J Biochem (Tokyo), 1995 Mar, 117(3), 654 - 60 Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme; Yoshimoto T et al.; A gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1 . The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M . lacunata . The expressed enzyme from E . coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration . The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5 . It had an optimum temperature of 35 degrees C for 5 min of reaction and was stable to up to 35 degrees C for 30 min at pH 7.0 . Its molecular weight was estimated to be 80,000 by SDS-PAGE and gel-filtration analyses . It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease . Its amino acid sequence was 38% homologous to that of the E . coli protease II . By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654 . The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant. J Biochem (Tokyo), 1995 Mar, 117(3), 535 - 44 Leader peptidase from Escherichia coli: overexpression, characterization, and inactivation by modification of tryptophan residues 300 and 310 with N-bromosuccinimide; Kim YT et al.; We have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme . This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40 degrees C . Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5 . The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS) . When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate . These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis . This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase . On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5,5'-dithiobis(2-nitrobenzoic acid), succinic anhydride, or 2,4,6-trinitrobenzenesulfonate . Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues . Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents . However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide . Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity. J Biochem (Tokyo), 1995 Mar, 117(3), 495 - 8 In vivo effect of GroESL on the folding of glutamate racemase of Escherichia coli; Ashiuchi M et al.; The overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells . The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form . We isolated the active enzyme and purified it effectively. J Biochem (Tokyo), 1995 Mar, 117(3), 480 - 8 A strategy for testing the suitability of cysteine replacements in dihydrofolate reductase from Escherichia coli; Iwakura M et al.; Amino acid sequences in proteins can contain residues which complicate biochemical, biophysical, or protein engineering studies but which are not essential for folding or activity . Their replacement with other naturally-occurring amino acids which are not subject to such complications but which maintain essential properties of the protein is a desirable goal . A simple strategy for testing various mutants for their suitability is described for a pair of cysteine residues in dihydrofolate reductase (DHFR) from Escherichia coli . Using a reconstructed gene which preserves the amino acid sequence and introduces a variety of unique restriction sites, the cysteines at positions 85 and 152 were replaced by site-directed and cassette mutagenesis . The enzymatic activity, stability, and folding mechanism of six double mutant DHFR proteins were examined with the purpose of identifying a suitable alternative to wild type DHFR . The Cys85-->Ala and Cys152-->Ser double mutant DHFR was found to retain the four channel folding mechanism and have activity and stability which are comparable to the wild type enzyme . The replacement of the cysteines improved the resistance of DHFR to the irreversible loss of activity at high temperature. J Biochem (Tokyo), 1995 Mar, 117(3), 475 - 9 An improved method for extraction of mRNA and protein from the fungus Chaetomium gracile with anhydrous hydrogen fluoride; Oshiro S et al.; We previously reported that DNAs directly applicable to restriction analyses and transformation of Escherichia coli can be extracted from fungi and yeasts by use of anhydrous hydrogen fluoride (HF) under a mild condition, 5 min at 0 degrees C {Oshiro, S., Katsura, N., Kitada, K., and Gunge, N . (1987) FEBS Lett . 220, 383-386} . In the present investigation, we examined whether this improved method is also applicable to extraction of RNA and protein from the fungus Chaetomium gracile . The RNA and protein were effectively extracted from the fungus after anhydrous hydrogen fluoride (HF) treatment for a short time (1 min) at 0 degrees C . The extracted poly(A)-enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly(U) directed not only the incorporation of {3H}glycine into polypeptides but also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extracted from the fungus by HF . Analyses by gel filtration and polyacrylamide electrophoresis under native conditions showed that dextranase extracted from the fungus by HF under the same conditions had the same molecular weight and electrophretic mobility as the enzyme excreted into the medium . This suggests that the mRNAs and proteins extracted by this method are also applicable to protein synthesis directed in a cell-free system and to enzyme purification from a fungus insusceptible to lytic enzymes . This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification within 1 h. Mol Microbiol, 1995 Mar, 15(6), 1151 - 64 Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture; Cox DL et al.; Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens . To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T . pallidum molecular architecture . Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X-100 . Intact, encapsulated treponemes were not labelled by monospecific antisera directed against four major T . pallidum lipoproteins or a candidate T . pallidum outer membrane protein (TpN50) with C-terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum . Each of these immunologic reagents, however, labelled encapsulated treponemes co-incubated with detergent . In contrast, antibodies generated against isolated T . pallidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze-fracture electron microscopy . In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T . pallidum cell envelope constituents . They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes. Mol Microbiol, 1995 Mar, 15(6), 1139 - 50 The biogenesis of c-type cytochromes in Escherichia coli requires a membrane-bound protein, DipZ, with a protein disulphide isomerase-like domain; Crooke H et al.; A mutant of Escherichia coli K-12, JCB606, which lacks all five c-type cytochromes synthesized during anaerobic growth in the presence of nitrite or trimethylamine-N-oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity . The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO . The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co-ordinate 4425 on the E . coli chromosome, the gene order being nrf-fdhF-mel-dipZ-ampC . Recombinant plasmids that restored Nrf activity to test-tube cultures of the mutant were isolated from a cosmid library . A 2.7 kb EcoRV-SmaI fragment (co-ordinates 4443 to 4446 kb on the physical map of the E . coli chromosome) was found potentially to encode three genes arranged in at least two operons . The second gene, dipZ, was sufficient to complement the JCB606 mutation . The translated DNA sequence predicts that DipZ is a 53 kDa integral membrane protein with a 37 kDa N-terminal domain including at least six membrane-spanning helices and a 16 kDa carboxy-terminal hydrophilic domain which includes a protein disulphide isomerase-like motif . It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues. Mol Microbiol, 1995 Mar, 15(6), 1127 - 37 Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K-12; Fong ST et al.; The cutA locus, presumably involved in copper tolerance in Escherichia coli, was characterized by a mutation leading to copper sensitivity . Copper-accumulation measurements with radioactive 64Cu2+ showed increased uptake by cutA copper-sensitive mutant cells, and reduced uptake when the cutA mutation was complemented in trans . The locus was mapped using complementation of the cutA mutant to partial copper tolerance with wild-type chromosomal fragments . The 3.2 kb DNA region involved in cutA was sequenced and analysed, revealing three significant open reading frames, none of which had been previously published . The products of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of about 50 kDa, 24 kDa, and 13 kDa, consistent with the sizes predicted from the DNA sequences . The 50 kDa and 24 kDa polypeptides were found in the bacterial inner membrane, and the 13 kDa polypeptide with the cytoplasmic fraction . In addition to being required for copper tolerance, cutA affects tolerance levels to zinc, nickel, cobalt and cadmium salts . Transcriptional fusions of cutA with the lux operon showed induction by copper, zinc, nickel, cobalt and, to a lesser extent, cadmium, manganese and silver salts. Mol Microbiol, 1995 Mar, 15(6), 1069 - 79 Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ; Sanna MG et al.; CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway . The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming . The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY . The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation . We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins . Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein . Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants . CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ . These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates . Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch. Mol Microbiol, 1995 Mar, 15(6), 1031 - 7 F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions; Bahassi EM et al.; The ccd locus of the F plasmid codes for two gene products, CcdA and CcdB, which contribute to the plasmid's high stability by post-segregational killing of plasmid-free bacteria . Like the quinolones, the CcdB protein is a poison of the DNA-topoisomerase II complexes, while CcdA acts as an antidote against CcdB . In addition to these poison-antipoison properties, the CcdA and CcdB proteins act together at transcription level to repress their own synthesis . In this work, we have isolated, in vivo, and characterized several non-killer CcdB mutants . All missense mutations which inactivate CcdB killer activity are located in the region coding for the last three C-terminal residues . However, the resulting mutant CcdB proteins retain their autoregulatory properties . We conclude that the last three C-terminal residues of CcdB play a key role in poisoning but are not involved in repressor formation. J Ethnopharmacol, 1995 Mar, 45(3), 193 - 7 Biological activities of a Turkish medicinal plant, Prangos platychlaena; Ulubelen A et al.; Prangos platychlaena has been used in traditional medicine in eastern Turkey . It stops bleeding and heals the scars when applied externally . When the isolated coumarins were tested against bacterial strains, only a slight activity was obtained. Curr Genet, 1995 Mar, 27(4), 359 - 66 NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans; Pieterse CM et al.; The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization . NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source . The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD . The P . infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies . The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A . nidulans . However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant . This suggests that there is no functional expression of the introduced niaA gene in A . nidulans . In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay . Plasmids containing chimaeric genes with the promoter of the P . infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A . nidulans protoplasts . No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A . nidulans . Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A . nidulans. Curr Genet, 1995 Mar, 27(4), 309 - 11 DNA insertion system for complex yeast shuttle vectors; Daniel J; A DNA insertion system, termed marked homologous recombination, was devised for the construction of complex yeast shuttle plasmids . This system, which is efficient, rapid and easy to use, should contribute to our understanding of gene-gene interactions in yeast cells. Genetika, 1995 Mar, 31(3), 324 - 32 {Analysis of a chromosomal DNA fragment isolated from a hybrid autonomous plasmid of Chlamydomonas reinhardtii}; Sizova IA et al.; The ability of a previously cloned fragment of nuclear DNA of Chlamydomonas reinhardtii (a 2.4-kb BamH 1-EcoR I fragment) to function as the origin of replication of plasmid pARG7.8.2.4, carrying the homologous arg7 marker gene of prototrophy or arginine in cells of C . reinhardtii transformants, was studied . As shown by Southern blotting, plasmid pARG7.8.2.4 integrates at several sites of chromosomal DNA of chlamydomonas without autonomous maintenance in cells . Results obtained using Southern blotting and plasmid rescue techniques demonstrated that plasmid pARG7.8, containing only the arg7 gene, is able both to integrate into the chromosome and to be maintained, in some cases, in the autonomous state in Arg+ transformants of alga . Analysis of the nucleotide sequence of the DNA fragment under study revealed several types of GC-enriched repeats that manifested homology with the nucleotide sequence of the arg7 gene in introns 10 and 12. Electrophoresis, 1995 Mar, 16(3), 377 - 88 Sequence dependent migration behavior of double-stranded DNA in capillary electrophoresis; Berka J et al.; Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence-dependent migration of double-stranded DNA fragments . It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices . Compared to linear DNA-ladder standards, both anomalously slow and rapid DNA fragments were observed, with the degree of anomalous migration depending on the electric field strength, polymer concentration, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA-intercalating dyes) . By selecting a combination of electrophoretic conditions (e.g . 3% T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), molecular weight dependent separations were closely restored. Bioorg Med Chem, 1995 Mar, 3(3), 313 - 20 CMP-KDO synthetase: overproduction and application to the synthesis of CMP-KDO and analogs; Sugai T et al.; CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC 2.7.7.38) has been cloned and overexpressed in Escherichia coli . The structure gene was amplified from the total DNA of E . coli K-235 through the primer-directed polymerase chain reaction . The gene was then cloned into lambda ZAP vector at the EcoRI and XbaI restriction sites and overexpressed in E . coli Sure strain at a level approximately 400 times as much as that produced in the host strain . Application of the enzyme to the synthesis of cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid (CMP-KDO) and analogs was studied . Of several KDO analogs tested, 5-fluoro-2-keto-3,5-dideoxyoctulosonic acid (5-FKDO) was found to be a good substrate of the enzyme, and the product (CMP-5-FKDO) was prepared and characterized, representing the first stable CMP-KDO analog prepared enzymatically to date . The natural enzyme product, CMP-KDO, was however quite unstable (t1/2 = 19 min, in 50 mM MgCl2, 0.2 M Tris buffer, pH 9.0) . A mechanism for the decomposition of CMP-KDO involving the hydrogen bonding interactions between the OH groups of C-5 and C-7 (and/or C-8) and the phosphate oxygens was proposed. Vet Immunol Immunopathol, 1995 Mar, 45(1-2), 45 - 54 Endogenous tumor necrosis factor (TNF) production and modification of pathological lesions in experimental Escherichia coli endotoxemia of piglets; Nakajima Y et al.; We examined the kinetics of tumor necrosis factor (TNF) production induced by Escherichia coli lipopolysaccharide (LPS) in relation to LPS tolerance and endotoxemic lesions of piglets . The plasma of piglets demonstrated cytotoxicity to TNF-sensitive L929 cells between 0.5 and 4 h after inoculation with 200 micrograms kg-1 of LPS . This cytotoxicity was neutralized by anti-bovine TNF serum . These piglets had disseminated intravascular coagulation (DIC) and meningoencephalitis . However, if piglets were first treated with three doses of 40 micrograms kg-1 of LPS, both TNF production and the occurrence of DIC were inhibited when 200 micrograms kg-1 of LPS was inoculated into these piglets . Repetitive inoculation with increasing doses of LPS induced fibrinoid vasculitis, meningoencephalitis and pneumonitis, while hemorrhage was minimal . A very low amount of TNF activity was detected from most of the samples of a piglet after repeated LPS inoculation . These results suggested that severity of the hemorrhagic and thrombotic lesions might relate to the amount of endogenous TNF activity, and that LPS tolerance might relate to inhibition of TNF production. Eur J Surg, 1995 Mar, 161(3), 147 - 55 Breakdown of adenine nucleotides, formation of oxygen free radicals, and early markers of cellular injury in endotoxic shock; Jabs CM et al.; OBJECTIVE: To study the influence of shock on muscle and plasma adenine nucleotide and creatine pools and their metabolites, and to identify early markers of cellular injury in shock . SETTING: Surgical research laboratory, Kuwait and UAE . DESIGN: Experimental study . MATERIAL: 19 New Zealand rabbits . INTERVENTIONS: 15 rabbits were injected with Escherichia coli endotoxin, and an additional 4 rabbits acted as controls . MAIN OUTCOME MEASURES: Blood and muscle energy metabolites, platelet count, arterial blood gas tensions, and arterial pressure were followed until the animals died . RESULTS: Five minutes after injection of endotoxin muscle ATP, creatine phosphate, and total adenine purine concentration decreased . This decrease was later reversed, but again decline to a critical level in the terminal phase . Loss of the muscle creatine pool indicated cellular damage after 3 hours . Plasma hypoxanthine, creatine, and lactate concentrations increased continuously throughout the study . CONCLUSION: Hypoxanthine formation is a possible source of oxygen free radicals in shock . The rise of hypoxanthine, creatine, and lactate concentrations in plasma during septic shock may reflect early high energy nucleotide failure, membrane injury, and anaerobic metabolism, respectively. Ostomy Wound Manage, 1995 Mar, 41(2), 36 - 40 Case study: abscess of the labia; Wood S et al.; A 68 year old female with no history of perianal abscess was examined in the Emergency Department of the hospital verbalizing complaints of swelling and tenderness in the left inguinal area . Physical examination revealed redness and swelling of the left labial area . The patient was admitted to the hospital and, following surgical incision and drainage by the physician, wound exploration revealed tunneling extending into the perirectal and vaginal areas . ET Nurse consultation was requested to establish a wound treatment regimen . The system of dressing used were a sterile, rayon/polyester dressing impregnated with 15 percent crystalline sodium chloride to cleanse the wound of slough and debris, in a ribbon form to facilitate packing of tunneling; a sterile 0.9 percent sodium chloride solution in gel form to protect the wound bed and keep it moist during granulation and reepithelialization; and an absorbent pad to collect drainage . This system of dressings addressed the patient's specific needs, was easy to use and proved easy to teach to a family member managing the patient's wound care at home . During the 10 1/2 weeks of treatment, wound healing progressed steadily, odor diminished rapidly and granulation of the wound bed progressed to healing with no maceration of the surrounding skin. Surg Endosc, 1995 Mar, 9(3), 341 - 3 Spilled gallstones--complications of abdominal-wall abscesses . Case report and review of the literature; Carlin CB et al.; Laparoscopic cholecystectomy has become the preferred method for removal of the diseased gallbladder . While its morbidity and mortality rates are lower than those of the open technique, it does have associated complications which may cause significant morbidity . The morbidity associated with spilled gallstones is not well studied and little can be found in the literature on this subject . We encountered a patient who developed abscesses within the abdominal wall following laparoscopic cholecystectomy . We recommend that spilled gallstones be removed when possible and that surgeons be aware of this possible complication. Plasmid, 1995 Mar, 33(2), 101 - 12 Competition between parental and recombinant plasmids affects the measure of recombination frequencies; Bierne H et al.; Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells") . Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid . Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements . To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid . We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid . The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling . The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells . Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells . This effect can be as high as several orders of magnitude. Mol Microbiol, 1995 Mar, 15(5), 883 - 93 Action of receiver and activator modules of UhpA in transcriptional control of the Escherichia coli sugar phosphate transport system; Webber CA et al.; Induction of the sugar-phosphate transport system in Escherichia coli by external glucose-6-phosphate is regulated by the UhpABC regulatory proteins . UhpA protein is required for uhpT transcription and is related to response regulators of two-component regulatory systems . UhpA and its homologues appear to be composed of two modules: the receiver module which contains the putative site of phosphorylation, and the activation module whose predicted helix-turn-helix motif is related to that present in many transcription activators . The roles of the two modules were examined by analysis of the regulatory consequences of uhpA deletion mutations generated by in vitro manipulations and missense mutations selected for independence from the requirement for UhpB kinase activity . Deletion of even seven amino acids from the C-terminus resulted in complete loss of transcription activation at the uhpT promoter . Overexpression of all C-terminal truncations that left intact the receiver module (residues 1-120) exhibited strong dominant-negative interference with a chromosomal uhpA+ allele . The genetic requirements for interference indicated that the overexpressed receiver module competed with intact UhpA for phosphate residues carried on UhpB . The site of phosphorylation of UhpA is not necessary for uhp activation by overexpressed UhpA but is necessary for UhpA action at normal levels of UhpA or for interference by the truncated species. Mech Ageing Dev, 1995 Mar 1, 78(2), 155 - 62 Elevated promotion of prostacyclin production by synthetic lipid A analogs in aged human endothelial cells in culture; Hasegawa N et al.; We examined the effects of E . coli lipopolysaccharide (LPS) and synthetic analogs of lipid A, a bioactive moiety of LPS, on the prostacyclin (PGI2) production by young and old human endothelial cells in vitro . PGI2 production by endothelial cells has been shown to decrease during in vitro cellular senescence as well as in vivo . LPS and all the analogs tested in this study did not stimulate PGI2 production by young endothelial cells more than twofold . However, LPS and the majority of the lipid A analogs examined stimulated the PGI2 production by old cells more than twofold (approximately two- to sixfold) . These results indicate that the responses to certain stimuli sometimes differ markedly between young and old cells, and this should be carefully considered when evaluating the biological effects of various compounds . Furthermore, these results suggest that certain synthetic lipid A analogs can be used as drugs to prevent some age-related vascular diseases. Jpn Heart J, 1995 Mar, 36(2), 127 - 77 Cyclic GMP as a second messenger in the cardiovascular system; Imai S; To delineate the importance of cyclic GMP (cGMP) as a second messenger for signal transduction within the cell, catalytic and regulatory features of the proteins involved in the synthesis and degradation of cGMP, i.e., guanylate cyclases and phosphodiesterases, are described . Characteristic features of cGMP-dependent protein kinases as intracellular receptors for cGMP and the contribution of this enzyme to the physiological functions of cGMP in the cardiovascular system are discussed. J Membr Biol, 1995 Mar, 144(1), 31 - 42 Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording; Cui C et al.; Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli . The pressure sensitivity of the protoplasts was studied . Two different unit conductance mechanosensitive channels, 1100 +/- 25 pS and 350 +/- 14 pS in 400 mM symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically . The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate- . Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient . Both of the channels were voltage sensitive . Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization . The mechanosensitive channels were reversibly blocked by gadolinium ion . Also they could reversibly be inhibited by protons . Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly . This indicates a potential modulation of these channels by KefA. Eur Cytokine Netw, 1995 Mar-Apr, 6(2), 109 - 12 Hemozoin differentially modulates the production of interleukin 6 and tumor necrosis factor in murine malaria; Prada J et al.; When murine peritoneal macrophages were loaded in vitro with Plasmodium vinckei hemozoin and stimulated for 24 hours with interferon-gamma and/or Escherichia coli lipopolysaccharide, the production of interleukin 6 (IL-6) was drastically reduced, whereas the secretion of tumor necrosis factor (TNF) was increased . In addition, non-radioactive in situ hybridizations in spleen sections of P . vinckei infected mice showed more TNF than IL-6 gene expression in the red pulp around hemozoin accumulation . These results provide evidence that IL-6 and TNF are differentially modulated by hemozoin and that subsequently, the secretion of IL-6 seems to be independent of the TNF production during murine malaria. Biochem Cell Biol, 1995 Mar-Apr, 73(3-4), 163 - 70 Enzymatic characterization of purified recombinant human renin; Pilote L et al.; Renin is a highly specific aspartyl protease of the renin-angiotensin system initially synthesized as preprorenin . Recombinant human prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line . The equivalent of 10-15 mg of recombinant human renin was secreted in the supernatant from each cell factory . Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity . This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I.mg protein-1.h-1 was used for kinetic analysis . The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1-Asn14, at their respective optimum pH of 5.5 and 6.8 . Although there was a six-fold increase in both Km and kcat values for the peptidic substrate (13.3 microM and 8.1 s-1, respectively), when compared with values for the natural substrate (2.04 microM and 1.41 s-1), the catalytic efficiency (0.69 microM-1.s-1) of the enzyme for both substrates was the same . However, the kcat/Km value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5 . The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin. Biochem Cell Biol, 1995 Mar-Apr, 73(3-4), 147 - 53 Physiological role of GlpB of anaerobic glycerol-3-phosphate dehydrogenase of Escherichia coli; Varga ME et al.; Anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli is encoded by an operon of three genes, glpACB . The promoter distal gene, glpB, encodes a 44-kilodalton polypeptide that is not part of the purified soluble dehydrogenase . By recombinant plasmid complementation, in a strain harboring a chromosomal deletion of glpACB, we found that all three genes were essential for anaerobic growth on glycerol-3-phosphate (G3P) . By isolation of inner membrane preparations we confirmed the cytoplasmic membrane localization of GlpB . GlpB displayed an electron paramagnetic resonance spectrum that suggested the presence of iron-sulfur center(s) within GlpB . We used this spectrum to show that the center(s) were reduced by the artificial reductant dithionite and by the physiological substrate G3P but not by lactate or formate . The center(s) were oxidized by fumarate . These data indicated that GlpB mediates electron transfer from the soluble GlpAC dimer to the terminal electron acceptor fumarate via the membrane-bound menaquinone pool. J Endod, 1995 Mar, 21(3), 128 - 30 Effects of lipopolysaccharides on human dental pulp cells; Nakane A et al.; Human dental pulp cells were treated with 1, 10, and 100 micrograms/ml of lipopolysaccharide (LPS) . The effects of treatment were examined by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells . LPS samples were purified from Porphyromonas gingivalis, Porphyromonas endodontalis, and Fusobacterium nucleatum isolated from root canals, and Escherichia coli 0111:B4 LPS was used as a positive control . At a concentration of 1 microgram/ml, none of the LPSs caused any change in the production of DNA or protein, whereas the amount of DNA was increased at 10 micrograms/ml and inhibited at 100 micrograms/ml . Protein synthesis was decreased by LPSs at both 10 and 100 micrograms/ml . Alkaline phosphatase activity was not changed at any concentration of LPS tested. Mol Microbiol, 1995 Mar, 15(6), 1017 - 29 Identification of an mRNA element promoting rate-limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme similar to RNase E; Fritsch J et al.; We have identified an mRNA element that is involved in the initial cleavage of the pufBALMX mRNA species in Rhodobacter capsulatus . This endoribonuclease recognition site, the first to be identified in a bacterial species other than Escherichia coli, shows strong similarities to mRNA sequences cleaved by the endoribonuclease E in E . coli . The presence of an RNase E-like enzyme in R . capsulatus is further supported by in vitro cleavage of E . coli transcripts by R . capsulatus extracts at sites attributed to RNase E and by the cross-reaction of a polypeptide from R . capsulatus with antisera against E . coli RNase E . Our data provide evidence that mRNAs are degraded in different bacterial species by enzymes with similar recognition sequences and activities . We present a model that attributes the segmental differences in stability of the polycistronic puf transcript to a specific distribution of mRNA decay-promoting and mRNA decay-impeding elements. Br J Pharmacol, 1995 Mar, 114(6), 1273 - 81 Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat; Wu CC et al.; 1 . We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock . 2 . Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite caused by LPS (1 microgram ml-1) . In contrast, pretreatment of macrophages with tetraethylammonium (10(-4) to 10(-2) M for 30 min), a non-selective inhibitor of potassium channels, did not affect the rise in nitrite caused by LPS . At the highest concentration (10(-5) M) used, cromakalim, an opener of ATP-sensitive potassium channels, caused a small, but significant inhibition of nitrite formation in macrophages activated with LPS, while lower concentrations (10(-7) to 3 x 10(-6) M) were without effect . 3 . The inhibition by glibenclamide (3 microM) of the increase in nitrite induced by LPS in J774.2 macrophages was weaker when glibenclamide was given several hours after LPS, indicating that glibenclamide inhibits the induction, but not the activity, of iNOS . In contrast, the degree of inhibition of nitrite formation caused by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was similar when this agent was given up to 10 h after LPS.(ABSTRACT TRUNCATED AT 250 WORDS) Vet Immunol Immunopathol, 1995 Mar, 45(1-2), 185 - 93 Characterization of ovine lentivirus envelope glycoprotein expressed in Escherichia coli cell and baculovirus systems; Kwang J et al.; The ovine lentivirus (OLV) envelope protein NH2- and COOH-terminal subunits gp70 and the NH2-terminal subunit gp40 were expressed in Escherichia coli cell . The entire gp70 envelope protein was also expressed in insect cells by the recombinant baculovirus . Guinea pigs were immunized with each bacterially expressed recombinant protein, and a serum neutralization assay was used to determine their capacity to neutralize OLV . These results showed that the major neutralization epitopes are located in the NH2-terminal half of the gp70 . The baculovirus expressed gp70 was found on the surface of insect cells and was immunobiologically active . Virus neutralization activity was also produced in sheep immunized with the baculovirus expressed recombinant protein. J Periodontal Res, 1995 Mar, 30(2), 116 - 23 Lipopolysaccharides from periodontal pathogens prime neutrophils for enhanced respiratory burst: differential effect of a synthetic lipid a precursor IVA (LA-14-PP); Aida Y et al.; When neutrophils are incubated with bacterial lipopolysaccharide (LPS), they become primed for enhanced release of superoxide anion (O2-) in response to stimulation by FMLP . We investigated the human neutrophil-priming activity of LPS from the periodontal pathogens, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Actinobacillus actinomycetemcomitans (Aa) in comparison with that of LPS from Escherichia coli (E . coli) . The optimum conditions for LPS to prime neutrophils were assessed for every LPS and found to be as follows: Neutrophils were incubated with LPS in the presence of 10% heat-inactivated plasma and 1 mM EDTA at 37 degrees C for 30 min and then stimulated with 1 microM FMLP at 37 degrees C for 7 min . Under these conditions, half-maximum priming was observed at 6.2 ng/ml Pg-LPS, 45 ng/ml Pi-LPS, 1.5 ng/ml Aa-LPS and 1.5 ng/ml E . coli-LPS . The priming activity of each LPS was neutralized by polymyxin B . Anti-CD14 monoclonal antibody inhibited priming by all LPS . The priming by Aa-LPS and E . coli-LPS was inhibited by LA-14-PP, a synthetic lipid A precursor IVA, but that by Pg-LPS and Pi-LPS was not . Priming by tumor necrosis factor alpha was not affected by polymyxin B, anti-CD14 antibody or LA-14-PP . Gelation of Limulus amebocyte lysate occurred at 10 pg/ml Pg-LPS, 30 pg/ml Pi-LPS, 3 pg/ml Aa-LPS and 3 pg/ml E . coli-LPS . Thus LPS from different periodontal pathogens primed neutrophils with different efficacy.(ABSTRACT TRUNCATED AT 250 WORDS) J Med Virol, 1995 Mar, 45(3), 273 - 81 Localization and reactivity of an immunodominant domain in the NS3 region of hepatitis C virus; Claeys H et al.; Analysis of the amino acid sequences of the nonstructural region 3 (NS3) of the hepatitis C virus type 1 revealed four points with a high average hydrophilicity (Ah) . Two of these potential antigenic sites were expressed in E . coli as short fragments . The first fragment of 91 residues (NS3f3: residues 1359-1449) harbors the hexapeptide K-K-K-C-D-E with an Ah of 2.33; the second fragment is 73 residues long (NS3f4: residues 1460-1532) and encompasses the heptapeptide R-S-N-R-R-G-R with an Ah of 1.79 . Both fragments were expressed with truncated hepatitis B core (tHBc) as a carrier protein . The fusion proteins were purified from the bacterial lysates by affinity chromatography on immobilized monoclonal antibodies against HBc, and evaluated as antigens in an enzyme immunoassay for the detection of HCV antibodies . In a specificity control panel, reactivity with NS3f3 was only found in proven HCV carriers, while reactivity with NS3f4 was weak in HCV carriers but accounted for some of the nonspecific serological reactions . In a group of 48 genotyped HCV-infected volunteer blood donors, antibodies against NS3f3 were detected in 90% (27/30) of HCV-type 1 infections and in all HCV-type 4 infections (5/5).(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Physiol, 1995 Mar, 105(3), 385 - 401 Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise; Andersen C et al.; LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes . The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar . The on and off reactions of sugar binding cause an increase of the noise of the current through the channel . The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose . The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site . The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose . The kinetics for sucrose movement was considerably slower . The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures . The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed. Eur J Immunol, 1995 Mar, 25(3), 847 - 51 Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity; Schultz U et al.; Crude preparations of chicken interferon (ChIFN) from various sources contain both antiviral and macrophage-activating factor (MAF) activity . Previous serological data indicated that unlike mammals, birds might express only a single type of IFN in response to viruses and mitogens that exhibits both activities . We have now expressed a complementary DNA for virus-induced ChIFN in transfected COS cells and in Escherichia coli . Purified recombinant ChIFN is a powerful antiviral agent and has high Mx promoter-inducing activity . However, as the sole agent, recombinant ChIFN lacks MAF activity: it does not induce the secretion of nitric oxide in primary monocyte-derived chicken macrophages . A neutralizing antiserum prepared against cloned ChIFN blocks most of the antiviral and Mx promoter-inducing activity present in preparations of natural ChIFN, but does not inhibit the MAF activity . These results demonstrate that chicken cells can be induced to secrete a novel cytokine which probably represents the avian homolog of mammalian IFN-gamma. Gut, 1995 Mar, 36(3), 401 - 6 Expression of binding of plasminogen, thrombospondin, vitronectin, and fibrinogen, and adhesive properties by Escherichia coli strains isolated from patients with colonic diseases; Shen W et al.; Escherichia coli strains isolated from patients with colonic disorders (n = 27) and strains isolated from the rectal mucosa of healthy subjects (n = 24) were compared with respect to expression of cell surface hydrophobicity, carriage of intestinal virulence factors, adhesion to tissue culture cells, and expression of binding of extracellular matrix proteins and plasma proteins . Strains isolated from patients with colonic disease did not express a more hydrophobic cell surface than strains from healthy subjects . Few strains from both groups carried genes encoding for recognised virulence factors of E coli . Only one strain, carrying the eae gene induced actin polymerisation in tissue culture cells . Strains from patients with colonic diseases adhered to HT29 cells, which are of intestinal origin, to a higher extent than E coli from healthy subjects . Significantly more strains from patients with colonic disorders than E coli from healthy subjects expressed binding of fibronectin, collagens, laminin, vitronectin, plasminogen, throbospondin, and fibrinogen . Expression of binding of these proteins may influence the pathogenesis of colonic disease by mediating binding to ulcerated tissue, preventing complement induced lysis of bacteria and by exerting proteolytic activity . There was no correlation between serotype, expression of cell surface hydrophobicity, and binding of extracellular matrix and plasma proteins. Genes Dev, 1995 Mar 1, 9(5), 626 - 37 Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA; Dammel CS et al.; A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA . RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures . Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant . The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation. Am J Physiol, 1995 Mar, 268(3 Pt 1), L501 - 8 Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators; Gutierrez HH et al.; Nitric oxide (.NO) is a short-lived mediator that can be induced by different cytokines and lipopolysaccharide (LPS) in a variety of cell types and produces many physiological and metabolic changes in target cells . In the current study, we show that a combination of cytokines, LPS, and zymosan-activated serum (ZAS; called for convenience cytomix Z) induces production of high concentrations of the NO oxidation products nitrite (NO2-) and nitrate (NO3-) by cultured rat fetal lung epithelial type II cells in a time-dependent fashion . Interferon-gamma and tumor necrosis factor-alpha alone did not significantly affect .NO synthesis, whereas ZAS, LPS, and interleukin-1 beta caused only a modest increase in formation of .NO oxidation products . Production of NO2- and NO3- was inhibited by NG-monomethyl-L-arginine and cyclohexmide . After exposure of these cells to a combination of the above cytokines, Escherichia coli LPS, and ZAS (cytomix Z), enhanced inducible nitric oxide synthase (iNOS) expression was indicated by an elevation in steady-state mRNA specific for iNOS (via Northern blot analysis) and increased immunofluorescence for iNOS after cell permeabilization, incubation with anti-iNOS antibody, and treatment with Cy3.18-conjugated rabbit-specific antibody . The extent of inflammatory mediator-induced.NO production by alveolar epithelium, which exceeds that of other lung cell types, reveals new insight into mechanisms of pulmonary host defense and pathways of free radical-mediated lung injury. J Gen Virol, 1995 Mar, 76 ( Pt 3), 519 - 27 Antibodies to parvovirus B19 NS-1 protein in infected individuals; von Poblotzki A et al.; Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease) . The infection is usually benign and self-limiting, but in adults cases of severe arthritis which may persist for years have been reported . Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection . The immune response against the third major protein, the non-structural protein NS-1, of parvovirus B19 has not been characterized so far . We cloned and expressed the full-length NS-1 protein and fragments thereof in Escherichia coli . The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection . Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA . Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1 . The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein. J Pharmacol Exp Ther, 1995 Mar, 272(3), 1141 - 50 Nitric oxide synthase inhibitor and lipopolysaccharide effects on reactivity of guinea pig airways; Fedan JS et al.; The in vivo and in vitro effects of nitric oxide (NO) synthase inhibitors and lipopolysaccharide (LPS) on reactivity of guinea pig airways were examined . In isolated, perfused tracheas from untreated animals, the NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME; 10(-4)M), NG-methyl-L-arginine (L-NMMA; 10(-4) M) and aminoguanidine (10(-4) M) had no effect or inhibited reactivity to extraluminally (EL) or intraluminally (IL) applied methacholine and histamine . L-NMMA (10(-4) M) did not appreciably contract resting or metacholine-contracted preparations (+/- 3 x 10(-4) M L-arginine) and L-arginine only weakly relaxed contracted tracheas (+/- L-NMMA) . Sodium nitroprusside and S-nitroso-N-penicillamine elicited relaxant responses and were more potent extraluminally than intraluminally . Methylene blue (10(-5) M) antagonized relaxation to sodium nitroprusside . Incubation with Escherichia coli LPS (10 micrograms/ml; 30 min incubation) alone in the EL and IL baths depressed methacholine and histamine concentration-response curves . In the presence of LPS, L-NAME potentiated responses to intraluminally applied methacholine but did not affect responses to extraluminally added methacholine . Four days after i.p . injection of animals with LPS (4 mg/kg), L-NAME potentiated responses to IL methacholine, and L-arginine acquired greater relaxant activity . LPS injection increased sensitivity to intraluminally added but not extraluminally added isoproterenol . LPS given by i.p . injection or by inhalation did not affect basal specific airway resistance of conscious animals or reactivity to methacholine aerosol during a postexposure period of 6 to 72 h . NO seems to have little role in regulating reactivity of guinea pig airways to bronchoconstrictor agonists, except after in vitro or in vivo exposure to LPS . After LPS injection the in vitro changes suggestive of NO synthase induction are not associated with altered airway reactivity to inhaled methacholine. Mutat Res, 1995 Mar, 327(1-2), 113 - 20 Mutations induced by dacarbazine activated with cytochrome P-450; Mudipalli A et al.; The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system . In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC . The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min . SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria . Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH . Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%) . Among the substitutions, transversions and transitions were in the ratio of 1:0.7 . Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots. Infect Immun, 1995 Mar, 63(3), 794 - 8 Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages; Nunoshiba T et al.; Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages . Pure NO . gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion . The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E . coli from the cytotoxicity of NO.-generating macrophages . Previous experiments involved exposing E . coli to a bolus of NO . rather than the steadily generated gas expected of activated macrophages . We show here detectable induction of soxS transcription by NO . delivered at rates as low as 25 microM/h . Maximal induction was observed at 25 microM NO . per h under anaerobic conditions but at 125 microM/h aerobically . After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure . This in vivo induction was almost completely eliminated by the NO . synthase inhibitor NG-monomethyl-L-arginine . The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E . coli after phagocytosis, which suggests that induction of the soxRS regulon by NO . can counteract most of the cytotoxic effects of NO . production by the macrophages . We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages. RNA, 1995 Mar, 1(1), 89 - 94 A mutation at the universally conserved position 529 in Escherichia coli 16S rRNA creates a functional but highly error prone ribosome; Santer UV et al.; A base substitution of G to U was constructed at position 529 in Escherichia coli 16S rRNA . The U529 mutant ribosomes were functional and present on polysomes but were highly error prone and caused a progressive loss of cell viability . They displayed elevated levels of readthrough of stop codons and frameshifting, and an increase in thermal sensitivity of beta-galactosidase, suggestive of missense errors . These results demonstrate that the university conserved G529 is involved in tRNA selection at the A site during protein synthesis. RNA, 1995 Mar, 1(1), 79 - 88 Translational control of maturation-protein synthesis in phage MS2: a role for the kinetics of RNA folding? Groeneveld H, Thimon K, van Duin J. The gene for the maturation (A) protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt . Secondary structure of the leader was deduced by phylogenetic comparison and by probing with enzymes and chemicals . The RNA folds into a cloverleaf, i.e., three stem-loop structures enclosed by a long-distance interaction (LDI) . This LDI is essential for translational control . Its 3'moiety contains the Shine-Dalgarno region of the A-protein gene, whereas its complement is located 80 nt upstream, i.e., about 30 nt from the 5'-terminus of the RNA chain . Mutational analysis shows that this base pairing represses expression of the A-protein gene . We present a model in which translational starts can only take place on nonequilibrated RNA, in which base pairing between the complementary regions has not yet taken place . We suggest that this pairing is kinetically delayed by the intervening sequence, which contains the three hairpins of the cloverleaf . The model is mainly based on the observation that reducing the length of the intervening sequence reduces expression, whereas increasing the length has the opposite effect . In addition, further stabilization of the LDI by a stronger base pair does not lead to a decrease in A-protein synthesis . Such a decrease is predicted to occur if translation would be controlled by the equilibrium structure of the leader RNA . These and other observations fit a kinetic model of translational control by RNA folding. Protein Eng, 1995 Mar, 8(3), 283 - 91 Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity; Wolf E et al.; A mutant of bovine pancreatic DNase I containing two additional residues in a loop next to C173 has been expressed in Escherichia coli, purified and characterized biochemically . Modelling studies suggest that the inserted arginine and glutamate side chains of the modified loop sequence C173-R-E-G-T-V176 could contact the bases 3' to the cleaved bond in the major groove of a bound DNA, and that up to 10 bp could interact with the enzyme and potentially influence its cutting rate . The loop insertion mutant has an 800-fold lower specific activity than wild-type and shows overall cleavage characteristics similar to bovine pancreatic DNase I . Compared with the wild-type enzyme, the mutant shows a strongly enhanced preference for cutting the inverted repeat: (formula: see text) or close variants thereof . Unexpectedly for a minor groove binding protein, the preferred cutting sites in opposite strands are staggered by 1 bp in the 5' direction, causing the cleavage of a TA and a TT step, respectively . This finding demonstrates that the sequence context is relatively more important for the cutting frequency than the nature of the dinucleotide step of the cleaved bond, and clearly shows that base recognition is involved in determining the sequence selectivity of the mutant . The importance of the sequence 5' to the cleaved bond for the cutting rate suggests that the additional major groove contacts may require a distortion of the DNA associated with a higher energy barrier, resulting in an increased selectivity for flexible DNA sequences and a lower overall activity of the mutant enzyme. Protein Eng, 1995 Mar, 8(3), 275 - 82 Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity; Sloane DL et al.; Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes . By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified . Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase . An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase . Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculo-virus-insect cell culture system . This variant was purified to > 90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase . The variant enzyme has no change in its apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax . For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, as expected for an enzyme performing predominantly 12-lipoxygenation . The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme . Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation. Protein Eng, 1995 Mar, 8(3), 249 - 59 Second-generation octarellins: two new de novo (beta/alpha)8 polypeptides designed for investigating the influence of beta-residue packing on the alpha/beta-barrel structure stability; Houbrechts A et al.; The sequence of octarellin I, the first de novo (beta/alpha)8 polypeptide, was revised according to several criteria, among others the symmetry of the sequence, beta-residue volume and hydrophobicity, and charge distribution . These considerations and the overall conclusions drawn from the first design led to two new sequences, corresponding to octarellins II and III . Octarellin II retains perfect 8-fold symmetry . Octarellin III has the same sequence as octarellin II, except for the beta-strands which exhibit a 4-fold symmetry . The two proteins were produced in Escherichia coli . Infrared and CD spectral analyses of octarellins II and III reveal a high secondary structure content . Non-denaturing gel electrophoresis, molecular sieve chromatography and analytical ultracentrifugation suggest that both of these second-generation artificial polypeptides exist as a mixture of a monomer and a dimer form . Octarellins II and III are at least 10 times more soluble than octarellin I . Urea-induced unfolding followed by fluorescence emission suggests that the tryptophan residues, designed to be buried in the (beta/alpha)8, are indeed packed in the hydrophobic core of both proteins . However, octarellin III displays a higher stability towards urea denaturation, indicating that introducing 4-fold symmetry into the beta-barrel might be important for stability of the overall folding. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1739 - 43 Translocation of the Escherichia coli transcription complex observed in the registers 11 to 20: "jumping" of RNA polymerase and asymmetric expansion and contraction of the "transcription bubble"; Zaychikov E et al.; Translocation of DNA-dependent RNA polymerase along the DNA template during RNA synthesis encompasses continuous as well as discontinuous steps . This is demonstrated by chemical probing of transcription complexes stalled in consecutive registers of RNA synthesis at base positions +11, +12, +14, +16, +18, and +20 . The "transcription bubble" translocates by continuous opening of the downstream edge in tandem with the growing RNA chain and discontinuous closing at the upstream edge after at least nine steps of RNA synthesis . The position of the enzyme remains unchanged during extension of the transcription bubble and "jumps" 10 bp downstream simultaneously with collapse of the transcription bubble. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1699 - 703 Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor; Dahlman-Wright K et al.; A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity . The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro . This report describes a structural characterization of the tau 1 core peptide fragment . Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions . The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation . Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character . Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1644 - 8 Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants; Douce G et al.; A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed . By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine . This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin . In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin . Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1639 - 43 Two short autoepitopes on the nuclear dot antigen are similar to epitopes encoded by the Epstein-Barr virus; Xie K et al.; To understand the relationship between antibodies present in patients with anti-nuclear dot (ND) autoimmune disease and the proteins they recognize, epitopes that react with the autoantibodies were mapped . A panel of fusion proteins containing different portions of the ND protein were overproduced in Escherichia coli . Immunoblot analysis with anti-ND antibodies revealed that most (10 of 12) sera recognize two major autoepitopes that are each a maximum of 8 amino acids long . The other two sera recognize one of the two epitopes . In addition to the short linear autoepitopes, a conformational epitope appears to be present on the ND antigen . Each of the two linear epitope sequences shares sequence similarities with those of several viral proteins found in the databases . Furthermore, two fusion proteins containing short Epstein-Barr virus (EBV) protein sequences that are similar to the ND epitopes were recognized by the human autoimmune sera, indicating that the autoepitopes are present in EBV protein sequences . Our results are consistent with the hypothesis that ND autoimmune disease might be associated with EBV infections. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1352 - 6 The dif resolvase locus of the Escherichia coli chromosome can be replaced by a 33-bp sequence, but function depends on location; Tecklenburg M et al.; The dif locus (deletion-induced filamentation) of Escherichia coli is a resolvase site, located in the terminus region of the chromosome, that reduces chromosome multimers to monomers . In strains in which this site has been deleted, a fraction of the cells is filamentous, has abnormal nucleoid structure, and exhibits elevated levels of the SOS repair system . We have demonstrated that a 33-bp sequence, which is sufficient for RecA-independent recombination and which shows similarity to the cer site of pColE1, suppresses the Dif phenotype when inserted in the terminus region . Flanking sequences were not required, since suppression occurred in strains in which dif as well as 12 kb or 173 kb of DNA had been deleted . However, location was important, and insertions at a site 118 kb away from the normal site did not suppress the Dif phenotype . These sites were otherwise still functional, and they exhibited wild-type levels of RecA-independent recombination with dif-containing plasmids and recombined with other chromosomal dif sites to cause deletions and inversions . It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region. Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1337 - 41 Leukemia inhibitory factor protects against experimental lethal Escherichia coli septic shock in mice; Waring PM et al.; Leukemia inhibitory factor (LIF) has recently been associated with septic shock in humans . In this study we sought to determine, in mice, the role of LIF in septic shock . During sublethal endotoxemia, serum LIF levels, as determined by radio-receptor competition assay, peaked at 2 h and were low (3 ng/ml), whereas in lethal Escherichia coli septic shock serum LIF levels rose progressively (> 30 ng/ml) in the premorbid phase coincident with the development of tissue injury . Single i.v . injections of high doses (up to 50 micrograms per mouse) of recombinant murine LIF had no obvious acute detrimental effects, whereas continued i.p . administration (30 micrograms per mouse per day) for 3-4 days induced a fatal catabolic state without evidence of preceding hemodynamic collapse or shock . Simultaneous or subsequent administration of high doses of LIF had no effect on mortality from sublethal and lethal E . coli septic shock, whereas prior administration conferred significant protection against lethality (P << 0.001 by log-rank test), an effect that was dose and interval dependent . This protective effect resembled endotoxin tolerance and was characterized by suppression of E . coli-induced serum tumor necrosis factor concentration (P < 0.05), reduction in the number of viable bacteria (P < 0.05), and prevention of sepsis-induced tissue injury . These observations suggest that systemic LIF production is part of the host response to both endotoxin and sepsis-induced tissue injury. Biochemistry, 1995 Feb 28, 34(8), 2710 - 23 Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications; Reyes VM et al.; Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A . All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit {Bolin, J . T., Filman, D . J., Matthews, D . A., Hamlin, R . C., & Kraut, J . (1982) J . Biol . Chem . 257, 13650-13662} . A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule . There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme {Davies, J . F., II, Delcamp, T . J., Prendergast, N . J., Ashford, V . A., Freisheim, J . H., & Kraut, J . (1990) Biochemistry 29, 9467-9479}, does not favor protonation at N8 . Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction . When the ecDHFR.NADPH complex (space group P3221; M . R . Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer {Wu, Y . D., & Houk, K . N . (1987) J . Am . Chem . Soc . 109, 906-908, 2226-2227} . In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation . This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds . Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2701 - 9 Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli; Parry L et al.; A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli . Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl beta-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer . The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to N1-acetylspermidine, much of which was excreted . Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer . The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine . These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted . Cells expressing large amounts of SSAT were much more sensitive to the growth inhibitory action of the antitumor agent N1,N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions . SSAT was readily purified to homogeneity from extracts of DH5 alpha cells containing pINSAT2 . The purified enzyme had a similar specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2610 - 20 Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS; Resing KA et al.; MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos . The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously {Alessi, D . R., et al . (1994) EMBO J . 13, 1610-1619} . Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218 . Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218 . Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos . Major autophosphorylation sites were residues S298 and Y300 . Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25 . Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase . Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300 . Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle . The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions. Biochemistry, 1995 Feb 28, 34(8), 2592 - 8 Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis; Farres J et al.; To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase . The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable . The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities . To test for the role of cysteine 302 as a nucleophile, the residue was mutated to a serine, a poor nucleophile . this C302S mutant was active but was a much poorer catalyst, with a kcat/Km value 7 x 10(5) times lower than that of the recombinant native enzyme . Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to be the rate-limiting step . Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase. Biochemistry, 1995 Feb 28, 34(8), 2553 - 9 Expression of bovine heart fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis; Abe Y et al.; Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli . In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis . The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme . Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFru6P, a 36% increase in Vmax, and a 2-fold increase in Kcat/Km of the kinase . There was no change in the kinetic properties of the phosphatase activity . Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFru6P, a 2.5-fold increase in Vmax, a 12-fold increase in kcat/Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase . Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFru6P and activation of the kinase without affecting the phosphatase activity . Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid . The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms . Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea . All phosphorylated enzymes were more susceptible to urea inactivation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2537 - 44 Endonuclease III interactions with DNA substrates . 2 . The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching; Xing D et al.; We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs . Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan {Kuo, C.-F., McRee, D . E., Fisher, C . L., O'Handley, S . F., & Cunningham, R . P . (1992) Science 258, 434-440} . The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment . Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher . Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT) . The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT) . This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA . A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2528 - 36 Endonuclease III interactions with DNA substrates . 1 . Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site; O'Handley S et al.; The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays . Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the beta-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate . The Kobs for a 13-mer carrying a centrally located reduced AP site is (2 x 10(6)-(2 x 10(7) M-1, while the Kobs for a 13-mer with no damage is (4.5 x 10(3)-(3.2 x 10(4) M-1 (approximately a 500-fold difference) . Larger oligonucleotides would not enter a gel when endonuclease III was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly . Competition assays suggest that the Kobs measured for both damaged and undamaged 13-mers is a minimum value and that the Kobs for larger oligonucleotides could be an order of magnitude greater . Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10(7) M-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10(5) M-1 {Xing, D., Dorr, R., Cunningham, R . P., & Scholes, C . P . (1995) Biochemistry 34, 2537-2544} . Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2480 - 8 Trichodiene synthase . Identification of active site residues by site-directed mutagenesis; Cane DE et al.; Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with {methyl-14C}methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site . The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis . The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive . A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F . sporotrichioides enzyme . From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified . Three of these mutants were overexpressed and purified to homogeneity . The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat . In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204 . Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca . 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2471 - 9 Trichodiene synthase . Substrate specificity and inhibition; Cane DE et al.; The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes . A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase . 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70. Biochemistry, 1995 Feb 28, 34(8), 2447 - 54 Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase; Adams JA et al.; Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase . This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity . We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities . The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase . The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme . Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities . This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase {DeBont, H . L., et al . (1993) Nature 363, 595-602} and MAP kinase {Zhang, F., et al . (1994) Nature 367, 704-711} . The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically . A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins . These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Feb 28, 34(8), 2393 - 9 Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419; Parast CV et al.; Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate . Active enzyme contains a glycyl radical whose alpha-hydrogen undergoes rapid exchange with solvent (t1/2 approximately 5 min at 0 degree C) . We have investigated this exchange using site-directed mutagenesis and mechanism-based inactivation . Mutation of the active-site cysteine 419 into a serine, which renders the enzyme catalytically inactive, abolishes alpha-hydrogen exchange in the radical . This suggests that the exchange process is not an intrinsic property of the glycyl radical but is a consequence of its interaction with cysteine 419 . This residue is also demonstrated to be involved in the transfer of the radical to acetylphosphinate, a mechanism-based inactivator of the enzyme . In contrast, mutation of the other essential cysteine 418 to a serine has no effect on the hydrogen exchange or the transfer of the radical to acetylphosphinate . A mechanism for the hydrogen exchange catalyzed by cysteine 419 consistent with a redox role for this residue in the normal catalytic reaction is proposed. Biochemistry, 1995 Feb 28, 34(8), 2441 - 6 Stereochemistry of tRNA(m5U54)-methyltransferase catalysis: 19F NMR spectroscopy of an enzyme-FUraRNA covalent complex; Kealey JT et al.; The catalytic mechanism of tRNA(m5U54)-methyltransferase (RUMT) involves the formation of a covalent adduct between Cys324 of RUMT and C6 of Ura54 in tRNA . The covalent adduct is subsequently methylated at C5 by S-adenosyl-L-methionine (AdoMet) . We used an RNA substrate analog containing 5-fluorouracil (FUra) in place of Ura54 to trap the covalent complex and analyzed the adduct by 19F NMR spectroscopy . The 19F NMR spectrum of the adduct consisted of an overlapping doublet of quartets, with an H6-F coupling constant of 4 Hz and a CH3-F coupling constant of 22.4 Hz . On the basis of the magnitude of the H6-F coupling constant, we determined that Cys324 of RUMT and the methyl moiety from AdoMet added across the 5,6-double bond of FUra54 in cis fashion . We deduced that the nucleophilic addition was also cis in the normal enzymatic reaction and that the subsequent beta-elimination of the 5-H and catalytic cysteine was trans . Further, on the basis of chemical considerations, we proposed several conformational adaptations of enzyme-substrate complexes that must occur on the reaction pathway . Together with previous studies, this study enables the proposal of the complete stereochemical pathway for the RUMT-catalyzed methylation of Ura54 in tRNA. FEBS Lett, 1995 Feb 27, 360(2), 125 - 31 Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli; Shindo H et al.; The three-dimensional structure of the C-terminal domain (47 residues) obtained from the hydrolysis of H-NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations . It is composed of an antiparallel beta-sheet, an alpha-helix and a 3(10)-helix which form a hydrophobic core, stabilizing the whole structure . This domain has been found to bind to DNA . Possible DNA binding sites are discussed on the basis of the solution structure of the C-terminal domain of H-NS. Gene, 1995 Feb 27, 154(1), 7 - 14 A new dhfrVIII trimethoprim-resistance gene, flanked by IS26, whose product is remote from other dihydrofolate reductases in parsimony analysis; Sundstrom L et al.; A new plasmid-borne gene, dhfrVIII, encoding high-level trimethoprim resistance (TpR) was found in an intestinal Escherichia coli . It seems to be a widely occurring mediator of TpR . Among 973 examined TpR E . coli, the new resistance gene was found in 13 (1.3%) isolates from Sweden, Finland and Nigeria . The new gene was sequenced and found to code for a dihydrofolate reductase (DHFR) of 169 amino acids (M(r) 19005) . The dhfrVIII gene on the studied plasmid pLMO226 was observed to be flanked by IS26 elements . The dhfrVIII gene and a 3' unidentified open reading frame (ORF) seem to be borne on a compound transposon with IS26 at its ends, since the configuration of two IS26 flanking dhfrVIII and the unidentified ORF was conserved among the isolates that were probe-positive for the gene . Phylogeny parsimony analysis showed the dhfrVIII-encoded enzyme to be only remotely related to other known plasmid-mediated, drug-resistant DHFR . Only a few of the latter form well-supported monophyletic groups. Gene, 1995 Feb 27, 154(1), 47 - 50 Transcriptional activation of the origin of coliphage lambda DNA replication is regulated by the host DnaA initiator function; Wegrzyn G et al.; The initiator of phage lambda DNA replication, the lambda O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli . Both specifically recognize their origins of replication, ori lambda and oriC, respectively, and organize the assembly of specific replication complexes . However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC . Here, we demonstrate that lambda plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein . Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at ori lambda . DnaA may act by binding to DnaA-boxes, situated around the lambda pR promoter; there are no such sequences in ori lambda . Our results being to explain in molecular terms why lambda plasmid replication is DnaA-dependent {Kur et al., J . Mol . Biol . 198 (1987) 203-210} and why the initiation of phage lambda DNA replication is blocked (in E . coli devoid of prophage Rac) after inactivation of DnaA {Wegrzyn et al., Genetics (1995) in press}. Gene, 1995 Feb 27, 154(1), 31 - 7 A fruiting body-specific cDNA, mfbAc, from the mushroom Lentinus edodes encodes a high-molecular-weight cell-adhesion protein containing an Arg-Gly-Asp motif; Kondoh O et al.; A cDNA clone (designated mfbAc), encoding 2157 amino acids (aa), was isolated from a mature fruiting-body cDNA library of the edible mushroom Lentinus edodes . The mfbA transcript was abundant in mature fruiting bodies, detectable in immature fruiting bodies but absent in earlier developmental stages and in the vegetative mycelium . Although more abundant in the pileus than the stipe, only low levels were found in the gill tissue . The deduced MFBA protein (234.5 kDa) contained a cell-surface attachment-promoting Arg-Gly-Asp (RGD) motif . MFBA was produced in Escherichia coli using a maltose-binding protein (MBP) fusion vector, but it was cleaved into four fragments even in a protease-deficient host . A 425-aa MFBA peptide containing the RGD motif (named MFBA(582-1006) peptide) was successfully produced using the phage T7 expression system . This MFBA(582-1006) peptide exhibited a cell adhesion and spreading activity toward mammalian cells . This activity of the MFBA fragment was competitively inhibited by the Gly-Arg-Gly-Asp-Ser-Pro peptide but not by the Gly-Arg-Gly-Glu-Ser-Pro peptide, showing that the RGD motif of MFBA is essential for the cell-binding activity. Biochem Biophys Res Commun, 1995 Feb 27, 207(3), 927 - 32 Creation of a high cytotoxic active human tumor necrosis factor having the truncated and more basic amino terminus; Guo D et al.; In order to define the structure-functional relationship of tumor necrosis factor(TNF), a mutant TNF gene was created by site-specific mutagenesis based on the PCR technique . This gene was highly expressed in E.coli cells . The amount of the recombinant protein was up to about 80% of the total cellular proteins . Through one-step ion exchange chromatography, the mutant TNF could be purified to homogeneity . This mutein showed the molecular weight of a dimer but not a trimer . It bears the features of truncated amino terminus and increase of the basicity of amino terminal residues . Compared with the wild type TNF, the specific activity of mutant TNF was increased by fourfold. Gene, 1995 Feb 27, 154(1), 93 - 8 Transcriptional regulation in the Chlamydia trachomatis pCT plasmid; Ricci S et al.; We have analyzed transcriptional regulation of the chlamydial plasmid pCT . Transcription of a full-length 2.9-kb ORF1-ORF2 mRNA is likely to be regulated by the sigma 66 transcription factor which recognizes the TATAAT and TNGNCA sequences at the -10 and -35 DNA regions, respectively . RNA synthesis starts 39 nucleotides (nt) upstream from the ATG start codon of ORF1 and terminates within the downstream ORF3 DNA region . A 2.8-kb transcript transverses the ORF3-6 DNA region, while two transcripts of 2.2 and 1.9 kb cover the ORF4-6 DNA region . These mRNAs overlap two abundant transcripts which regulate the expression of the ORF3 and ORF4 genes . The accumulation of transcripts associated with these ORFs is likely to be regulated at the level of RNA synthesis by an unknown sigma factor which could select the RTTTAAA and TTYTTR sequences located at the -10 and -35 DNA regions, respectively . This new promoter consensus sequence could be unique to the gene expression machinery of Chlamydiae. Nucleic Acids Res, 1995 Feb 25, 23(4), 701 - 7 Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin; Conley DL et al.; Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells . We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication . Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA . A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity . Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons . Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function . Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA. Nucleic Acids Res, 1995 Feb 25, 23(4), 670 - 4 The construction and analysis of M13 libraries prepared from YAC DNA; Vaudin M et al.; Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive . We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA . Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA . We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones. Nucleic Acids Res, 1995 Feb 25, 23(4), 599 - 605 Role of CRP in transcription activation at Escherichia coli lac promoter: CRP is dispensable after the formation of open complex; Tagami H et al.; The role of cAMP receptor protein (CRP) in transcription activation at the Escherichia coli lac promoter was investigated focusing on the steps after the formation of open complex . Although CRP binding to the lac DNA is stabilized in the ternary open complex, a high concentration of heparin dissociates CRP from the open complex without affecting the interaction between RNA polymerase and promoter, resulting in a binary complex . The release of CRP is directly shown by Western blotting and DNase I footprinting . The binary complex exhibits a slightly increased gel mobility compared to the ternary complex . The binary complex retains the characteristics of the open complex in footprinting pattern which is essentially identical with that of the open complex of the lac UV5 promoter . The binary complex is competent for transcription . These results indicate that CRP is not necessary for the maintenance of active open complex . In addition, the removal of CRP does not increase the production of abortive RNAs . We conclude that the contact between CRP and RNA polymerase is not essential for transcription activation after the formation of the open complex at the lac promoter . In other words, the role of CRP in the lac promoter is restricted to the steps up to the formation of open complex. Nucleic Acids Res, 1995 Feb 25, 23(4), 683 - 8 Decoding with the A:I wobble pair is inefficient; Curran JF; tRNAs with inosine (I) in the first position read three codons ending in U, C and A . However, A-ending codons read with I are rarely used . In Escherichia coli, CGA/U/C are all read solely by tRNAICGArg . CGU and CGC are very common codons, but CGA is very rare . Three independent in vivo assays show that translation of CGA is relatively inefficient . In the first, nine tandem CGA cause a strong rho-mediated polar effect on expression of a lacZ reporter gene . The inhibition is made more extreme by a mutation in ribosomal protein S12 (rpsL), which indicates that ribosomal binding by tRNAICGArg is slow and/or unstable in the CGA cluster . The second assay, in which codons are substituted for the regulatory UGA of the RF2 frameshift, confirms that aa-tRNA selection is slow and/or unstable at CGA . In the third assay, CGA is found to be a poor 5' context for amber suppression, which suggests that an A:I base pair in the P site can interfere with translation of a codon in the A site . Two possible errors, frameshifting and premature termination by RF2, are not significant causes for inefficiency at CGA . It is concluded that the A:I pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare usage of codons read with the A:I base pair. Nucleic Acids Res, 1995 Feb 25, 23(4), 571 - 9 Bulged-out nucleotides protect an antisense RNA from RNase III cleavage; Hjalt TA et al.; Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs . We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides . Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides . Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced . The set of mutant and wild-type CopA variants was characterized structurally in vitro . The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III . In vitro cleavage rates were drastically increased when either the lower or both bulges were absent . This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo . The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed . In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo. J Mol Biol, 1995 Feb 24, 246(3), 388 - 400 Partition functions of mini-F affect plasmid DNA topology in Escherichia coli; Biek DP et al.; Efficient segregation of the low copy number plasmid mini-F is dependent on partition functions encoded by the plasmid sopABC genes . The sop region encodes proteins SopA and SopB and a cis-acting element, sopC, which may function as a centromere analog . The SopC segment contains 12 imperfect 43 bp repeats to which the SopB protein binds . We have found that mutations in the sop genes affect superhelicity of isolated plasmid DNA . Plasmids with mutations in sopB or a deletion of the sopC segment were more highly negatively supercoiled than normal . In contrast, a mutation in the autoregulatory SopA protein resulted in plasmid DNA that was more relaxed . The SopAB proteins provided in trans to a pBR322 plasmid carrying sopC resulted in the relaxation of negative supercoils . We suggest that binding of SopB protein to the cis-acting sopC segment in vivo, alone or in conjunction with other proteins, produced a change in DNA topology in which positive superhelical turns were introduced locally . This higher-order nucleoprotein structure may allow interaction of plasmid mini-F with the partition apparatus. J Mol Biol, 1995 Feb 24, 246(3), 374 - 81 Expression, purification and crystallisation of phosphorylase kinase catalytic domain; Owen DJ et al.; The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain . The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9) . A three-fold excess of magnesium to ATP was used for crystal growth . The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree . The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit . Data to 3 A resolution have been collected and structure determination is in progress. J Biol Chem, 1995 Feb 24, 270(8), 4076 - 87 Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli; Gary JD et al.; We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides . We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol . From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene . Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes . Using an E . coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity . We also examined the phenotype of two E . coli strains where this isoaspartyl dipeptidase gene was deleted . We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins . We found that the iadA deletion does not result in reduced stationary phase or heat shock survival . Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000 . The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells. J Biol Chem, 1995 Feb 24, 270(8), 4023 - 30 Characterization of the COOH terminus of non-muscle caldesmon mutants lacking mitosis-specific phosphorylation sites; Yamashiro S et al.; Phosphorylation of rat non-muscle caldesmon by cdc2 kinase causes reduction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomyosin ATPase . We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, because the COOH-terminal half of caldesmon contains all 7 putative Ser or Thr sites for cdc2 kinase . Codons for the 7 putative Ser or Thr residues have been mutated to Ala, and resultant mutants were bacterially expressed . Analyses of the phosphopeptide maps of these mutants have identified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method . Actin binding experiments have suggested that 5 sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are important for the pho