 50 micrograms/kg . Previous administration of BN 52021, a specific PAF antagonist at a dose of 50 mg/kg abolished the motor alterations induced by IP injection of PAF (25 micrograms/kg) and significantly (p less than 0.01) reduced by 61.2% those induced by IV endotoxin (50 micrograms/kg) . Indomethacin (10 mg/kg IP) as well as SC 19220 (5 mg/kg IV), a PGE2 antagonist, injected prior to endotoxin (50 micrograms/kg IV) or PAF (25 micrograms/kg IP) also reduced significantly (p less than 0.01) the duration of MMC inhibition . It is concluded that endogenous release of PAF is partly responsible for the intestinal motor alterations induced by endotoxin; these effects, strongly reduced after treatment with BN 52021, are also mediated through the release of prostaglandins. Microbiol Immunol, 1989, 33(5), 373 - 9 Binding of F41 and K99 fimbriae of enterotoxigenic Escherichia coli to glycoproteins from bovine and porcine colostrum; Lindahl M; F41 and K99 fimbriae of enterotoxigenic Escherichia coli were found to bind to periodate-sensitive oligosaccharides of glycoproteins from bovine and porcine colostrum . Only a minor component of casein fractions (kappa-casein) possessed receptors for one type of fimbriae (K99) . Both whey and fat globule membranes were rich in glycoproteins with receptor structures . Porcine colostrum seemed to contain a higher quantity of receptors than bovine colostrum. Intervirology, 1989, 30(2), 86 - 95 Characterization of southern African isolates of maize streak virus: typing of three isolates by restriction mapping; Clarke BA et al.; The genomic replicative form DNAs (RF-DNA) of three maize streak virus isolates (MSV-CT, MSV-PE, and MSV-SW) from widely separated locations in southern Africa were characterized by restriction endonuclease mapping in order to assess the feasibility of using the technique to determine genetic variability between isolates . The viruses were transmitted to and propagated in laboratory-grown maize by the leafhopper vector Cicadulina mbila (Naude) . MSV-PE produced more severe symptoms than MSV-CT and MSV-SW; the isolates were serologically identical in 'western' immunoblot tests, but distinct in 'sandwich' enzyme-linked immunosorbent assays . RF-DNA of all three isolates was prepared from infected maize; the RF-DNA of MSV-CT and MSV-PE was cloned in a plasmid vector in Escherichia coli . Restriction maps were generated from this cloned DNA and from the RF-DNA of MSV-SWA . The maps were similar in regions expected to be conserved, but there were also important differences between all isolates . The implications of these results, and of relationships amongst these and other sequenced isolates of MSV, are discussed. Can J Vet Res, 1989 Jan, 53(1), 43 - 7 Requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains; Broes A et al.; The requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains lacking the colonization factor antigens K88, K99, 987P and F41 was investigated using two encapsulated strains and their acapsular variants, one of which produced the fimbrial antigen CS1541 in vitro . None of the strains adhered in vitro to enterocytes isolated from newborn colostrum-deprived piglets . All of the strains caused diarrhea in orally infected, hysterotomy-derived, colostrum-deprived piglets although a great variability in the clinical response of the piglets was observed . Colonization of the small intestine of infected piglets by these strains was only moderate and no differences in the ability to colonize the small intestine was noted between the strains . All of the strains reacted in the indirect fluorescent antibody test with both CS1541 and 987P antisera when applied to organisms in the intestines of infected piglets . A control strain expressing the 987P fimbrial adhesin also reacted with the CS1541 antiserum applied to organisms in the intestines of an infected piglet . It was concluded that capsular antigen KX105 was not essential for intestinal colonization and production of diarrhea in hysterotomy-derived colostrum-deprived pigs, and that fimbrial antigen CS1541 does not promote in vitro adherence to enterocyte brush borders but could be important in bacterial colonization in vivo. J Med Chem, 1989 Jan, 32(1), 165 - 70 Synthesis of an analogue of tabtoxinine as a potential inhibitor of D-alanine:D-alanine ligase (ADP forming); Greenlee WJ et al.; The design and synthesis of a potential inhibitor of D-alanine:D-alanine ligase (ADP forming) (EC 6.3.2.4) are described . This enzyme, which catalyzes the second step in the biosynthesis of bacterial peptidoglycan, is believed to generate D-alanyl phosphate as an enzyme-bound intermediate . With tabtoxinine, a potent inhibitor of glutamine synthetase, as a model, beta-lactams 9R and 9S were synthesized as potential precursors of a D-alanyl phosphate mimic. Infect Immun, 1989 Jan, 57(1), 82 - 7 Age-specific colonization of porcine intestinal epithelium by 987P-piliated enterotoxigenic Escherichia coli; Dean EA et al.; Neonatal (less than 1-day-old), 3- and 7-day old, and older (3-week-old postweaning) pigs were challenged by intragastric inoculation with 987P-piliated (987P+) enterotoxigenic Escherichia coli (ETEC) 987 . Neonatal pigs were colonized (i.e., there were greater than or equal to 10(8) CFU of test strain per 10-cm ileal segment) and developed diarrhea . Intestinal colonization and the incidence and severity of diarrhea were lower in 3- and 7-day old pigs than in neonates . Older pigs were not colonized and did not develop diarrhea following oral inoculation with five strains of 987P+ ETEC . Strain 987 (987P+) adhered in vitro to intestinal epithelial cell brush borders isolated from both neonatal (sensitive) and older (resistant) pigs . The in vivo growth and expression of 987P pilus by strain 987 in ligated ileal loops created in neonatal and older pigs were similar . The in vivo adherence of 987P+ ETEC to intestinal epithelium in ligated ileal loops in neonatal and older pigs was compared . In neonatal pigs, most of the bacteria were in layers associated with the villous epithelium . In older pigs, most of the bacteria were associated with mucus-like material in the intestinal lumen . We concluded that swine develop an innate resistance to 987P+ ETEC by 3 weeks of age . This resistance does not appear to be due to an absence of 987P-specific receptors in the intestines of the older pig or to an inability of 987P+ bacteria to grow and express pili in the older pig . We hypothesized that the resistance of older pigs to 987P-mediated disease is due to release of 987P-specific receptors into the intestinal lumen, where these receptors facilitate bacterial clearance rather than bacterial adherence to intestinal epithelium and colonization. Infect Immun, 1989 Jan, 57(1), 76 - 81 Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli; Hoschutzky H et al.; The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens . Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16 . After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea . The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000 . The fimbrillins were smaller, their molecular weights being different with different F antigens . The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B . Lund, G . Lindberg, B.-I . Marklund, and S . Normark, Proc . Natl . Acad . Sci . USA 84:5898-5902) . An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared . The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads . The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl . The results indicate that the P adhesin(s) of uropathogenic E . coli represents a group of related proteins with conserved receptor recognition domains . The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc . Natl . Acad . Sci . USA 84:5898-5902; B . Lund, F . Lindberg, and S . Normark, J . Bacteriol . 170:1887-1894). G Batteriol Virol Immunol, 1989 Jan-Dec, 82(1-12), 17 - 24 Interferon release by LPS-treated macrophages from patients affected by neoplasia; Merendino RA et al.; Concerning the modulatory role of LPS on immunocompetent cells, the production of interferon by macrophages primed with human gamma interferon (HUIFN gamma), was studied . In particular the production of IFN from primed macrophages of patients affected by breast cancer, differentiated in presence of autologous serum or in serum from healthy donors, was compared with the IFN production from macrophages of healthy donors . The levels of endotoxin-induced IFN were enhanced by priming macrophages with HUIFN gamma . The "in vitro" treatment with Escherichia coli LPS restores the interferon production of primed macrophages, obtained from patients affected by breast cancer, differentiated in presence of serum from healthy donors . Moreover, LPS treatment of primed macrophages from patients, differentiated in autologous serum, did not influence the interferon production of these cells . Nevertheless, primed macrophages from healthy donors appeared to be more sensitive to LPS treatment in comparison with primed macrophages from patients affected by breast cancer. J Pharm Biomed Anal, 1989, 7(2), 233 - 8 Interferon-alpha: a gene family in therapeutic use; Lundgren E et al.; Several variants of interferon-alpha (IFN-alpha) were isolated and purified to homogeneity . They differed to various degrees in biological properties . However, three IFN-alpha 2 variants showed only minor differences from a variant called IFN-alpha 88 with regard to their ability to inhibit growth and to bind to specific receptors, tested on Daudi cells . Two monoclonal antibodies were studied, which showed overlapping specificity for at least one peptide obtained after HPLC separation of tryptic digests . The monoclonal antibodies could discriminate between sequence differences to a much higher degree than the receptor on Daudi cells . It is concluded that the receptor is degenerate and binds well to different structural variants of IFN and that for therapeutic use, several of the variants will probably have the same biological potency. Princess Takamatsu Symp, 1989, 20, 171 - 6 Phosphorylation of the tumor suppressor gene RB protein by M-phase specific histone H1 kinase; Taya Y et al.; We have noted the presence of the consensus amino acid sequence for phosphorylation by M-phase specific histone H1 kinase in six sites of the tumor suppressor gene retinoblastoma (RB) protein and determined whether or not RB protein is, in fact, phosphorylated by this kinase . Highly purified enzyme was used for this purpose . Human cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in Escherichia coli cells were shown to be phosphorylated by this kinase in vitro . Synthetic peptides for the six expected sites were also phosphorylated . These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase . We also noted the presence of putative phosphorylation sites by H1 kinase in a homologous region between the RB gene and L1 family repetitive sequences. J Cell Sci Suppl, 1989, 12, 171 - 82 Cellular and viral control of the initiation of DNA replication; Roberts JM et al.; Cell-free replication of SV40 DNA in extracts prepared from S phase cells is at least 20-fold more efficient than in extracts from G1 cells . The increased activity of S phase extracts correlates with the presence of an S phase-specific cellular factor that enhances DNA unwinding at the replication origin . This change in origin-DNA structure during the initiation of SV40 replication proceeds through at least three discrete steps which can be distinguished by their extent of topologic unwinding (linking differences of -1, -2 and -5) . Specific DNA elements flanking the core origin enhance replication in vivo and facilitate the formation of the pre-initiation complexes, indicating that formation of these underwound conformations may be the limiting step in the initiation of DNA synthesis . In addition, the factor that activates DNA replication in extracts from S phase cells also enhances the formation of the most highly underwound -5 pre-initiation complex . These observations suggest that during SV40 replication, formation of the rate-limiting pre-initiation complex is the focus of at least three regulatory elements . Two of these are DNA sequences flanking the replication origin and the third is a cellular factor specific to the S phase cell. Folia Biol (Praha), 1989, 35(6), 360 - 72 Structural and functional stability of foreign genes in transgenic tobacco plants; Vyskot B et al.; The Drosophila genomic