Int J Radiat Oncol Biol Phys, 1989 Apr, 16(4), 963 - 6 Assessment of the repair and damage of DNA induced by parent and reduced RSU-1069, a 2-nitroimidazole-aziridine; O'Neill P et al.; The cellular repair and damage of DNA induced by parent and reduced RSU-1069, a 2-nitroimidazole-aziridine, was assessed at both the molecular and cellular level . At the molecular level, after in vitro incubation with parent or reduced RSU-1069, plasmid DNA was transfected into Escherichia coli (AB1157) with subsequent selection for gene expression . For equivalent levels of DNA strand breakage following such treatment it is evident from the relative transformation frequencies that interactions with reduced RSU-1069 lead to DNA damage consistent with bifunctional action of a metabolite(s) . At the cellular level, the cytoxicity of RSU-1069 was determined for a series of repair deficient mutants of E . coli under both aerobic and hypoxic conditions . The differential aerobic:hypoxic cytotoxicity ratio is approximately 3 . We conclude that the repair of cellular DNA damage induced by RSU-1069 involves activation of the gene products under the control of the recA gene and not those under the control of the ada gene . The ability of cellular systems to repair damage induced by RSU-1069 may play a significant role in determining its efficiency to act as a hypoxic cell radiosensitizer and a hypoxia selective cytotoxin. Carcinogenesis, 1989 Apr, 10(4), 661 - 6 Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase; Graves RJ et al.; Self-complementary oligodeoxynucleotides have been synthesized containing O6-methylguanine (O6meG), O6-ethylguanine (O6etG), O6-isopropylguanine (O6iprG) and O4-methylthymine (O4meT) . They anneal in solution to give double-stranded DNA . These double helices have been used as substrates for the DNA repair protein O6-alkylguanine-DNA-alkyltransferase coded for by the ada gene of Escherichia coli . The repair followed second-order chemical kinetics . O6meG was repaired by the 19-kd transferase at a rate of 2.54 x 10(7) M-1 s-1 which is close to the theoretical limit for a diffusion-controlled reaction; O6etG and O4meT are repaired 1,000 and 10,000 times more slowly . The 39-kd alkyltransferase (which is precursor to the 19-kd form) and the 19-kd transferase repaired O6etG at similar rates . O6iprG was not repaired . The repair of oligomers containing O6meG was only slightly inhibited by the presence of nonalkylated oligomers . Oligomers containing O6etG were only slightly more effective as inhibitors of repair than the nonalkylated oligomers, indicating that the transferase does not bind selectively to alkylated DNA . Parallel structural studies have shown that O6-alkylguanine:C and O4-alkylthymine:A base pairs have a similar geometry with the alkylated base displaced into the major groove of the DNA in contrast to O6-alkylguanine:T and O4-alkylthymine:G base pairs which retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine . Measurement of the rate of repair of these different base pairs suggests that pairs with the alkyl group exposed in the major groove may be repaired more rapidly than those with the alkyl group more deeply buried in the helix. Blood, 1989 Apr, 73(5), 1202 - 6 Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin; Bolyard MG et al.; The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit . Recombinant B beta chains were expressed at 100 ng/mL in an IPTG-dependent manner . A first cistron sequence, inserted into the expression vector 5' to the B beta chain cDNA, was required to express the protein . Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers . The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase . Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1-42 fragment . The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli . However, these levels are sufficient to allow further characterization of this fibrinogen subunit. Arch Biochem Biophys, 1989 Apr, 270(1), 391 - 7 Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine; Kyogashima M et al.; Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E . coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans . The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria . These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively . E . coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids . Of the standard glycolipids tested in solid phase binding assays, E . coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside . The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4{Neu-Ac alpha 2-3}Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4{NeuAc alpha 2-3}Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested . N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans . Possibly this distribution of sialyl derivatives explains the host range of infection by the organism. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2291 - 5 Mutagenic specificity of a potent carcinogen, benzo{c}phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide, which reacts with adenine and guanine in DNA; Bigger CA et al.; Mutations were induced in the supF gene of the pS189 shuttle vector by treatment with optically active benzo{c}phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide in vitro and replication in human cells . The induced mutation frequency was 60-fold greater than the spontaneous rate, and most of the mutations analyzed were transversions (86%), which principally consisted of similar numbers of A.T----T.A and G.C----T.A changes . The unusual susceptibility of A.T pairs to mutation by this chemical agent is consistent with its chemical reactivity toward adenine and argues that the mutations are targeted to the adducts formed . The central base in the sequences 5'-AGA-3', 5'-AAC-3', and 5'-GAG-3' was particularly susceptible to mutation . Twelve "hotspots" in the supF gene accounted for most mutations seen . Some of these hotspots differed from those found by others for racemic benzo{a}pyrene dihydrodiol epoxide and, even when a hotspot was common, the mutagenic changes were not always the same . Although adenine insertion opposite a noninstructional lesion could account for most of the data, no single mutagenic mechanism could encompass all of it . The cellular machinery that converts chemical damage to mutations must determine the mutational result to a large extent, but the findings herein show that the chemical agent itself plays a large role in determining both the location and the nature of the mutations that arise. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2286 - 90 An essential yeast protein, encoded by duplicated genes TIF1 and TIF2 and homologous to the mammalian translation initiation factor eIF-4A, can suppress a mitochondrial missense mutation; Linder P et al.; We describe the isolation and characterization of two previously undescribed genes, TIF1 and TIF2, from Saccharomyces cerevisiae . The protein-encoding sequences of the two genes are highly conserved, resulting in two completely identical proteins, whereas the flanking regions show no obvious homology . The two yeast proteins are highly similar to the translation initiation factor eIF-4A from mouse . Elevated gene dosage of TIF1 or TIF2 results in the suppression of a missense mutation in the mitochondrial oxi2 gene, which codes for subunit III of cytochrome-c oxidase, although the sequence of the Tif protein indicates its cytoplasmic localization . Inactivation of either gene by gene disruption has no effect on cell viability or on mitochondrial functions . However, simultaneous inactivation of both genes is lethal to the cell. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2281 - 5 Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease; Heitman J et al.; We prepared a set of temperature-sensitive mutants of the EcoRI endonuclease . Under semipermissive conditions, Escherichia coli strains bearing these alleles form poorly growing colonies in which intracellular substrates are cleaved at EcoRI sites and the SOS DNA repair response is induced . Strains defective in SOS induction (lexA3 mutant) or SOS induction and recombination (recA56 and recB21 mutants) are not more sensitive to this in vivo DNA scission, whereas strains deficient in DNA ligase (lig4 and lig ts7 mutants) are extremely sensitive . We conclude that although DNA scission induces the SOS response, neither this induction nor recombination are required for repair . DNA ligase is necessary and may be sufficient to repair EcoRI-mediated DNA breaks in the E . coli chromosome. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2248 - 52 Binding of a soluble factor of Escherichia coli to preproteins does not require ATP and appears to be the first step in protein export; Watanabe M et al.; We have constructed a mutant form of the maltose binding protein precursor, termed preMBP*, that, unlike its wild-type counterpart preMBP, retains translocation competence after synthesis . In a homologous Escherichia coli translation/translocation system, preMBP* was translocated either co- or posttranslationally with virtually 100% efficiency into inverted vesicles (INV) derived from the E . coli plasma membrane . Translation of preMBP* mRNA in a wheat germ system and subsequent incubation with INV yielded no translocation . However, addition of increasing amounts of an E . coli postribosomal supernatant (PRS) to the wheat germ extract stimulated preMBP* translocation with virtually 100% efficiency occurring at 100 micrograms of PRS per 50 microliters of incubation mixture . The activity in the E . coli PRS appears to be identical to the previously described "export" factor . The soluble activity can bind to preMBP* posttranslationally and in the absence of ATP . Subsequent targeting to INV and/or translocation, however, requires ATP . Binding of the soluble activity to preMBP* thus appears to be the first step in a multistep translocation reaction. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2167 - 71 Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement; Holz M et al.; The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid . At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane . The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping . The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein . These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant . Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant . The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin . At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type . These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2157 - 61 Human immunodeficiency viral long terminal repeat is functional and can be trans-activated in Escherichia coli; Kashanchi F et al.; The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains the viral promoter, which is responsible for viral gene expression in eukaryotic cells . We have demonstrated that HIV LTR can also function as a promoter in Escherichia coli . A recombinant plasmid containing the HIV LTR linked to the chloramphenicol acetyltransferase gene can express the enzyme efficiently upon transformation into bacteria . Mung bean nuclease analysis mapped the bacterial transcriptional start site of the promoter to the U3 region of the LTR, in contrast to transcription in eukaryotic cells, which initiates in the U3-R boundary of the LTR . The HIV LTR, besides being fully functional in E . coli, can also be specifically trans-activated by the HIV tat gene product . Trans-activation is demonstrated by an increase in chloramphenicol acetyltransferase activity as well as an increase in the mRNA level of the enzyme . This trans-activation of HIV LTR by tat protein in bacteria offers a useful system to investigate further the specific interaction between tat protein with HIV LTR and the mechanisms of trans-activation. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2147 - 51 Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli; Fontecave M et al.; Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis . The active enzyme contains a tyrosyl free radical whose formation requires oxygen . Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E . coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen . We now conclude from isotope incorporation experiments that E . coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides . Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP . To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme . The reaction is stimulated by NADPH, Mg2+, and ATP . Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties . Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme . The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world." J Neurosurg, 1989 Apr, 70(4), 619 - 22 CSF production in acute ventriculitis; Breeze RE et al.; Clinically, there appears to be a significant reduction in cerebrospinal fluid (CSF) formation during acute ventriculitis--an observation that has not been well documented by experimental studies . To examine this phenomenon, an inoculum of Escherichia coli was injected into the lateral ventricles of New Zealand White rabbits . Approximately 18 hours later, the survivors (64%) underwent a 3-hour ventriculocisternal perfusion of carbon-14-dextran (MW 7 X 10(4)) as a reference marker for CSF formation . On the average, CSF formation in this experimental group was reduced by one-half to two-thirds of normal, confirming the clinical observation . Histologically, the stroma of the choroid plexus was the site of an extensive inflammatory infiltrate . Meningitis, ependymitis, and focal encephalitis completed the picture . Vasculitis was not present in the choroid plexus . The epithelium of the choroid plexus underwent patchy cellular swelling or frank necrosis and destruction . It is postulated that the changes in the choroid plexus caused by the inflammatory process were responsible for the diminished CSF formation in this acute setting . Reduced choroidal blood flow and/or enterotoxin may play a role in these alterations. J Cell Biol, 1989 Apr, 108(4), 1419 - 29 The SPA2 protein of yeast localizes to sites of cell growth; Snyder M; A yeast gene, SPA2, was isolated with human anti-spindle pole autoantibodies . The SPA2 gene was fused to the Escherichia coli trpE gene, and polyclonal antibodies were prepared to the fusion protein . Immunofluorescence experiments indicate that the SPA2 gene product has a sharply polarized distribution in yeast cells . In budded cells the SPA2 protein is present at the tip of the bud; in unbudded cells, it is localized to one edge of the cell . When a-cells are induced to form schmoos with alpha-factor, the SPA2 protein is found at the tip of the schmoo . These areas of SPA2 localization correspond to cellular sites expected to be involved in bud formation and/or cell growth . The SPA2 antigen is present in a-cells, alpha-cells, and a/alpha-diploid cells, but is absent in mutant cells in which the SPA2 gene has been disrupted . spa2 mutant cells are viable, but display defects in the direction and control of cell growth . Compared to wild-type cells, spa2 mutant cells have slightly altered budding patterns . Entry into stationary phase is impaired for spa2 mutants, and mutants with one particular allele, spa2-7, form multiple buds under nutrient-limiting conditions . Thus, SPA2 is a newly identified yeast gene that is involved in the direction and control of cell division, and whose gene product localizes to the site of cell growth. Microb Pathog, 1989 Apr, 6(4), 297 - 309 The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli; Hamers AM et al.; An oligonucleotide probe, derived from the N-terminal amino acid sequence of the CFA/I fimbrial subunit protein, was used to identify the gene encoding this protein within a cloned DNA fragment encoding CFA/I fimbriae . The gene (cfa b) was found and sequenced . Flanking it upstream was a gene (cfa a) encoding a protein of 206 amino acids and downstream a gene (cfa c) probably encoding an 85 kDa protein was found . This genetic organisation of the CFA/I operon differs from that of other fimbrial operons in Escherichia coli . All three proteins have signal peptides . The nucleotide sequence was analysed for homology with other sequences, secondary structure, ribosomal binding sites and possible promoter sequences . A region of dyad symmetry probably involved in the regulation of translation of the cfa c gene was found at the 5' end of this gene . A region of dyad symmetry was also observed within the cfa b gene . In front of the CFA/I operon part of insertion sequence IS2 was found . This IS2 sequence was found in a number of CFA/I plasmids, obtained from strains isolated from various geographic locations . The insertion of the IS2 element in the CFA/I operon therefore probably happened rather early during evolution of CFA/I producing Escherichia coli strains. FEMS Microbiol Lett, 1989 Apr, 49(2-3), 315 - 21 Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8 (P) fimbriae of Escherichia coli O18:K5:H5; Krallmann-Wenzel U et al.; DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pil) exist in a single copy on the chromosome of E . coli O18:K5 strain 2980 . In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined . The pil genes were mapped close to the gene clusters thr and leu controlling the biosynthesis of threonine and leucine, respectively . The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E . coli chromosomal linkage map. Photochem Photobiol, 1989 Apr, 49(4), 509 - 12 The enzymology and molecular biology of the Ca2+-activated photoprotein, aequorin; Cormier MJ et al.; Aequorin is a bioluminescent protein, isolated from the hydromedusan Aequorea victoria . A recombinant cDNA plasmid (pAEQ1) was shown to encode apoaequorin by detecting photoprotein activity in an extract of an E . coli strain containing pAEQ1 (Prasher et al., 1986, Biochem . Biophys . Res . Comm . 126, 1259-1268) . The nucleotide sequence of the pAEQ1 insert has been determined and is shown to differ significantly from the aequorin cDNA (AQ440) isolated by Inouye et al . (1985, Proc . Natl . Acad . Sci . USA 82, 3154-3158) . Comparisons of the coding regions of the two cDNAs show there are 52 nucleotide differences, 19 of which are responsible for 18 amino acid replacements . These differences explain the microheterogeneity observed at 17 positions during the sequencing of native apoaequorin . Five aequorin isotypes extracted from Aequorea tissue are observed on 2-dimensional gels and the E . coli-expressed apoaequorin is shown to co-migrate with one of these isotypes . The multiple isotypes could be caused by the presence of a multi-gene family since Southern blot analysis of Aequorea DNA suggests the presence of a minimum of four aequorin genes . Immunoblot analysis suggests that purified native aequorin is proteolytically cleaved during its purification from Aequorea . Comparison of the deduced cDNA translations and the protein sequence suggests the loss of seven residues from the amino terminal . Overexpression of the apoaequorin cDNA in E . coli now provides the means of obtaining gram quantities of a single isotype of the protein which can be converted to aequorin in the presence of coelenterate luciferin, oxygen and an appropriate thiol . Proper extraction procedures and a single chromatographic step provides apoaequorin which is greater than 95% homogeneous. Cell Tissue Res, 1989 Apr, 256(1), 167 - 73 Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues; Thaete LG et al.; Proliferating cell nuclear antigen is expressed in cells from late G1 through the S-phase of the cell cycle . Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells . Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei . We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro . A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes . Immunostaining in fetal and neonatal lung samples from mice was higher than in adults . Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue . In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates . This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E . coli DNA polymerase-III holoenzyme. Infect Immun, 1989 Apr, 57(4), 1186 - 91 Failure of pertussis toxin to inhibit activation of guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) in the T84 cell line; Crane JK et al.; The heat-stable enterotoxin (STa) of Escherichia coli causes intestinal secretion by stimulating guanylate cyclase, an enzyme believed to be distinct from the STa receptor . Pertussis toxin (PT) has been reported to block the ability of STa to stimulate guanylate cyclase in rat intestinal mucosa (S . A . Epstein, R . A . Giannella, and H . J . Brandwein, FEBS Lett . 