fragment Dm111 and the selectable dominant nptII gene were transferred via a Ti-vector into tobacco plants in order to check the structural and functional stability of transgenes in plants and their progeny . Southern blot analyses clearly showed that transgenes were integrated intact and did not suffer from any gross DNA rearrangements . Contrary to this structural stability, not all of the transgenic plants and their offspring displayed the original and stable expression of the nptII gene . The levels of the NPTII enzyme strongly varied in individual plants and did not depend on the copy number of the nptII gene . Both the non-stable nptII expression during the individual plant development in one original transgenic tobacco and some irregularities in segregation ratios after self-pollination indicated that epigenetic effects due to methylation of DNA modulated the expression of foreign genes in transgenic plants . This conclusion was supported by a spontaneous and 5-azacytidine-stimulated demethylation. Proteins, 1989, 6(3), 294 - 305 Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation; Todd AP et al.; Colicin E1 is an E . coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel . We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels . A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine . A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix . After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure. Proteins, 1989, 6(3), 249 - 58 The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA; Beran-Steed RK et al.; Limited digestion of E . coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme . This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme . It required around 400 mM NaCl for elution . A truncated topoisomerase that lacks this C-terminal domain was purified . It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl . Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration . Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme. Proteins, 1989, 6(3), 231 - 9 Peptide sequencing and site-directed mutagenesis identify tyrosine-319 as the active site tyrosine of Escherichia coli DNA topoisomerase I; Lynn RM et al.; Tyrosine 319 of E . coli topoisomerase I is shown to be the active site tyrosine that becomes covalently attached to a DNA 5' phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme . The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA-linked tryptic peptide . Site-directed mutagenesis converting Tyr-319 to a serine or phenylalanine completely inactivates the enzyme . The structure of the enzyme and its catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available information. J Cell Sci Suppl, 1989, 11, 115 - 37 Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity; Kozutsumi Y et al.; Immunoglobulin heavy chain binding protein (BiP) associates transiently with various proteins destined for the secretory pathway . To investigate the relationship between BiP and the 78K (K = 10(3) Mr) glucose-regulated protein (GRP78), we have determined a partial amino acid sequence of purified mouse BiP and isolated and sequenced a full-length cDNA clone encoding mouse GRP78 . The 26 amino-terminal residues of the mature BiP protein are identical to a sequence of amino acids located near the start of the open reading frame encoding GRP78 . A polyclonal antiserum raised against mouse GRP78 protein expressed in bacteria from the cloned GRP78 cDNA could immunoprecipitate complexes consisting of BiP and unfolded forms of immunoglobulin heavy chains . Furthermore, a monoclonal antibody raised against mouse BiP immunoprecipitated mouse GRP78 expressed in monkey CV-1 cells from an SV40-GRP78 recombinant vector . Finally, like the endogenous BiP of simian cells, mouse GRP78 associated with malfolded, non-glycosylated forms of influenza hemagglutinin (HA) when GRP78 and HA were co-expressed from SV40 vectors in CV-1 cells . These studies confirm that BiP is identical to GRP78 . Comparison of the nucleic acid and deduced amino acid sequence of mouse GRP78 with those of other rodent and human GRP78s revealed an extremely high degree of sequence identity . BiP/GRP78 is closely related (approximately 60% identity) to the cytoplasmic 70K heat-shock proteins . Surprisingly, the carboxy-terminal 29 amino acids of BiP/GRP78, which are not conserved in HSP70 proteins, are almost identical in sequence to the steroidogenesis activator peptide found in the cytoplasm of rat Leydig tumor cells . Possible relationships between these polypeptides are discussed. Free Radic Res Commun, 1989, 8(1), 37 - 45 Intracellular oxidative cleavage of DNA in Escherichia coli by the copper-1,10-phenanthroline complex; Lickl E et al.; Plasmid and chromosomal DNA of E . coli during exponential growth are cleaved after treatment with copper(II)-1, 10-phenanthroline complex (1:2) without providing any exogenous reductant . About 1500 copper molecules per cell are present as estimated by atomic absorption analysis . Within the cell the endogenous reducing substances may have participated in the sequential oxidative reactions, which lead to the damage of DNA . A portion of the resultant DNA fragments originates from plasmid DNA as demonstrated by hybridization tests. Zhongguo Yao Li Xue Bao, 1989 Jan, 10(1), 7 - 10 {Hypothermic effect of intracerebroventricular injections of taurine on endotoxin-induced fever in rabbits}; Chen YJ et al.; Taurine (Tau) was infused by intracerebroventricular (icv) injection 20 min after iv endotoxin (ET) . Cerebrospinal fluid was taken from the posterior cisterns at 0, 60, 210 and 390 min after Tau infusion . The concentrations of cAMP and PGE2 in the samples were determined by radioimmunoassay . In the control groups, an equivalent isotonic saline (NS) (non-pyrogenic) instead of Tau or ET was used respectively . The rectal and ear skin temperatures of the rabbits in a non-fasting state were recorded automatically . Tau icv 1.0 mg/50 microliters, followed by the slow infusion of Tau 0.06-0.22 mg/(kg.min) for about 20 min into rabbits, caused sedation and peripheral vasodilation (rise in ear skin temperature), and initially blocked the rise in rectal temperature induced by ET (iv, 1 micrograms/kg) . The results for the control groups were significantly different from those of the ET + Tau group . In the ET + NS group, the fluctuations in concentrations of PGE2 and cAMP paralleled the change in rectal temperature, but in the ET + Tau group, the changes in PGE2 and cAMP concentrations were similar to those of the ET + NS group, even though the fever was initially inhibited . There were no changes in the concentrations of these mediators in the NS + Tau group. Arch Virol, 1989, 107(1-2), 1 - 13 Release of mycoplasmavirus L1 upon transfection of Acholeplasma laidlawii with homologous and heterologous viral DNA; Just W et al.; This communication reports on the release of Mycoplasmavirus L1 after infection of Acholeplasma laidlawii with purified L3 virus . Release also occurred after transfection with certain restriction fragments from MV-L3 and MV-L1 genomes . Since circular molecules are efficiently taken up in polyethylene glycol-mediated transfection, inducing fragments were applied cloned in E . coli plasmids . Release was also observed after electroporation of cells incubated with MV-L1 replicative intermediate DNA and linear MV-L3 DNA isolated from virus particles, respectively . Released MV-L1 viruses were identified after virus plaque formation on indicator lawns according to plaque morphology and hybridization with labeled viral DNA probes as well as by DNA restriction analysis . Uninfected and untransfected cells from six laboratory strains of A . laidlawii (including a MV-L1 resistant one) were examined for the presence of MV-L1 DNA . They all bear MV-L1 DNA integrated in their genomes. Biomed Biochim Acta, 1989, 48(1), 69 - 76 DNA topoisomerase I from Diplococcus pneumoniae; Storl K et al.; A type I topoisomerase has been purified from Diplococcus pneumoniae using phosphocellulose and hydroxylapatite chromatography . The purified enzyme catalyses the relaxation of negatively supercoiled DNA . The relaxation requires Mg2+ and is favoured by 0.2 M monovalent cations . The enzyme does not exhibit catenating or supercoiling activities . Using circular pBR322 DNA from dam+- and dam- -hosts as substrates for the enzyme, the relaxation reaction proceeds with somewhat higher efficiency with plasmids containing methylated adenine in GATC sequences . Plasmids from dcm+- and dcm- -hosts show no difference in reactivity. Microbiol Immunol, 1989, 33(6), 449 - 57 Characterization of a nalidixic-acid-resistant mutant of Escherichia coli as a strict aerobe; Yoshizawa Y et al.; An Escherichia coli mutant, C18, which plates at an efficiency of 5.0 x 10(-4) under anaerobic condition, was isolated among spontaneous nalidixic-acid-resistant mutants . This strict aerobic mutation was mapped by P1 cotransduction with a gyrA linked transposon Tn10 and found to be at the gyrA gene . A low degree of superhelicity of pBR322 DNA isolated from C18 was demonstrated by agarose gel electrophoresis with various concentrations of ethidium bromide . The superhelical density of pBR322 isolated from C18 was 80% of the value of pBR322 isolated from wild-type bacteria cultured under aerobic condition, and 50% cultured under anaerobic condition . These results lead us to conclude that a certain mutation of the gyrA gene causes a decrease in DNA superhelicity and prevents anaerobic growth. Life Sci, 1989, 45(6), 509 - 15 Desensitization of alpha-1 adrenergic receptor mediated smooth muscle contraction in aorta from endotoxic rats; Wakabayashi I et al.; Desensitization of vascular smooth muscles in endotoxemia was studied using the aorta from intraperitoneally endotoxin-injected rats . The KCl- and phenylephrine-induced contractions were significantly decreased in the endotoxic aorta compared to the control . In the endotoxic aorta the phenylephrine-induced contracture showed a gradual tension decrease after reaching a plateau and was attenuated by prior exposure to high concentration of phenylephrine, while KCl produced a sustained contraction and it was not affected by prior exposure to phenylephrine . The phenylephrine- and KCl-induced contractures of the control aorta showed stable plateaus and were not affected by prior exposure to phenylephrine . Neither diminished contractile force nor in vitro desensitization of phenylephrine contracture of isolated aorta was prevented by pretreatment of endotoxic rats with an alpha-adrenergic antagonist, phentolamine . These findings suggest that the contractile response to phenylephrine is easily desensitized in the endotoxic aorta compared to the control and neither this in vitro desensitization nor the diminution of contractile force is caused by in vivo exposure of aorta to a high concentration of catecholamines during endotoxemia. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1989, 7(1), 12 - 4 {Report on a fungus parasitizing Entamoeba histolytica}; Cao CQ et al.; Infection of Entamoeba histolytica with chytridiaceous fungus Sphaerita was observed in some specimens obtained from a farmer and stained with iron-haematoxylin . The fungi were found in 78% of the cysts, mostly immature ones . Within the amoebae this parasite occurred singly, in groups, or in the form of a sporangium . It was located in the cytoplasm, the glycogen mass or the chromatoidal bars . In the same specimen, the parasitic fungus was also found in 18% of E . coli cysts; in 11% of E . nana cysts; while only one of 16 E . hartmanni cysts was parasitized . It is an interesting case of superimposed parasitism so far reported in China as well as a rare case of several species of amoebae being heavily involved with the same in the scientific literature. Mol Carcinog, 1989, 2(2), 107 - 15 Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli . II: A host-mediated approach to N-nitroso-N,N-dimethylamine and endogenous mutagenesis in vivo; Horsfall MJ et al.; An intrasanguineous host-mediated assay was used to determine the mutational specificity of the hepatocarinogen N-nitroso-N,N-dimethylamine metabolized in vivo . A total of 114 forward mutations in the lacl gene of Escherichia coli reisolated from the livers of treated Swiss albino mice were characterized at the DNA sequence level . Consistent with the methylating ability of this compound and the demonstrated mutagenic specificity of O6-methylguanine, the