203:44-48, 1986) . This suggested that a guanine nucleotide regulatory protein (G protein) coupled the STa receptor to guanylate cyclase, a function not previously recognized for G proteins . We sought to explore this phenomenon and, if possible, to identify this G protein . Initial experiments with the human colon carcinoma cell line T84 revealed that higher-than-expected concentrations (1 micrograms/ml) of PT were needed to intoxicate cells, as assessed by ADP-ribosylation of endogenous PT substrate, but that 99 to 100% intoxication could be achieved . Homogenates made from fully intoxicated cells did not differ from controls in basal or STa-stimulated guanylate cyclase activity, and cyclic GMP accumulation in intact T84 cells was not changed by PT treatment . We conclude that a PT-sensitive G protein is not involved in the stimulation of cyclic GMP production by the enterotoxin STa. Infect Immun, 1989 Apr, 57(4), 1126 - 30 Molecular cloning and nucleotide sequence of the colonization factor antigen I gene of Escherichia coli; Karjalainen TK et al.; The colonization factor antigen I (CFA/I) gene has been isolated and sequenced . The amino acid sequence of CFA/I deduced from the nucleotide sequence is composed of 170 amino acids . The first 23 amino acids are considered to be the signal peptide of the CFA/I protein since they are not present in the protein sequence . Among the remaining amino acids, only two are different from the protein sequence: amino acid position 76 is an aspartic acid instead of an asparagine, and position 97 is a serine instead of an alanine . The CFA/I gene has a typical Shine-Dalgarno sequence located 10 base pairs (bp) upstream from the initiation codon . The sequence TACAAT located 48 bp upstream from the initiation codon was tentatively designated the -10 sequence of the CFA/I gene promoter . No sequences homologous to the consensus -35 promoter sequence was found . A pair of inverted repeat sequences followed by a stretch of eight A's are located 45 bp downstream from the termination codon of the CFA/I gene; this region may be a rho-independent transcriptional terminator. Development, 1989 Apr, 105(4), 707 - 14 Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice; Kothary R et al.; Transgenic mice have been generated that express the E . coli beta-galactosidase gene under the control of the promoter from the mouse heat-shock gene, hsp68 . Sequences from -664 to +113 relative to the start of transcription of the hsp68 gene were sufficient to direct stress-induced expression of the beta-galactosidase gene in adult tail tissue and various tissues of fetal stages of development . Expression was detected in situ by staining with the chromogenic substrate, X-gal . The hybrid gene was refractory to induction in preimplantation embryos until the blastocyst stage of development, as reported for the endogenous hsp68 gene . No constitutive expression was observed by in situ staining or Northern analysis at any stage of development, even in tissues that constitutively express the endogenous hsp68 gene . We conclude that the hsp68 promoter region included in the construct contains sufficient sequence information for heat and arsenite inducibility, but it does not contain sequences controlling tissue-specific expression during development . This tightly regulated inducible promoter may provide a useful tool for short-term inducible gene expression in transgenic mice. Mol Microbiol, 1989 Apr, 3(4), 531 - 40 DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus; Dorman CJ et al.; The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase . Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA . We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA . At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes . The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene . However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants . tolC insertion mutations alter the distribution of plasmid topoisomers in vivo . This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier . As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change . The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed. Mol Microbiol, 1989 Apr, 3(4), 459 - 68 The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth; Betermier M et al.; We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome . We have identified this protein as Fis, a factor involved in several site-specific recombinational switches . Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant . DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site . Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda. Genetika, 1989 Apr, 25(4), 753 - 5 {Methyl-cytosine specific restriction in Escherichia coli K-12}; Povilenis PI et al.; Experiments on transformation of Escherichia coli K-12 cells by plasmids carrying RM systems with different recognition sites containing 5-methylcytosine have shown that the gene mcrB determines the function of restriction . The data obtained made it possible to believe that E . coli possesses no restriction system recognizing specifically cytosine methylated in position 4. Genetika, 1989 Apr, 25(4), 614 - 25 {Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66}; Orekhov AV et al.; The shuttle Escherichia coli - Streptomyces plasmids were used to transform S . lividans 66 . Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E . coli . Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E . coli and has the property of R +/- M+ mutant . Restriction in S . lividans 66 correlates with the appearance in DNA from E . coli of the sites susceptible to Scg2I restriction endonuclease . The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S . griseus Kr . 15 and S . coelicolor A3(2) . Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase . The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase . So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence . Neither restriction of plasmid DNA in S . lividans 66 is dependent on dcm modification in E . coli, though its dependence on dam modification is not excluded . It is assumed that the restriction in S . lividans 66 is specified by endonuclease analogous to Scg2I. Genetika, 1989 Apr, 25(4), 605 - 13 {Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9}; Mukhamedshin EK et al.; HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated . The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants . The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold . The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain . This mutation does not stimulate excision of Tn5 and Tn10 . LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold . The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed. Mol Gen Genet, 1989 Apr, 216(2-3), 475 - 83 The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: in vivo experiments; Masters M et al.; The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes . This sequence also occurs at many other sites on the E . coli chromosome including three sites within the essential cell division genes ftsQ and A . Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression . Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se . It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation . Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains. Mol Gen Genet, 1989 Apr, 216(2-3), 217 - 23 dam methylation and the initiation of DNA replication on oriC plasmids; Landoulsi A et al.; Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants . Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts . The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant . However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids . We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region. EMBO J, 1989 Apr, 8(4), 1105 - 10 A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity; Marshall MS et al.; The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 . To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli . The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E . coli . Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E . coli . These results define a distinct catalytic domain at the C terminus of GAP . In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products . This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein. EMBO J, 1989 Apr, 8(4), 1041 - 7 Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor; Glickman JN et al.; Adaptors mediate the interaction of clathrin with select groups of receptors . Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network . Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail . Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor) . In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors . This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant . This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes. Hinyokika Kiyo, 1989 Apr, 35(4), 593 - 6 {Clinical effects of intravesical instillation of polymyxin B in patients with acute cystitis}; Suzuki Y et al.; The effectiveness of intravesical instillation of Polymyxin B was studied in 17 patients suffering from acute cystitis . The 17 patients, all female, had an average age of 42 years . The average duration between the onset of symptoms and the initial visit to the hospital was 4.6 days . The causative organisms were 15 strains of E . coli and 1 each of P . rettegeri and K . pneumonia, respectively . Improvement of symptoms was observed in 76% of the cases but pyuria was cleared in only 47% of the cases . Clinical results were excellent in 8 cases (47%), good in 6 cases (35%) and poor in 3 cases (18%) . The overall effectiveness rate was 82% . No appreciable amount o Polymyxin B, was detected in the serum of 4 patients examined. Am J Physiol, 1989 Apr, 256(4 Pt 1), G721 - 6 Electrolyte transport in rabbit cecum . I . Effect of RDEC-1 infection; Tai YH et al.; To investigate the characteristics of intestinal ion and fluid secretion induced by the adherent, effacing enteropathogenic Escherichia coli strain RDEC-1, we infected weanling rabbits with 10(7)-10(8) RDEC-1 organisms and then studied cecal ion transport under short-circuit conditions in Ussing chambers . Results in tissues with confluent adherent organisms were compared with those in uninfected ceca and in ceca stimulated with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) . The short-circuited cecum normally absorbed Na and Cl, secreted bicarbonate (as represented by the residual ion flux), and displayed a high rate of nondiffusional Na and Cl transport . RDEC-1 infection did not alter the short-circuit current (Isc), but it increased the conductance (Gt), decreased the potential difference (PD), abolished net Na absorption, and reversed Cl absorption to secretion . The changes in Na and Cl net fluxes may be explained by inhibition of a Na-Cl linked absorptive process . In contrast, DBcAMP significantly increased the Isc, PD, and Gt, decreased net Na flux, and abolished net Cl absorption by stimulating electrogenic Cl secretion . These results suggest that RDEC-1-induced changes in cecal ion transport are not mediated by cAMP . The reduction in Na-Cl linked absorption is consistent with anatomic changes in the apical surfaces of absorptive epithelial characteristic of effacing enteroadherence, whereas the increased conductance is consistent with tight junction disruption seen with RDEC-1 infection. J Bacteriol, 1989 Apr, 171(4), 2128 - 35 Identification of Escherichia coli DNA helicase IV with the use of a DNA helicase activity gel; Trieu VN et al.; A DNA helicase activity gel was developed based on the assumption that DNA helicases could unwind double-stranded DNA in a polyacrylamide matrix . The production of single-stranded DNA was detected by staining the activity gel with acridine orange and visualizing the gel under long-wave UV light . The products of DNA helicase activities appeared as red bands within a green fluorescent background . A novel DNA helicase, called helicase IV, was detected in crude extracts of Escherichia coli with the use of the helicases activity gel assay . The new DNA helicase was purified to near homogeneity . The chromatographic properties and the sequence of its 11 amino-terminal residues proved that helicase IV was distinct from all of the previously described DNA helicases from E . coli. J Bacteriol, 1989 Apr, 171(4), 2066 - 74 Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF; Biek DP et al.; Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101 . We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production . The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling . A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication . IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains . In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains . A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function . Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance. J Bacteriol, 1989 Apr, 171(4), 2056 - 65 Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF; Biek DP et al.; Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis . One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions . Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine . The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants. J Bacteriol, 1989 Apr, 171(4), 1904 - 14 Nucleotide sequences required for Tn3 transposition immunity; Kans JA et al.; The Tn3 transposon inserts at a reduced frequency into a plasmid already containing a copy of Tn3, a phenomenon known as transposition immunity . The cis-acting site on Tn3 responsible for immunity was mapped by deletions from each side to be within the terminal 38-base-pair sequence that is inversely repeated at the ends of Tn3 . Two palindromic sequences are present in the essential part of this region . Some deletions conferred only partial immunity, and others conferred negative immunity . Multiple copies of partially immune ends conferred additional immunity . No other part of Tn3 was necessary for immunity. Arch Biochem Biophys, 1989 Apr, 270(1), 23 - 32 Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2; Oesch F et al.; Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli . Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein . With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins . Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes . Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2 . This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1 . A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2 . Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively . These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily. Surg Gynecol Obstet, 1989 Apr, 168(4), 323 - 31 New approach to endotoxic and septic shock by means of polymyxin B immobilized fiber; Hanasawa K et al.; Endotoxin shock therapy has not been successfully dealt with pharmacologically . Circulation of endotoxins in the blood is an important factor in the pathogenesis and clinical symptoms of endotoxic shock . We have recently developed polymyxin immobilized fiber (PMX-F) as a biomaterial for selectively detoxifying endotoxins . In ex vivo experiments, direct hemoperfusion (DHP) by PMX-F was performed on dogs injected with purified endotoxin . Only one of eight survived in the control group, but ten of 12 survived in the group receiving DHP with PMX-F . Mortality in the treated group decreased remarkably . Thus, the results indicate the efficacy of PMX-F in neutralizing endotoxins . By the same method as already mentioned, DHP with PMX-F was carried out on live dogs with Escherichia coli induced sepsis . All of the dogs in the control group died within 18 hours after bacterial infusion . All of the dogs in the treated group, however, survived for more than three days . Two of the five dogs are still alive . One of the remaining two survived for seven days and the other, 14 . We found that PMX-F treatment prolonged or increased the survival rate in the endotoxic and septic dogs. Mutat Res, 1989 Apr, 211(2), 193 - 203 Mutational spectrum analysis of umuC-independent and umuC-dependent gamma-radiation mutagenesis in Escherichia coli; Sargentini NJ et al.; gamma-Radiation mutagenesis (oxic versus anoxic) was examined in wild-type, umuC and recA strains of Escherichia coli K-12 . Mutagenesis {argE3(Oc)----Arg+} was blocked in a delta (recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair . Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 (+1 frameshift) reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen . While the umuC strain showed the gamma-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but not all oxygen-dependent frameshift mutagenesis . For anoxically irradiated cells, the yields of GC----AT {i.e., at the supB and supE (Oc) loci} and AT----GC transitions (i.e., at the argE3 and hisG4 loci) were essentially umuC independent, while the yields of (AT or GC)----TA transversions (i.e., at the supC, supL, supM, supN and supX loci) were heavily umuC dependent . These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to gamma-radiation mutagenesis. J Biomol Struct Dyn, 1989 Apr, 6(5), 1013 - 26 Linguistics of nucleotide sequences . I: The significance of deviations from mean statistical characteristics and prediction of the frequencies of occurrence of words; Pevzner PA et al.; Mathematical models of the generation of genetic texts appeared simultaneously with the first sequencing DNA . They are used to establish functional and evolutionary relations between genetic texts, to predict the number and distribution of specific sites in a sequence and to identify "meaningful" words . The present paper deals with two problems: 1) The significance of deviations from the mean statistical characteristics in a genetic text . Anyone who has addressed himself to the statistical analysis of sequenced DNA is familiar with the question: what deviations from the expected frequencies of occurrence of particular words testify to the "biological" significance of those words? We propose a formula for the variance of the number of word's occurrences in the text, with allowance for word overlaps, making it possible to assess the significance of the deviations from the expected statistical characteristics . 2) A new method for predicting the frequencies of occurrence of particular words in a genetic text using the statistical characteristics of "spaced" L-grams . The method can be used for predicting the number of restriction sites in human DNA and in planning experiments on the physical mapping and sequencing of the human genome. J Biol Regul Homeost Agents, 1989 Apr-Jun, 3(2), 50 - 4 Construction of a modified murine interferon alpha with increased stability; Li BL et al.; The mutant {Ser86} murine interferon alpha A was constructed by oligodeoxyribonucleotide-directed site-specific mutagenesis and expressed under the control of lambda phage PL promoter in Escherichia coli . The effect of the substitution of serine for the cysteine residue at position 86 in the murine interferon alpha A was studied . It was observed that the mutant Ser86 murine interferon alpha A which does not contain the free cysteine residue in the peptide was inactivated more slowly than its parent with a free cysteine residue at position 86 . Heat-inactivated murine interferon alpha A cannot be reactivated, but the heat-inactivated Ser86 derivative can be reactivated significantly to about 27.5 per cent of the original activity in the presence of sodium dodecylsulfate . These results suggest that the free cysteine in the peptide is a factor contributing to the instability of the protein. Mol Gen Genet, 1989 Apr, 216(2-3), 446 - 54 UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli; Verma M et al.; Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication . This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect) . However, by introducing an unirradiated E . coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon . We therefore conclude that UV-irradiation of E . coli leads to a trans-acting inhibition of initiation of replication . The inhibition is transient and does not appear to be an SOS function. Kansenshogaku Zasshi, 1989 Apr, 63(4), 369 - 75 {A study of neutrophil functions in patients with chronic respiratory tract diseases}; Yamaguchi T et al.; We tested blood neutrophil functions in the patients with chronic respiratory tract diseases to study the mechanism of susceptibility to bacterial infections . Peripheral blood neutrophils were obtained from 15 healthy subjects and 14 patients including diffuse panbronchiolitis, bronchiectasis, chronic emphysema and chronic bronchitis . Seven patients suffered from the chronic P . aeruginosa infection . Firstly, neutrophil chemotaxis was determined by the method of Boyden Chamber assays using FMLP as a neutrophil chemoattractant . The number of migrated neutrophils were 239.2 +/- 65.6 cells/50 HPF in the patients group and 256.6 +/- 49.0 cells/50 HPF in the control group . Secondly, neutrophil phagocytosis against P . aeruginosa, E . coli and K . pneumoniae was determined by phagocytic activity (PA) and phagocytic index (PI) . PA against each bacteria was 44.1 +/- 13.2% (P . aeruginosa), 44.8 +/- 12.3% (E . coli) and 35.8 +/- 13.6% (K . pneumoniae) in the patients group and 42.3 +/- 10.6% (P . aeruginosa), 43.0 +/- 11.9% (E . coli) and 36.3 +/- 16.0% (K . pneumoniae) in the control group . PI against each bacteria was 2.2 +/- 0.6 (P . aeruginosa), 2.1 +/- 0.3 (E . coli) and 2.6 +/- 0.9 (K . pneumoniae) in the patients group and 2.2 +/- 0.6 (P . aeruginosa), 2.2 +/- 0.4 (E . coli) and 2.5 +/- 0.6 (K . pneumoniae) in the control group . Thirdly, neutrophil bacteriocidal activity was determined by superoxide production and intracellular killing efficiency . Superoxide produced from OPZ-triggered neutrophils was 17.8 +/- 6.5 nmol/3.5 X 10(6) cells/20 min in the patients group and 20.2 +/- 5.8 nmol/3.5 X 10(6) cells/20 min in the control group, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Biochimie, 1989 Apr, 71(4), 545 - 50 Conformational changes in the active site of tryptophanase revealed by the circular dichroism method; Zakomirdina LN et al.; Tryptophanase from E . coli displays positive CD in the coenzyme absorption bands at 337 and 420 nm . Breaking of the internal coenzyme-lysine imine bond upon reaction with hydroxylamine or amino-oxyacetate is accompanied by a strong diminution of the positive CD . Interaction of tryptophanase with L-threonine and beta-phenyl-DL-serine(threo form) leads to a decrease in absorbance at 337 nm and to an increase at 425 nm . This is associated with inversion of the CD sign, i.e . with disappearance of the positive CD in the 420-nm band and its replacement by a negative CD . L-Phenylalanine, alpha-methyl-DL-serine and D-alanine cause an increase in absorbance at 425-430 nm and a diminution of the positive CD in this band . In the presence of D-alanine and indole a negative CD appears in the 400-450 nm region . It is inferred that an external coenzyme-quasisubstrate aldimine is formed on interaction of the above amino acids with the enzyme . L-Alanine and oxindolyl-L-alanine evoke an intense narrow absorption band at 500 nm ascribed to a quinonoid intermediate; a positive CD is observed in this band . The dissymmetry factor delta A/A in the 500-nm band is much smaller than that in the absorption bands of the unliganded enzyme . Inversion of the CD sign on formation of the external aldimine and diminution of the dissymmetry factor in the quinonoid band indicate that reorientations of the coenzyme occur in the course of the catalytic action of tryptophanase. Biochimie, 1989 Apr, 71(4), 533 - 43 A physical-chemical and immunological comparison shows that native and renatured Escherichia coli tryptophan synthase beta 2 subunits are identical; Murry-Brelier A et al.; An acid-denaturation of the beta 2 subunit of Escherichia coli tryptophan synthase has been recently described . In the present study, renaturation yield of acid-denaturated beta 2, and the influence of temperature, protein concentration and presence of ligands are investigated . It is also demonstrated that 3 forms of the protein are obtained at the end of the renaturation process: one is fully active, and is identical to native beta 2, as indicated by some of its chemical and physical properties, as well as by its immunological reactivity towards monoclonal antibodies specific for the native protein . A second form is composed of high molecular weight insoluble and inactive aggregates . A third form consists of low molecular weight soluble and inactive aggregates . The results obtained for the immunochemical reactivity of these small aggregates indicate that they are formed with partly correctly folded beta monomers assembled by specific but incorrect quaternary interactions . The capacity of monoclonal antibodies to detect such incorrect structures and to characterize renatured proteins is particularly emphasized. Biochimie, 1989 Apr, 71(4), 509 - 19 Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of L- and D-tryptophan and beta-mercaptoethanol with Escherichia coli tryptophan synthase; Drewe WF Jr et al.; The reactions of the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase with D- and L-Trp and the presteady-state reaction of L-Ser and beta-mercaptoethanol under different premixing conditions have been investigated by rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy . The reaction of alpha 2 beta 2 with L-Ser and beta-mercaptoethanol occurs in 3 detectable relaxations with rates similar to the 3 relaxations seen in the partial reaction with L-Ser and in the reaction with L-Ser and indole . The presteady-state phase of the reaction of beta-mercaptoethanol with the alpha-aminoacrylate intermediate is characterized by 2 relaxations . The RSSF spectra for this reaction show that the spectral changes that take place in these 2 phases are essentially identical . The L-Trp reaction is biphasic, and the spectral changes occurring in each phase of the reaction also are identical . The 2 new spectral bands formed (lambda max congruent to 420 nm and congruent to 476 nm) are assigned as the L-Trp external aldimine (Schiff's base) and L-Trp quinonoid intermediates, respectively . The reaction of D-Trp also is biphasic . Analysis of first and second derivatives of the RSSF spectral changes give evidence for the formation of spectral bands with lambda max of approximately 423 nm, approximately 450 nm, and approximately 478 nm . The positions and shapes of these bands suggest a D-Trp external aldimine structure (423 nm) and a quinonoidal species (450 and 478 nm) . However, product studies do not support this latter assignment . The behavior of the D- and L-Trp reactions and the reaction of beta-mercaptoethanol with the alpha-aminoacrylate strongly indicate the pre-existence of 2 slowly equilibrating forms of the internal aldimine and of the alpha-aminoacrylate. Biochimie, 1989 Apr, 71(4), 427 - 38 Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances; Yagi T et al.; The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E . coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride . A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells . The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values . In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract . Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme . The modified forms were stable in E . coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal . The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase . The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E . coli cells. Mol Gen Genet, 1989 Apr, 216(2-3), 455 - 61 Cloning and characterization of an Anacystis nidulans R2 superoxide dismutase gene; Laudenbach DE et al.; The E . coli iron superoxide dismutase gene (sodB) was utilized as a heterologous probe to isolate a superoxide dismutase (sod) gene from Anacystis nidulans R2 . Nucleotide sequence analysis revealed a 603 bp open reading frame with deduced amino acid sequence similar to other sod genes and to cyanobacterial superoxide dismutase amino-terminal sequences . Assuming proteolytic cleavage of the initial methionine residue, the molecular mass of the mature A . nidulans R2 sodB polypeptide is 22,000 daltons . Only a single copy of the superoxide dismutase sequence was detected in the A . nidulans R2 genome using Southern hybridization . Northern hybridization analysis indicated a single, monocistronic RNA transcript of approximately 720 bases . Primer extension mapping localized the transcription start site to 46 bases upstream from the initial methionine residue . A single orientation of a 2.1 kb PstI fragment containing the entire sod gene cloned into pUC18 was able to complement E . coli sodAsodB mutants . Complementation of the E . coli mutants was based on the ability of the cells to grow aerobically on minimal glucose medium . Growth curves of the complemented E . coli sodAsodB mutants showed that these cells exhibited levels of resistance to paraquat comparable to that of the wild-type E . coli phenotype. AIDS, 1989 Apr, 3(4), 215 - 20 Use of synthetic peptides for the detection of antibodies against the nef regulating protein in sera of HIV-infected patients; Sabatier JM et al.; Human sera were tested for the presence of anti-nef antibodies by radioimmunoassay (RIA), with recombinant radiolabelled nef expressed in E . coli . Of the 300 HIV-positive sera tested by RIA, 70 +/- 5.3% were found to be anti-nef positive . Anti-nef antibodies bound to nef with a high affinity (K 0.5 = 2.2 x 10(-9) M) . In 31 of the sera, the specificity of anti-nef antibodies was further analysed by enzyme-linked immunosorbent assay (ELISA) with large synthetic peptides ranging from 31 to 66 amino acid residues and spanning the total sequence of nef from HIV-1 . The results obtained showed that the immunodominant antigenic sites of nef were located close to the N- and C-terminal regions of the molecule. J Protein Chem, 1989 Apr, 8(2), 239 - 49 Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum by hybridization of site-directed mutants; Soper TS et al.; The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity . Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit . Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987) . This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain) . To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue . Coexpression in Escherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually . Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule . This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation . In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stabilized by elevated ionic strengths or by glycerol. J Photochem Photobiol B, 1989 Apr, 3(2), 247 - 58 Extent of formation of a dimeric adenine photoproduct in polynucleotides and DNA; Sharma ND et al.; Quantum yields are reported for the formation of a dimeric adenine photoproduct, A = A, in adenine homopolymers and DNA irradiated at 254 nm . The A = A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A = A on acid hydrolysis . Acid hydrolysates derived from DNA radiolabelled with {14C} 2'-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate . Although A = A is formed with a relatively high quantum yield of 1.6 X 10(-3) mol einstein-1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA).poly(dT) and undetectable in poly(rA).poly(dT) . Respective quantum yields of 6 X 10(-5) and 9 X 10(-6) were estimated for the formation of A = A in single- and double-stranded E . coli DNA implying that the photoproduct has very limited biological significance . From studies with d(ApG), d(GpA), ApG, GpA, d(A)20 and d(A4G)4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A = A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets. Exp Eye Res, 1989 Apr, 48(4), 561 - 7 Dimethylthiourea inhibits the inflammatory response to intravitreally-injected endotoxin; Fleisher LN et al.; Dimethylthiourea, a potent scavenger of toxic oxygen metabolites such as the hydroxyl radical, hypochlorous acid, and hydrogen peroxide, was tested for its ability to inhibit an experimentally induced inflammatory response . Inflammation was induced in one eye of male New Zealand white rabbits by intravitreal injection of 10 ng Escherichia coli endotoxin; the contralateral eye received an equal volume of pyrogen-free saline vehicle . Dimethylthiourea was administered intraperitoneally to these animals at 0, 300, 450 and 600 mg kg-1 . At 24 h post-endotoxin injection, all vehicle-injected eyes appeared normal with the exception of a small, but significant increase in aqueous humor protein concentration in the 600 mg kg-1 dimethylthiourea group . In endotoxin-injected eyes, treatment with dimethylthiourea, especially at the highest dose, significantly reduced iridal hyperemia, aqueous humor cell number and protein and prostaglandin-E concentrations, and the ex vivo release of prostaglandin-E from the lens . The ability of dimethylthiourea to significantly inhibit the inflammatory response to intravitreally-injected endotoxin suggests that toxic oxygen metabolites may play an important role in the initiation and/or propagation of this form of acute anterior uveitis . Furthermore, the data are consistent with an important interaction between toxic oxygen and arachidonic acid metabolites. Eur J Biochem, 1989 Apr 1, 180(3), 577 - 85 Studies of the functional topography of Escherichia coli RNA polymerase . A method for localization of the sites of affinity labelling; Grachev MA et al.; A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase . The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids . The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide . The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain . Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides . The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit . Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide . In order to increase reliability and resolution of the method, partial trypsinolysis may be employed . The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E . coli RNA polymerase. Mutat Res, 1989 Apr, 222(4), 311 - 6 Prophage induction by 4 antimalaria drugs (daraprim, fansidar, nivaquine and camoquine) and in combination with aflatoxin B1; Fasunon OD et al.; The abilities of 4 antimalaria drugs (Daraprim, Fansidar, Nivaquine and Camoquine) on their own and in combination with aflatoxin B1 to induce prophage in tester strains E . coli D21 and D22 were studied . The 4 drugs were found to induce prophage in the tester strains; Daraprim and Fansidar without the need for activation by liver mixed function oxidases (S9 microsomal fraction), while Nivaquine and Camoquine did require activation by this system . Aflatoxin B1 was used as a positive control in all the tests . The combined effects of each of the drugs with aflatoxin B1 were lower than that of the individual drugs acting alone . From these results, it may be concluded that the drugs may, on their own, pose a carcinogenic hazard to the population in the Nigerian environment exposed to them . In addition, the depression of prophage induction observed when the drugs were combined with aflatoxin B1 may be indicative of a common target site of action in the tester strains. Ann Surg, 1989 Apr, 209(4), 448 - 54 Low protein diets improve survival from peritonitis in guinea pigs; Peck MD et al.; Enteral diets with different protein content were tested to determine their effect on outcome in a model of protracted bacterial peritonitis . Hartley guinea pigs were provided with gastrostomies, and 1 week later, osmotic pumps were implanted into the peritoneal cavity to allow for continuous release of live bacteria over the course of 1 week . Three days after pump implantation, the animals began receiving isocaloric enteral diets that contained 5%, 10%, 15%, or 20% of total calories as protein . After 2 weeks of observation, the survivors were killed . All animals lost weight during the 2-weeks period, but there was no difference in weight lost . Nitrogen balance correlated with dietary protein . The mortality rate was significantly higher in the groups that received 15% and 20% of total calories compared with the group that received 5% (p less than 0.05) . Although dietary protein in the 5% group was insufficient for meeting the nutritional needs of the animal, survival was best in this group . Possible explanations are that protein restriction in this model may either augment host defence or impair bacterial virulence. Appl Environ Microbiol, 1989 Apr, 55(4), 984 - 93 Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili; Thiry G et al.; A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed . Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker . The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation . Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili . One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats . The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production . The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies. Acta Med Okayama, 1989 Apr, 43(2), 81 - 8 Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells; Mori S et al.; To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized . SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution . The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin . Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates . The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III . The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl . Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process . The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s). Acta Med Okayama, 1989 Apr, 43(2), 73 - 80 A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA; Seki S et al.; A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established . The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions . Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form . A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I), ATP, T4 DNA ligase and four deoxynucleoside triphosphates . After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy . The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired . When the priming factor was omitted, DNA repair did not occur . The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen. Hybridoma, 1989 Apr, 8(2), 209 - 21 Monoclonal antibodies against human basic fibroblast growth factor; Seno M et al.; Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF . Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b . All these MAbs bound the complete form of hbFGF produced in E.coli . Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E . coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs . MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF . MAb52 and MAb98 recognized the one located between the amino acid residue no . 14 and 40 . None of MAbs bound bovine acidic FGF (aFGF) . Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed . This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF . Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column. Eur J Biochem, 1989 Apr 1, 180(3), 555 - 61 Gene synthesis, expression in Escherichia coli and purification of immunoreactive human insulin-like growth factors I and II . Application of a modified HPLC separation technique for hydrophobic proteins; Hummel M et al.; We have expressed synthetic genes encoding human insulin-like growth factors I and II in large quantities in Escherichia coli as fusion proteins with the 300 N-terminal amino acids of the E . coli trpE gene product . The resulting hybrid proteins were purified from the insoluble fraction of crude bacterial lysates using a rapid HPLC separation procedure employing a C8 reversed-phase column and a gradient of 2-propanol in formic acid . With the procedure we obtained in volatile solvents highly purified fusion proteins that were used for further biochemical and immunological procedures . Here we describe biochemical characteristics of the bacterially expressed fusion proteins and demonstrate that these proteins share substantial antigenic properties with the native polypeptides allowing the establishment of highly specific monoclonal antibodies. J Bacteriol, 1989 Apr, 171(4), 2166 - 72 Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli; Miller KW et al.; The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity . After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70% . The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems . SLPI proved to be a potent inhibitor of translation in vitro . When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75% . The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA-metabolizing enzymes, such as RNase . In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61 . Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin . On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA . Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPI/DNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E . coli . These results indicate that, in general, high-level expression of basic recombinant proteins in E . coli may be problematic. J Bacteriol, 1989 Apr, 171(4), 1854 - 61 Transcription of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli; Bognar A et al.; The folC gene of Escherichia coli is cotranscribed with an upstream gene from two promoters located in the noncoding region 5' to the coding sequence of the upstream gene . Virtually all of the expression of the folC gene product, folylpolyglutamate synthetase-dihydrofolate synthetase, is therefore due to the upstream gene promoters . No promoter activity was found in the coding sequence of the upstream gene or in the 72-base-pair noncoding region between the two genes . It is shown that a third gene, which may overlap the coding sequence of the folC gene by 8 base pairs at the 3' end, nevertheless, has an promoter independent from that of the upstream gene-folC operon . These results contrast with those presented by Nonet et al . (M . L . Nonet, C . C . Marvel, and D . Tolan, J . Biol . Chem., 262:12209-12217, 1987), who concluded that folC was cotranscribed with the gene at its 3' end and the gene upstream to folC was cotranscribed with the gene(s) further upstream . A stable stem-loop structure resembling a rho-independent terminator is present within the noncoding region between the upstream gene and the folC gene . Folypolyglutamate synthetase expression is 6- to 15-fold lower than that of the upstream gene product, suggesting that the stem-loop terminates some of the transcription from the upstream gene promoter . We found by deletion mutagenesis and cloning sequences containing the stem-loop structure into a termination reporter plasmid that this stem-loop does not act as an effective terminator of transcription . We also found that the stem-loop does not protect the upstream gene message from degradation, since expression of the upstream gene product in maxicell experiments is the same whether the stem-loop structure is present or deleted. Infect Immun, 1989 Apr, 57(4), 1192 - 9 Characterization of the F41 fimbrial antigen of enterotoxigenic Escherichia coli by using monoclonal antibodies; van Zijderveld FG et al.; We produced monoclonal antibodies (MAbs) from 23 different murine hybridoma cell lines against the F41 fimbrial antigen of bovine and porcine enterotoxigenic Escherichia coli . Cell lines were created by fusing myeloma cells and spleen cells of mice that were immunized with either purified F41 or with Formalin-killed whole cells . The specificity of the MAbs to the F41 antigen was proven by enzyme-linked immunosorbent assays (ELISAs) and radioimmunoprecipitation tests . Epitope analysis with a competition ELISA revealed that the 23 MAbs recognized at least five epitopes . These results were corroborated by those of immunodiffusion tests, in which all possible combinations of two MAbs were tested against ultrasonically disintegrated F41 antigen . In a double-antibody sandwich ELISA, all peroxidase-conjugated MAbs bound to the F41 antigen of all 182 bacterial strains that were tested . Apparently, the epitopes recognized by the MAbs are highly conserved . Immunoelectron microscopy revealed that the MAbs were directed to fimbrial structures 3 to 4 nm in diameter and that the epitopes were equally distributed along the fimbriae . Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed . The results of the radioimmunoprecipitation assay suggested that F41 fimbriae are composed of a single repeating 29,000-dalton protein subunit; however, we could not exclude the possibility of the existence of minor fimbrial components. Cancer Res, 1989 Apr 1, 49(7), 1763 - 7 Isolation and characterization of complementary DNA to proliferating cell nucleolar antigen P40; Reddy AB et al.; Proliferating cell nucleolar antigen P40 is a late G1-specific protein, which was found in a variety of human tumors (A . Chatterjee, J . W . Freeman, and H . Busch . Cancer Res., 47: 1123-1129, 1987) . Two overlapping complementary DNA clones for antigen P40 were isolated by immunoscreening a lambda gt11 human expression library . The complete nucleotide sequence of the clones was determined . The complementary DNAs encode the Mr 30,000 portion of the COOH-terminal portion of the protein . The mRNA for P40 was 2.8 kilobases long and was expressed maximally in G1 cells in cell cycle . A series of deletion mutants of the expressed peptide was constructed and the deletion mutants were expressed in Escherichia coli . Using these mutants, the epitope region of P40 recognized by a P40-specific monoclonal antibody was identified . The hydropathy plot based on the protein sequence revealed that this region of the protein is largely hydrophilic . This protein is unique and differs in sequence from other proliferating cell nuclear/nucleolar antigen proteins of similar molecular weight such as protein B23 and cyclin. Biochem Biophys Res Commun, 1989 Mar 31, 159(3), 1397 - 403 DNA polymerase alpha activity is not affected by protein kinases or alkaline phosphatase; Prussak CE et al.; Recent studies with crude or partially purified cell extracts have suggested that DNA polymerase alpha activity may be regulated by enzymatic phosphorylation . To further investigate these findings, we have examined the effects of protein kinases and phosphatases on highly purified DNA polymerase alpha from mouse cells . Incubation of DNA polymerase alpha with a variety of protein kinases, including protein kinase C, had no effect on polymerase activity . In addition, treatment of the polymerase with soluble calf intestinal alkaline phosphatase had no effect on DNA polymerase alpha activity, further indicating that phosphorylation does not have a direct role in modulating polymerase activity . In contrast, incubation of DNA polymerase alpha with calf intestinal alkaline phosphatase crosslinked to agarose beads resulted in a time dependent disappearance of polymerase activity . This loss of DNA polymerase activity was dependent on phosphatase activity, as the alkaline phosphatase inhibitors, potassium phosphate or levamisole, prevented the loss of polymerase activity in the presence of the beaded phosphatase . The loss of DNA polymerase alpha activity following beaded phosphatase treatment was not a general phenomena as the large fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase or mouse primase were not affected by similar treatment . The decreased DNA polymerase activity following incubation with phosphatase beads correlated with the binding of the DNA polymerase polypeptides, p185 and p68, to the agarose beads and this binding could not be reversed by either 150 mM potassium chloride or sodium sulfate . The binding of the polymerase to the agarose beads was dependent on the phosphatase activity, as the polymerase could be first treated with soluble calf intestinal phosphatase and subsequently bound to added Sepharose 4B beads . Surprisingly, Sepharose CL4B, a highly desulfated agarose preparation, did not bind the phosphatase-treated polymerase suggesting that sulfated polysaccharides are required for polymerase binding . The physiological correlate of this binding is unknown, but it has been reported that sulfated polysaccharides exist in a variety of intracellular compartments . It would be interesting to speculate that phosphorylation controls the intracellular compartmentalization of DNA polymerase alpha. Biochem Biophys Res Commun, 1989 Mar 31, 159(3), 991 - 8 Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6 proteins and inhibition of proliferation; May LT et al.; Interleukin-6 (IL-6) is a cytokine which is not only produced by a wide variety of different cells but one which also affects the function of diverse tissues . We have studied the expression of the IL-6 gene in freshly explanted human umbilical vein endothelial cells (HUVEC) and have also evaluated the effect of IL-6 on HUVEC proliferation . Cytokines like interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF) as well as bacterial products such as the lipopolysaccharide (LPS) rapidly enhance production of biologically active IL-6 by HUVEC (IL-6 bioassay: increase in alpha 1-antichymotrypsin secretion by Hep3B2 cells and its neutralization by antiserum to E . coli-derived human IL-6) . The two inducible RNA start sites in the IL-6 gene that are used in cytokine-induced fibroblasts (at +1 and -21) are also used in the same relative proportion (+1 greater than -21) in cytokine or LPS-induced HUVEC as determined by S1-nuclease protection assays for IL-6 transcripts . Immunoaffinity chromatography followed by Western blotting shows that IL-6 species secreted by IL-1 alpha-induced HUVEC are of molecular mass 23-25, 27-30 and 45 kDa as judged by SDS-PAGE under reducing conditions . Finally, rIL-6 inhibits {3H}-thymidine incorporation by HUVEC in a dose-dependent manner . Thus IL-6 is not only produced by HUVEC but may also affect its proliferation . The ability of the vascular endothelium to rapidly secrete IL-6 in response to inflammation-associated cytokines is of strategic value since it generates a circulatory signal which helps mobilize the acute phase plasma protein response and enlists the immune system in host defence. Biochem Biophys Res Commun, 1989 Mar 31, 159(3), 927 - 32 50 residues coded by exon 2 of chicken lysozyme carry residual catalytic activity; Kuchinke W; The generally accepted hypothesis that exons code for fundamental polypeptide structures was tested with a fusion protein consisting of almost the entire polypeptide coded by exon 2 of chicken lysozyme fused to the N-terminus of beta-galactosidase of E.coli . Exon 2 encodes residues 28-81 of lysozyme . It thus contains Glu 35 and Asp 52, which are essential for hydrolysis of glycosidic bonds . The exon 2-beta-galactosidase fusion protein hydrolysed the substrate 4-methylumbelliferyl-N,N',N''-triacetyl-chitotrioside with a reaction rate about 1/40,000 of that of native lysozyme . The low hydrolysis rate of exon 2-peptide is partially caused by its low affinity to its substrate. Biochem Biophys Res Commun, 1989 Mar 31, 159(3), 1128 - 34 A new compound, withangulatin A, promotes type II DNA topoisomerase-mediated DNA damage; Juang JK et al.; Withangulatin A, a new compound with a known chemical structure and from the antitumor Chinese herb Physalis angulata L, was found to act on topoisomerase II to induce topoisomerase II-mediated DNA damage in vitro . It has two effective dosage ranges of approximate 0.5 and 20 microM, with about one-third the activity of 20 microM VM-26. Biochem Biophys Res Commun, 1989 Mar 31, 159(3), 969 - 75 A novel cleavage product of beta-thromboglobulin formed in cultures of stimulated mononuclear cells activates human neutrophils; Walz A et al.; A neutrophil-activating peptide, NAP-2, was found to be produced in cultures of human mononuclear cells in the presence of E . coli lipopolysaccharide or phytohaemagglutinin . NAP-2 induced the release of elastase from cytochalasin B-treated human neutrophils . Amino- and carboxy-terminal sequencing and electrophoretic analysis showed that NAP-2 is a single peptide of 70 amino acids (Mr 7,628, IEP 8.7) corresponding to a carboxyterminal fragment of beta-thromboglobulin . NAP-2 is homologous to NAF/NAP-1 . When aligned on the basis of their two first cysteines, 13 out of 20 amino-terminal residues are identical . The overall homology between the two peptides is 46%. J Immunol Methods, 1989 Mar 31, 118(2), 153 - 60 Epitope characterization by modifications of antigens and by mapping on resin-bound peptides . Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13; Syu WJ et al.; The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report . Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin . Three of these MAbs react with the C-terminal peptide S13(84-117) which has five Lys residues clustered within the last 16 amino acids . Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50% . Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs . A 23-residue peptide corresponding to S13(95-117) was synthesized by a modified Merrifield solid phase method . Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling . The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides . The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13(97-117) . The third MAb binds to S13(98-117) but binds more strongly when the sequence is lengthened . Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized . Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide . The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13(2-22) and bound more strongly to the synthetic S13(1-22). FEBS Lett, 1989 Mar 27, 246(1-2), 89 - 93 Cloning and expression of an interleukin-1 beta precursor and its conversion to interleukin-1 beta; Dalboge H et al.; A gene coding for a N-terminal precursor of interleukin-1 beta (IL-1 beta) was cloned and expressed in E . coli . The isolated Met-Glu-Ala-Glu-IL-1 beta precursor was enzymatically converted to IL-1 beta by means of dipeptidylaminopeptidase (DAP I) . This method ensured a correct N-terminal residue and the often observed expression of Met-IL-1 beta was thus avoided . The pure and physically homogeneous product exhibited the characteristic properties of natural IL-1 beta . The in vitro biological activity was measured in the lymphocyte-activating factor assay and was compared to that of natural IL-1 beta isolated from stimulated monocyte culture using exactly the same purification procedure . The specific biological activity of both products was 2 x 10(-8) U/mg indicating that the recombinant product exhibits full biological activity. FEBS Lett, 1989 Mar 27, 246(1-2), 140 - 4 The 23 KDa polypeptide of the photosynthetic oxygen-evolving complex from mustard seedlings (Sinapis alba L.) . Nucleotide sequence of cDNA and evidence for phytochrome control of its mRNA abundance; Wenng A et al.; The nucleotide sequence of a cDNA from mustard shows 78% homology in deduced amino acid sequence for the mature protein compared to the sequence for the 23 kDa protein of the oxygen-evolving complex from spinach {(1987) FEBS Lett . 