predominant mutation was the G:C----A:T transition . These were recovered, on average, seven times more frequently at guanines flanked (5') by a purine residue than at those preceded by a pyrimidine residue--a specificity similar to that reported for many direct-acting SN1 alkylating agents . This nitrosamine appears to be distinguished from related N-nitroso methylating compounds by the induction of additional mutational events . Here, the exceptions consisted of four A:T----G:C transitions, four A:T site transversions, and a single G:C----T:A transversion . In addition, the DNA sequence alterations of 34 I- mutants of E . coli reisolated from otherwise untreated mice were identified . The predominant mutation was the G:C----A:T transition, which accounted for almost half of all background mutations . The sites at which these mutations were recovered appear to indicate that some of these mutations may have arisen as a result of an accelerated rate of cytosine deamination . These data suggest that many of the additional "spontaneous" mutations observed under in vivo conditions resulted from genotoxic events occurring during the host-defense (immune) reaction. Gene, 1989, 76(2), 359 - 62 The HU protein is not essential for generating deletions adjacent to transposon Tn3; Morita M et al.; Escherichia coli mutants deficient in the HU protein were used to test whether this protein is essential for generating DNA deletions adjacent to the ends of Tn3 . There were no significant differences in the frequencies of the adjacent DNA deletions in the isogenic series which differ only in the HU loci (hupA or hupB), indicating that the HU protein is not essential for this reaction. Gene, 1989, 76(2), 187 - 93 Characterization of in vitro constructed IS30-flanked transposons; Stalder R et al.; In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro . Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit . In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9} transposition activity in vivo . By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element . These two insertion sites were used several times independently by Tn2700 and Tn2702 . This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30. Clin Physiol Biochem, 1989, 7(1), 34 - 9 Estradiol and catecholestradiols as possible genotoxic carcinogens; van Aswegen CH et al.; Estradiol and other estrogens are not classified as genotoxic carcinogens, but rather as tumor promoters in the multistage process of carcinogenesis . This study is a reexamination of the carcinogenic status of estradiol and the catecholestradiols (2-OHE2 and 4-OHE2) with the recently developed bacterial assays for genotoxic carcinogens, the Chromotest . The bacterial kits revealed estradiol and catecholestradiols as biphasic and potential genotoxic carcinogens with the following SOS inducing potency values: E2 43,265 (SD 8,300); 2-OHE2 30,153 (SD 2,500), and 4-OHE2 68,939 (SD 4,500) . The differences between these values are statistically highly significant (p less than 0.0005) . These results were confirmed by studies on Escherichia coli, which showed an increase in cell number and a stimulation of DNA content in the presence of the estrogens . It is therefore concluded that estradiol and the catecholestradiols are possible genotoxic carcinogens which probably act as tumor inducers rather than tumor promoters. Ann Ist Super Sanita, 1989, 25(1), 69 - 73 Molecular mechanisms of hydrogen peroxide cytotoxicity; Cantoni O et al.; The molecular mechanisms of H2O2 toxicity have been investigated in both mammalian or bacterial cells . DNA breakage mediates cytotoxicity by low concentrations of H2O2 in mammalian cells, but DNA lesions do not appear as a direct consequence of the action of the hydroxyl radical; rather, these radicals may disturb intracellular Ca2+ homeostasis, which results in secondary reactions ultimately leading to DNA strand breakage and cytotoxicity . Studies that have used Escherichia coli (E . coli) as a cellular system have indicated that the two modes of killing detectable in cells exposed to increasing concentrations of H2O2 are mediated by different radical species . Mode-one killing seems to be produced by the superoxide anion whereas mode-two killing seems to be a consequence of the hydroxyl radical attack. Mol Biol (Mosk), 1989 Jan-Feb, 23(1), 306 - 14 {Selective inhibition of 3'-5'-exonuclease activity of DNA-polymerase I from Escherichia coli by a fluoride ion}; Mikhailov VS et al.; The effect of NaF on the enzymatic activities of the large fragment of E . coli DNA polymerase I (Klenow enzyme-KE) with different DNA-substrates was studied . It was shown that fluoride ion at concentrations of 5-10 mM efficiently inhibits the 3'----5' exonuclease activity of KE but does not affect the polymerase activity of the enzyme . Selective inhibition of the 3'----5' exonuclease activity of KE is Mg-dependent and is observed with double- or single-stranded DNAs . In reaction with the 14-mer oligonucleotide annealed with single-stranded phage M13 DNA the enzyme was found not only to perform the exonucleolytic hydrolysis of the primers but to catalyse also a limited elongation of some primers, adding a few nucleotide residues in the absence of exogenous dNTP . The primer elongation is inhibited by inorganic pyrophosphatase and is stimulated by micromolar concentrations of exogenous pyrophosphate thus suggesting a possible role of PPi contamination in dNTP generation via pyrophosphorolysis . Traces of precursors in DNA preparations obtained by generally employed methods may serve as another source of nucleotides for the primer elongation. Gene Anal Tech, 1989 Jan-Feb, 6(1), 17 - 20 Quick screening of plasmid deletion clones carrying inserts of desired sizes for DNA sequencing; Xie WQ et al.; Plasmids pWQX001 and pWQX005, constructed from pGEM-4 with an insert of 6.5 kb, were unidirectionally digested with exonuclease III and exonuclease VII . The DNA digests were ligated and used to transform competent cells of Escherichia coli DH5-alpha . The size of the deletion plasmid carried by each transformant was estimated through agarose gel electrophoresis of crude lysates without any purification of the plasmid DNA . Colonies carrying plasmid DNAs with different deletions of the insert were grown and their DNAs were purified through a miniprocedure . The size of each purified plasmid DNA was determined accurately after linearization of the plasmid with an appropriate restriction endonuclease . The remainder of the DNA preparation was sufficiently pure to be sequenced using Sanger's dideoxynucleotide chain termination method . An easy, quick procedure is described for the preliminary selection of templates for DNA sequencing after construction of deletion clones of recombinant plasmid DNA using exonuclease III and exonuclease VII . This procedure permits a rapid screening of large numbers of colonies and selection of those carrying plasmid DNAs with inserts of the desired sizes for sequencing . This procedure does not require purification of the deletion plasmid DNA. J Biol Regul Homeost Agents, 1989 Jan-Mar, 3(1), 13 - 9 Production and in vivo biologic actions of recombinant mouse interferon alpha 2; Dron M et al.; We have expressed a recombinant mouse interferon alpha (r.Mu-IFN alpha 2) in Escherichia coli under the control of a tryptophan promoter using a synthetic adaptor formed by annealing two partially complementary oligonucleotides which introduced an ATG start codon and re-established the complete coding sequence of the mature IFN alpha 2 protein in the expression vector . Levels of up to 10(7) reference units of Mu-IFN alpha 2 per liter of culture were obtained using this construction . This recombinant mouse interferon alpha 2 exhibited antiviral activity in mice infected with EMC virus and antitumor activity in mice inoculated with Friend leukemia cells in a manner similar to that of natural mouse interferon alpha/beta. Free Radic Res Commun, 1989, 6(1), 47 - 55 Role of hydroxyl radicals in Escherichia coli killing induced by hydrogen peroxide; Brandi G et al.; Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing . In this paper we have found that hydroxyl radicals (OH.) generated by H2O2 and intracellular divalent iron are not involved in the induction of mode one lethality (i.e . cell killing produced by concentrations of H2O2 lower than 2.5 mM) . In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxamine, did not affect the survival of cells exposed to 2.5 mM H2O2 . In addition cell vulnerability to the same H2O2 concentration was independent on the intracellular iron content . In contrast, mode two lethality (i.e . cell killing generated by concentrations of H2O2 higher than 10 mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content . It is concluded that OH . are required for mode two killing of E . coli by hydrogen peroxide. Izv Akad Nauk SSSR Biol, 1989 Jan-Feb, (1), 148 - 51 {Coprecipitation of proteins and DNA-protein complexes with magnesium sarcolysate: research on an association of topoisomerase I and RNA polymerase with DNA}; Vasetskii ES et al.; A specific cosedimentation of proteins and their complexes with DNA at low temperature (M-band technique) has been demonstrated . Model experiments with reconstituted SV40 DNA-topoisomerase I and SV40 DNA-E . coli RNA polymerase complexes demonstrated the potential and capacities of the method . It allows fractionation of DNA-protein complexes from naked DNA and has greater range, higher reproducibility and lower background values than similar methods. Int J Immunopharmacol, 1989, 11(1), 45 - 55 Phospholipid metabolism in polymorphonuclear leukocytes from rheumatoid arthritis patients: effects of non-steroidal anti-inflammatory agents and clotrimazole; Smith DM Jr et al.; Arachidonic acid (AA) metabolism and phospholipase A2 (PLA2) activity were measured in the peripheral blood polymorphonuclear leukocytes (PMNL) from ten patients with rheumatoid arthritis (RA) on treatment with various non-steroidal anti-inflammatory agents (NSAIA) . AA metabolism and PLA2 activity were measured both initially and after treatment with either placebo or Clotrimazole, a broad spectrum anti-mycotic agent, as a possible anti-rheumatic drug . AA metabolism was also measured in PMNL from ten patients with active RA untreated with any NSAIA and ten normal volunteers . Using 3H-AA prelabeled cells, we show that there was a significantly higher (P less than 0.025) production of 3H-LTB4 in response to stimulation with the calcium ionophore A23187 in untreated RA patients than in normal volunteers (mean +/- S.D.:4.8 +/- 1.6% and 3.1 +/- 1.0%, respectively) . The production of 3H-LTB4 by PMNL from patients on NSAIAs was less elevated (mean +/- S.D.:4.1 +/- 1.5%) and was not significantly different from normal controls . Concurrently we examined PLA2 activity in PMNL-sonicates from ten of our study patients using autoclaved {14C}oleate-labeled E . coli biomembranes as an exogenous substrate . Using linear regression analysis, we demonstrate a significant correlation between in vitro PLA2 activity and the release of 3H-AA from the cellular phospholipids (deacylation) in response to A23187 stimulation (r = -0.526, P less than 0.025) . We also demonstrate significant correlations between the overall clinical state of the RA patient, as evaluated by a modified rheumatoid activity index (MRAI), and both the release of 3H-AA from the cellular phospholipids and its production of total {3H}eicosanoids (r = -0.557, P less than 0.025 and r = 0.644, P less than 0.005, respectively) . This data suggests that: PLA2 activity may, in part, account for the higher generation of LTB4 by RA PMNL; NSAIAs may be capable of modulating this abnormality; and Clotrimazole may affect the clinical or laboratory data of RA patients already on treatment with NSAIA. Free Radic Biol Med, 1989, 6(2), 123 - 9 Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA; Denq RY et al.; Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites . The latter may be residues of thymine glycol . Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas bovine serum albumin was much less effective . Mannitol and benzoate, added as scavengers of HO., and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected . These results indicate a metal-catalyzed interaction of O2- with H2O2, which produces HO . which, in turn, causes DNA strand scission and oxidation of thymine residues to thymine glycol . Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O2- prevented the damage seen with the aerobic cells . Genetic defects in AP endonucleases rendered E . coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O2- production . Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O2- was protective . These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O2- plus H2O2, both in vitro and in vivo. Vet Med (Praha), 1989 Jan, 34(1), 1 - 12 {Objective evaluation of the immunogenic effectiveness of polyvalent vaccines against enteral infections of newborn calves under enzootic conditions}; Mensik J et al.; For evaluating the protective effect of polyvalent vaccine against diarrhoea in new born calves caused by rotaviruses, coronaviruses and enterotoxigenic E . coli the method was selected of mathematical and statistical analysis of the set of data characterizing the most important clinical symptoms of the disease during the first three weeks after birth . In two large-scale breeds with a mass occurrence of diarrhoea of the known etiology the state of health of calves before the vaccine application was compared with the state of health of calves born in the period after the vaccination . The following traits were selected for evaluating the severity of the disease: the age of calf at the beginning of the disease, character and intensity of diarrhoea, length of disease and results of treatment . In all the selected indicators, statistically significant differences were determined pointing to the fact that the vaccine used for increasing the immunity against enteropathogenic viruses and bacteria, applied in high pregnant cows and heifers before parturition, protected the sucking calves and calves fed the milk of their mothers against the disease in the first week after birth . The diarrhoea, occurring in the calves of the vaccinated cows and heifers mostly during the second week of age was easy to treat therapeutically and ended in most cases after a short period of a tea diet . After the vaccination, the death loss in both localities decreased more than eight times. Protein Eng, 1989 Jan, 2(5), 379 - 86 Synthetic genes for human muscle-type adenylate kinase in Escherichia coli; Kim HJ et al.; An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter . The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length . Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein . The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene . The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1 . Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity . These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity . Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site. Genetics, 1989 Jan, 121(1), 47 - 55 The Tc3 family of transposable genetic elements in Caenorhabditis elegans; Collins J et al.; We describe genetic and molecular properties of Tc3, a family of transposable elements in Caenorhabditis elegans . About 15 Tc3 elements are present in the genomes of several different wild-type varieties of C . elegans, but Tc3 transposition and excision are not detected in these strains . Tc3 transposition and excision occur at high frequencies, however, in strain TR679, a mutant identified because of its highly active Tc1 elements . In TR679, Tc3 is responsible for several spontaneous mutations affecting the unc-22 gene . Tc3-induced mutations are unstable, and revertants result from precise or nearly precise excision of Tc3 . Although Tc3 is very active in TR679, it is not detectably active in several other mutator mutants, all of which exhibit high levels of Tc1 activity . Tc3 is 2.5 kilobases long, and except for sequences near its inverted repeat termini, it is unrelated to Tc1 . The termini of Tc3 are inverted repeats of at least 70 base pairs; the terminal 8 nucleotides of Tc3 are identical to 8 of the terminal 9 nucleotides of Tc1. J Surg Res, 1989 Jan, 46(1), 41 - 8 The effect of Escherichia coli endotoxin on the adrenergic control of lipolysis in the human adipocyte; Forse RA et al.; We investigated the effect of Escherichia coli O127:B8 endotoxins on the adrenergic control of lipolysis in the human adipocyte . Adipose tissue was incubated in vitro with isoproterenol to stimulate the beta-1 receptors, clonidine to stimulate the alpha-2 receptors, and theophylline to stimulate the subreceptor mechanism . Using a dual radioisotope technique, a lipolysis factor was calculated for each sample . The basal lipolysis factor was significantly (P less than 0.006) decreased 31% with endotoxin . beta-1 adrenergic receptor stimulation (isoproterenol, 1 X 10(-8) to 1 X 10(-4) M) was significantly decreased an average of 31% with E . coli endotoxin . The beta-1 receptor responsiveness was also significantly (P less than 0.02) decreased but not the receptor sensitivity . This indicated an alteration in the post beta receptor mechanism . The various components of the post beta-1 adrenergic mechanism were stimulated including the beta-1 receptor, the G protein, adenylase cyclase, and the lipase phosphorylase . The results indicated a significant 24.2% reduction of the beta-1 receptor and a 25.4% reduction in G protein stimulation . Thus the E . coli endotoxin effect on the beta adrenergic mechanism is at the G protein . The endotoxin had no effect on the alpha-2 receptor stimulation nor the theophylline stimulation of the subreceptor lipolysis . This study indicates that E . coli endotoxin (O127:B8) decreases in vitro beta adrenergic stimulation of human adipocyte lipolysis, and this effect can be partially reversed by theophylline. J Bacteriol, 1989 Jan, 171(1), 99 - 103 Altered induction of the adaptive response to alkylation damage in Escherichia coli recF mutants; Volkert MR; Escherichia coli recF mutants are hypermutable when treated with methyl methanesulfonate (G . C . Walker, Mol . Gen . Genet . 152:93-103, 1977) . In this study, methylation hypermutability of recF mutant strains was examined, and it was found that recF+ is required for normal induction of the adaptive response to alkylation damage . Although this regulatory effect of recF mutations results in reduced levels of enzymes that specifically repair methyl lesions in DNA, it only partially explains the hypermutability . Further examination showed that methylation hypermutability of recF mutant strains required a functional umuDC operon, a component of the SOS response . These results lead to the hypothesis that methylation hypermutability results from the effects of recF mutations on the induction of both the SOS response and the adaptive response. J Bacteriol, 1989 Jan, 171(1), 616 - 9 Export-defective lamB protein is a target for translational control caused by ompC porin overexpression; Click EM et al.; Overexpression of OmpC protein from an inducible plasmid vector reduced the amount of the precursor form of LamB protein in LamB signal sequence mutants . The stability of the precursor form of LamB protein was not affected, indicating that the effect of OmpC overexpression was on the synthesis of the precursor rather than on degradation . These results indicate that a functional signal sequence is not required on an outer membrane protein for it to be a target for translational control. J Bacteriol, 1989 Jan, 171(1), 577 - 80 Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene; Matsuyama A et al.; The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned . The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold . The complete nucleotide sequence of the ackA gene was determined . It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297 . The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome. J Bacteriol, 1989 Jan, 171(1), 402 - 9 Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide; Fikes JD et al.; Mutations previously designated prlD were described that suppressed malE signal sequence mutations and were located in the vicinity of the secA gene on the Escherichia coli chromosome . In this study, we demonstrated that four such independently isolated prlD mutations represented three unique single-base substitutions in secA, resulting in alterations at residues 111, 373, and 488 of the 901-residue SecA protein . Heretofore, the only mutations that had been described for secA were located early in the gene and resulted in a general protein export defect . Insertion mutations in the cloned gene X-secA operon that reduced or eliminated suppression by a prlD mutation also have been obtained . The properties of these suppressor and insertion mutations provide some insight into the role of SecA in the protein export process. J Bacteriol, 1989 Jan, 171(1), 360 - 8 Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene; Brandner JP et al.; A Rhodobacter sphaeroides mutant (CYCA1) lacking cytochrome c2 (cyt c2) was previously constructed (T . J . Donohue, A . G . McEwan, S . Van Doren, A . R . Crofts, and S . Kaplan, Biochemistry, 27: 1918-1924, 1988) by a combination of in vivo and in vitro molecular genetic techniques . CYCA1 was incapable of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCA1 contained significant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of approximately 554 nm which had previously gone undetected under these physiological conditions in wild-type cells . In addition, the PS- phenotype of CYCA1 can be complemented in trans with stable low-copy-number (approximately 5 to 9 per R . sphaeroides genome) broad-host-range plasmids containing the wild-type cyt c2 structural gene (cycA) and upstream regulatory sequences . cyt c2 and cycA-specific mRNA levels were elevated in both the wild type and CYCA1 derivatives harboring intact cycA genes in trans, presumably as a result of increased gene dosage . Although photosynthetically grown wild-type cells contained approximately twofold more cycA-specific transcripts than chemoheterotrophically grown cells, there was an approximately four- to sevenfold increase in cyt c2 levels under photosynthetic conditions . Similarly, complemented CYCA1 strains contained between 1.3- and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6- to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions . These data are discussed in terms of possible posttranscriptional control over cyt c2. J Bacteriol, 1989 Jan, 171(1), 303 - 7 Mutations in uvrD induce the SOS response in Escherichia coli; Ossanna N et al.; We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage . Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies . Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants . The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity . The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication . We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication . We believe that defective DNA replication in these mutants increases SOS expression. J Bacteriol, 1989 Jan, 171(1), 213 - 21 Nucleotide sequence of traQ and adjacent loci in the Escherichia coli K-12 F-plasmid transfer operon; Wu JH et al.; The F tra operon region that includes genes trbA, traQ, and trbB was analyzed . Determination of the DNA sequence showed that on the tra operon strand, the trbA gene begins 19 nucleotides (nt) distal to traF and encodes a 115-amino-acid, Mr-12,946 protein . The traQ gene begins 399 nt distal to trbA and encodes a 94-amino-acid, Mr-10,867 protein . The trbB gene, which encodes a 179-amino-acid, Mr-19,507 protein, was found to overlap slightly with traQ; its start codon begins 11 nt before the traQ stop codon . Protein analysis and subcellular fractionation of the products expressed by these genes indicated that the trbB product was processed and that the mature form of this protein accumulated in the periplasm . In contrast, the protein products of trbA and traQ appeared to be unprocessed, membrane-associated proteins . The DNA sequence also revealed the presence of a previously unsuspected locus, artA, in the region between trbA and traQ . The artA open reading frame was found to lie on the DNA strand complementary to that of the F tra operon and could encode a 104-amino-acid, 12,132-dalton polypeptide . Since this sequence would not be expressed as part of the tra operon, the activity of a potential artA promoter region was assessed in a galK fusion vector system . In vivo utilization of the artA promoter and translational start sites was also examined by testing expression of an artA-beta-galactosidase fusion protein . These results indicated that the artA gene is expressed from its own promoter. J Bacteriol, 1989 Jan, 171(