216, 234-240} . There is also a high degree of homology between the premature protein sequences concerning the hydrophobic domain and its distance from the suggested processing site . The accumulation of mRNA for the 23 kDa protein in mustard was stimulated by continuous far-red light and reversal experiments by means of red/far-red light pulse treatment show the involvement of phytochrome in controlling the mRNA abundance for the 23 kDa polypeptide in mustard . The accumulation of the mRNA can be inhibited in white light if the seedlings are treated with the herbicide Norflurazon. FEBS Lett, 1989 Mar 27, 246(1-2), 13 - 6 The nucleotide sequence of the genes coding for the S19 and L22 equivalent ribosomal proteins from Halobacterium halobium; Mankin AS; The primary structure of the two ribosomal protein genes of archaebacterium Halobacterium halobium has been determined . The encoded polypeptides are homologous to the Escherichia coli ribosomal proteins S19 and L22 . The two genes constitute part of an operon whose organization is analogous to that of the 'S10' operon of E . coli. FEBS Lett, 1989 Mar 27, 246(1-2), 21 - 4 Identification of single-strand initiation signals in the terC region of the Escherichia coli chromosome; Hiasa H et al.; On the basis of clear-plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome . At least two single-strand initiation signals were identified from the terC region . The nucleotide sequences of these two signals were determined . Sequence homologies, variations of the consensus of n' protein recognition sites, 5'-GAAGCGG-3', were found within these signals . A novel conserved sequence was also found within these signals . Their initiation activities were measured both by the infection growth assay and by the ability to convert the single-stranded DNA to the duplex replicative form DNA in vivo. FEBS Lett, 1989 Mar 27, 246(1-2), 109 - 12 On the biosynthesis of free and covalently bound PQQ . Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein; van der Meer RA et al.; Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule . This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition . Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC . Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ . Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor . Implications for the role of PQQ in eukaryotic cells are discussed. FEBS Lett, 1989 Mar 27, 246(1-2), 197 - 201 Differences in 23 S rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex . Probing by bifunctional Pt(II)-containing reagent; Chistyakov PG et al.; rRNA-protein cross-links in free E . coli 35S-labeled 70 S ribosomes and in the initiation complex 35S-labeled 70 S ribosome.AUGU6.fMet-tRNA(fMet) were studied with the aid of a new type of binuclear Pt(II) compound - dichlorotetra-ammine(1,6-hexamethylenediaminediplatinum++ +) dichloride . The use of this reagent allowed us to reveal differences in the rRNA-protein neighbourhood in free 70 S ribosomes and in the initiation complex . Proteins L3, L6, L23 and L25 were shown to cross-link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16, L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex . 16 S rRNA was found to be cross-linked preferentially to a single protein, S1, in both states of the ribosomes. FEBS Lett, 1989 Mar 27, 246(1-2), 149 - 52 Formation of an ion transport supercomplex in Escherichia coli . An experimental model of direct transduction of energy; Bagramyan KA et al.; Hydrogen gas production was observed to occur during ATP-driven H+/K+ exchange in anaerobically grown E . coli . Neither process was found in aerobically grown cells or anaerobic cells grown on nitrate medium or when the osmotic pressure was decreased or K+ removed, or finally when DCCD, arsenate or CCCP was applied . Dithiothreitol restored the process even in the presence of CCCP but not in other cases of inhibition . A model of a multienzyme transport super-complex is proposed . The supercomplex consists of three genetically independent mechanisms: F0F1 H+-ATPase to provide energy, the K+-transporting Trk system as energy sink and formate-hydrogen lyase as donor of reducing equivalents . Within this supercomplex direct transduction of energy is accomplished via oxidation of 2 SH to S-S. FEBS Lett, 1989 Mar 27, 246(1-2), 137 - 9 Protein synthesis inhibitors and catalytic RNA . Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P; Vioque A; RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl-, peptidyl- or free tRNAs in the case of ribosomes . In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA) . Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P. J Biol Chem, 1989 Mar 25, 264(9), 4896 - 900 Polypeptide structure of germin as deduced from cDNA sequencing; Dratewka-Kos E et al.; Synthesis of a relatively rare glycoprotein (germin) signals the onset of growth in germinating wheat embryos . Germin mRNA (1075 nucleotide residues) has an 85-residue 5'-untranslated sequence, a 69-residue sequence that can encode a 23-residue signal-peptide sequence, a 603-residue sequence that can encode a 201-residue mature-protein sequence, and a 318-residue 3'-untranslated sequence that begins with a UAA-terminator codon, ends with a 63-residue polyadenylate tract, and has three polyadenylation (and other, related) signals (AAUAAN etc.) . One polyadenylation signal is just 9 nucleotides from the polyadenylation site, the shortest stretch of nucleotides yet found between polyadenylation signal and site in any animal or plant mRNA . The mature-protein coding sequence in germin mRNA contains an unusually high proportion (87%) of G + C in the third positions of its codons . The amino acid sequence of germin does not have extensive internal homologies or repetitions, and it is not characterized by regions of unusually high charge density, as is nucleoplasmin, another water-soluble homopentameric protein with otherwise closely related structural properties . Germin does, however, contain a stretch of 34 uncharged amino acid residues and these may possibly mediate its homopentameric structure and its remarkable resistance to enzymic proteolysis . In view of a possible association of germin with cellular membranes, the most interesting relatedness of the germin sequence to the sequences of other proteins is an 80% homology between a decapeptide sequence in mature germin and a decapeptide sequence in Escherichia coli glycerol-3-phosphate acyltransferase . The relation of germin-gene structure to overall gene regulation during early plant growth is discussed. J Biol Chem, 1989 Mar 25, 264(9), 4948 - 52 Preliminary X-ray crystallography studies of recombinant human interleukin-1 alpha . Purification and structural characterization; Hassell AM et al.; Human interleukin-1 alpha, cloned and expressed in E . coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry . The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent . The space group is P2(1)2(1)2(1) . Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees . The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection . Native diffraction data were collected . Screens for heavy atom derivatives have been initiated. Vet Rec, 1989 Mar 25, 124(12), 305 - 8 Flunixin meglumine and flurbiprofen in cows with experimental Escherichia coli mastitis; Lohuis JA et al.; The effect of flunixin meglumine and flurbiprofen on the course of experimental Escherichia coli mastitis was examined . Nine cows (within one month post partum) were inoculated intramammarily with 20 x 10(5) viable E coli in both rear quarters . Three cows remained untreated (controls); three cows received three injections of flunixin meglumine and three cows received flurbiprofen as two intravenous infusions . Flunixin meglumine and flurbiprofen were initially given before clinical signs were observed . Treatment was repeated if the cows' temperature increased by more than 1 degree C . In the untreated cows, rectal temperature and heart rate increased from three hours after infection, and rumen motility (both frequency and amplitude) decreased from four hours after infection . Treatment with flunixin meglumine or flurbiprofen almost completely abolished the febrile response during the first nine hours after infection, and the decrease in rumen motility was less pronounced in the treated animals . These results suggest that the decrease in rumen motility during E coli mastitis is at least partly due to a mechanism involving prostaglandin. Vet Rec, 1989 Mar 25, 124(12), 297 - 9 Natural infection with an attaching and effacing Escherichia coli in the small and large intestines of a calf with diarrhoea; Pearson GR et al.; Attaching and effacing Escherichia coli were identified in the small and large intestine of a calf with naturally occurring diarrhoea . The organisms were associated with intestinal lesions and were identified by immunoperoxidase staining and transmission and scanning electron microscopy, but they did not produce Shiga-like toxin (verotoxin). J Biol Chem, 1989 Mar 25, 264(9), 5260 - 8 Genetically engineered polymers of human CuZn superoxide dismutase . Biochemistry and serum half-lives; Hallewell RA et al.; CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons . Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits . The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene . The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence . Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli . The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface . The polymers are soluble, thermostable, and of near normal specific activity . Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits . The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active . Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats . The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min. J Biol Chem, 1989 Mar 25, 264(9), 5226 - 32 The primary structure of Escherichia coli L-threonine dehydrogenase; Aronson BD et al.; The complete primary structures of Escherichia coli L-threonine dehydrogenase has been deduced by sequencing the cloned tdh gene . The primary structure so determined agrees with results obtained independently for the amino acid composition, the N-terminal amino acid sequence (20 residues), and a short sequence at the end of an internal peptide of the purified enzyme . The presence of a predicted Asp-Pro bond at residues 148 and 149 was confirmed by treatment of purified threonine dehydrogenase with dilute acid and subsequent analysis of the resulting cleavage products . The primary structure of L-threonine dehydrogenase from E . coli has been examined for possible homology to other NAD+-dependent dehydrogenases; indications are that this enzyme is a member of the zinc-containing long-chain alcohol/polyol dehydrogenase family. J Biol Chem, 1989 Mar 25, 264(9), 5128 - 33 Heparin cofactor IIOslo . Mutation of Arg-189 to His decreases the affinity for dermatan sulfate; Blinder MA et al.; Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor