Escherichia coli

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 phosphorylation-dependent reduction in actin binding . Characterization of a mutant lacking all 7 Ser or Thr sites (7-fold mutant) has revealed that 7-fold mutation eliminates all phosphorylation sites by cdc2 kinase . While the in vitro properties of the 7-fold mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase, are very similar to those of nonmutated protein, such properties are not affected by the treatment with cdc2 kinase in contrast to nonmutated protein . This mutant should thus be useful to explore the functions of the mitosis-specific phosphorylation of caldesmon.

J Biol Chem, 1995 Feb 24, 270(8), 3926 - 31
Subunit interactions in dimeric kinesin heavy chain derivatives that lack the kinesin rod; Young EC et al.; The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular "heads"; the heads are attached to a "rod" domain which is a two-stranded alpha-helical coiled-coil . Interaction between the heads is thought to be important to kinesin function . The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T.-G., Suhan, J., and Hackney, D . D . (1994) J . Biol . Chem . 269, 16502-16507) . However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear . To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, A., van Dyke, M., and Stock, J . (1991) Science 252, 1162-1164) to lack the rod . Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a 2-residue linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment . Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility . Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and K448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers . Derivative self-association is non-covalent but extremely stable with an association constant > or = 2 x 10(8) M-1 . Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.

J Biol Chem, 1995 Feb 24, 270(8), 3816 - 22
MH1, a second-site revertant of an Escherichia coli mutant lacking Na+/H+ antiporters (delta nhaA delta nhaB), regains Na+ resistance and a capacity to excrete Na+ in a delta microH(+)-independent fashion; Harel-Bronstein M et al.; The Escherichia coli mutant delta nhaA delta nhaB (EP432), which lacks the two specific Na+/H+ antiporter genes, is incapable of efficiently excreting Na+ . Accordingly at low K+ (6 mM) medium, its intracellular Na+ concentration is only slightly lower (1.5-2x) than the extracellular concentration (50 mM), explaining the high sensitivity to Na+ (> or = 30 mM) of the mutant . This Na+ sensitivity is shown to be a powerful selection for spontaneous second-site suppressor mutations that allow growth on high Na+ (< or = 0.6 M) with a rate similar to that of the wild type . One such mutation, MH1, maps at 25.7 min on the E . coli chromosome . It confers Na+ but not Li+ resistance upon delta nhaA delta nhaB cells and exposes a Na(+)-excreting capacity, maintaining a Na+ gradient of about 8-10 (at 50 mM extracellular Na+), which is similar to that of the wild type . Although lower, Na+ excretion capacity is also observed in the delta nhaA delta nhaB mutant when grown in medium containing higher K+ (70 mM) . This capacity is accompanied with a shift in the sensitivity of the mutant to higher Na+ concentrations (> or = 300 mM) . Whereas Na+ excretion by a wild type carrying delta unc is uncoupler sensitive, that of MH1 delta unc is dependent on respiration in an uncoupler-insensitive fashion . It is concluded that under some conditions (high K+ in the medium or in MH1-like mutants), a primary pump driven by respiration is responsible for Na+ extrusion when the Na+/H+ antiporters are not active.

J Biol Chem, 1995 Feb 24, 270(8), 3765 - 71
T4 endonuclease V protects the DNA strand opposite a thymine dimer from cleavage by the footprinting reagents DNase I and 1,10-phenanthroline-copper; Latham KA et al.; The glycosylase/abasic lyase T4 endonuclease V initiates the repair of ultraviolet light-induced pyrimidine dimers . This enzyme forms an imino intermediate between its N-terminal alpha-NH2 group and C-1' of the 5'-residue within the dimer . Sodium borohydride was used to covalently trap endonuclease V to a 49-base pair oligodeoxynucleotide containing a site-specific cyclobutane thymine dimer . The bound and free oligonucleotides were then subjected to nuclease protection assays using DNase I and a complex of 1,10-phenanthroline-copper . There was a large region of protection from both nucleases produced by endonuclease V evident on the strand opposite and asymmetrically opposed to the dimer . Little protection was seen on the dimer-containing strand . The existence of a footprint with the 1,10-phenanthroline-copper cleavage agent indicated that endonuclease V was interacting with the DNA predominantly via the minor groove . Methylation by dimethyl sulfate yielded no areas of protection when endonuclease V was covalently attached to the DNA, indicating that the protein may closely approach the DNA without direct contact with the bases near the thymine dimer . The Escherichia coli proteins Fpg and photolyase display a very different pattern of nuclease protection on their respective substrates, implying that endonuclease V recognizes pyrimidine dimers by a novel mechanism.

J Biol Chem, 1995 Feb 24, 270(8), 3625 - 30
Expression and kinetic characterization of recombinant human stomach alcohol dehydrogenase . Active-site amino acid sequence explains substrate specificity compared with liver isozymes; Kedishvili NY et al.; A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library . The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli . The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg . Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity . Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal . This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid . Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency . The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased . There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH . For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site . The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.

J Biol Chem, 1995 Feb 24, 270(8), 3487 - 90
The Bcl-2 oncoprotein functions as a pro-oxidant; Steinman HM; The mechanism by which the bcl-2 oncogene exerts its anti-apoptotic and antioxidant action is unknown . We found that expression of bcl-2 in superoxide dismutase-deficient (SOD-) Escherichia coli resulted in increased transcription of the KatG catalase-peroxidase, a 13-fold increase in KatG activity and a 100-fold increase in resistance to hydrogen peroxide . In addition, mutation rate was increased 3-fold, and katG and oxyR, a transcriptional regulator of katG induction, were required for aerobic survival . These data indicate that Bcl-2 acts as a pro-oxidant in E . coli, i.e . Bcl-2 generates reactive oxygen intermediates . In support of a pro-oxidant mechanism in eukaryotic cells, we found a 73% increase in superoxide dismutase activity in a murine B-cell line overexpressing Bcl-2 . Increases in reduced glutathione and in oxyradical damage to DNA, previously observed in other overexpressing cell lines, are additional evidence for a pro-oxidant mechanism . Thus, Bcl-2 does not appear to be an antioxidant . Instead, Bcl-2 appears to influence levels of reactive oxygen intermediates that induce endogenous cellular antioxidants . This activity of Bcl-2 may control entry into apoptosis.

Biochim Biophys Acta, 1995 Feb 23, 1243(2), 157 - 60
Characterization of the okra mucilage by interaction with Gal, GalNAc and GlcNAc specific lectins; Wu AM et al.; A bio-active polysaccharide, which was the major component of the extract of the common okra, Hibiscus esculentus, was isolated from the extract by precipitation with ethanol between 28.5 to 45% . According to a previous report (Whistler, R.L . and Conrad, H.E . (1954) J . Am . Chem . Soc . 76, 1673-1674), this polysaccharide contains the Gal alpha 1-->4Gal sequence, which is the ligand for the uropathogenic Escherichia coli and toxic lectins . Analysis of the binding property of the okra polysaccharide by precipitin assay with Gal, GalNAc and GlcNAc specific lectins showed that this okra mucilage reacted best with Mistletoe toxic lectin-I (ML-I) and precipitated over 80% of the ML-I nitrogen (5.1 micrograms N) added . It also precipitated well with Abrus precatorius (APA), Momordica charantia (MCA) and Ricinus communis (RCA1) agglutinins, but poorly with other lectins . The results obtained suggest that this polysaccharide is a valuable reagent to differentiate Gal specific lectins from the GalNAc and/or GlcNAc specific series.

Biochim Biophys Acta, 1995 Feb 23, 1243(2), 151 - 6
Cross-linking of porin with glutardialdehyde: a test for the adequacy of premises of cross-linking theory; Azem A et al.; Exposure of porin from Escherichia coli to glutardialdehyde followed by SDS-gel electrophoresis in 3% polyacrylamide yielded three bands that were identified in order of decreasing mobility as monomers and two and three cross-linked polypeptide chains . The distribution of protein among the three species for different extents of reaction showed a remarkably good agreement with corresponding values predicted from cross linking theory for an oligomer composed of three identical subunits arranged according to a 3-fold rotation axis . Electrophoresis performed in 7.5% polyacrylamide yielded four bands that were assigned to polypeptide chain monomers, dimers, and two types of trimers carrying two and three intersubunit cross-links . Our findings provide evidence that the central premise of cross-linking theory, viz . that the intersubunit cross-links formed upon exposure of a protein to a bifunctional reagent be governed by the symmetry of the molecule, is valid . Careful interpretation of cross-linking experiments thus proves an effective method to assess the oligomeric structure of a protein and reveal the symmetry underlying the spatial arrangement of the subunits within the molecule.

Nature, 1995 Feb 23, 373(6516), 718 - 21
Failure of a single-headed kinesin to track parallel to microtubule protofilaments; Berliner E et al.; Kinesin, a two-headed motor enzyme molecule, hydrolyses ATP to direct organelle transport along microtubules . As it moves along a microtubule, kinesin remains associated with, or 'tracks', microtubule protofilaments . We have prepared truncated kinesin derivatives that contain either two mechanochemical head domains or only a single head . Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme frequently fails to track protofilaments, suggesting that it detaches from microtubules during movement . In this way, the one-headed kinesin derivative is similar to the motor enzyme myosin, which frequently detaches from the actin filament during movement . For myosin (which has two heads), the consequence of this detachment is that single molecules do not appear to drive continuous movement along the filament . Our observations suggest that the ability of single two-headed kinesin molecules to drive continuous movement results from a 'hand-over-hand' mechanism in which one head remains bound to the microtubule while the other detaches and moves forwards.

Nature, 1995 Feb 23, 373(6516), 671 - 6
Pathway of processive ATP hydrolysis by kinesin; Gilbert SP et al.; Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle . The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration . Kinesin dissociates from the microtubule after ATP hydrolysis . This step is rate-limiting . The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state . These results provide an explanation for the motility differences between skeletal myosin and kinesin.

Biochim Biophys Acta, 1995 Feb 22, 1247(1), 159 - 62
The structure and physicochemical properties of rat liver macrophage migration inhibitory factor; Nishihira J et al.; We expressed rat macrophage migration inhibitory factor (rMIF) in E . coli using the cDNA isolated from a rat liver cDNA library . rMIF specifically bound glutathione (dissociation constant = 500 microM) . We purified rMIF homogeneously on SDS-PAGE by S-hexylglutathione Sepharose affinity column chromatography and Sephadex G-100 column chromatography . The amino-acid sequence of rMIF was highly homologous to that of human MIF from a T-cell line; only a single amino-acid residue was substituted if conservative amino-acid substitutions were involved . The molecular weight of rMIF was calculated to be 12.4 kDa and 23.6 kDa by SDS-PAGE and analytical ultracentrifugation, respectively . Thus, it was concluded that the native rMIF formed a homodimeric structure . Proton nuclear magnetic resonance (1H-NMR) study revealed that rMIF was less thermostable (the denaturing temperature was from 50-60 degrees C) than human MIF (the denaturing temperature is about 80 degrees C (Nishihira et al . (1993) Biochem . Mol . Biol . Int . 31, 841-850) . The secondary structure of rMIF evaluated by 1H-NMR experiments revealed that the contents of alpha-helix, beta-strand, and coil were 13.8%, 55.6%, and 30.6%, respectively.

Biochim Biophys Acta, 1995 Feb 21, 1260(3), 276 - 84
Induction and processing of promutagenic O4-ethylthymine lesion in specific gene segments of plasmid DNA; Musarrat J et al.; High affinity antibodies were used for the quantitative assessment of the miscoding O4-ethylthymine (O4-EtThy) base lesion in nanogram amounts of membrane transblotted restriction fragments of ENU treated DNA . The polyclonal antibody (TB3) specifically recognized attomoles of the alkylation adducts in modified DNA with no cross-reactivity to an excess of unmodified DNA . The sensitivity of the immuno-quantitative method was determined to be in the range of 76 attomoles to 2.43 fmol, corresponding to 0.24 x 10(-7) to 7.9 x 10(-7) adducts per nucleotide in plasmid DNA . Modification levels in ras and tk genes were estimated as 0.025 and 0.014 adducts respectively . Specific antibody binding was proportional to the dose of ENU and size of the DNA fragments . In differentially ethylated ras gene, the amount of O4-EtThy was quantified as 0.026, 0.08 and 0.13 adducts per gene fragment . A DNA concentration dependent antibody binding was observed with large (23.13 and 9.41 kb) and smaller (2.02 kb) fragments of HindIII digested ENU treated phage lambda DNA . To monitor the repair of O4-EtThy lesions in specific segments, damage was assessed in sequences of plasmid DNA established in various Escherichia coli strains . The loss of antibody binding to O4-EtThy adducts in ethylated DNA fragments of 6.4 kb ras gene and 3.6 kb tk gene occurred with an approximate t1/2 of 45 and 35 min, respectively, in the repair proficient wild type E . coli . On the contrary, no repair was seen in the alkyltransferase deficient double mutant ada-ogt- strain . The results specifically demonstrate the sensitivity of the immunological technique and the unique ability of the O4-EtThy specific antibodies to scan this promutagenic base lesion and its repair in very small amounts of selected gene segments in DNA.

Biochim Biophys Acta, 1995 Feb 21, 1260(3), 259 - 68
A monoclonal antibody generated against a recombinant peptide fragment of the B3 domain of carcinoembryonic antigen reacts with intact carcinoembryonic antigen; Hagendorff G et al.; The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-B3 domain of carcinoembryonic antigen (CEA) was achieved . The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues . The fusion protein lacZMF-ceaB3 represented approx . 30% of total proteins expressed after induction . The fusion protein was formed as inclusion bodies . Simple washing steps led to an insoluble fusion protein which was of approx . 80% purity . Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector . Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbp-ceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogeneous form after affinity chromatography employing amylopectin . Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha . A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes . The mAB CEA/HK2 specifically recognized CEA in immunoblots . The described experimental strategy should be generally applicable for generation of fusion proteins . These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.

Biochim Biophys Acta, 1995 Feb 21, 1260(3), 245 - 58
Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor; Kotani E et al.; Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies . We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin . The sequence (10477 bp) encoded 3133 amino acids . The characteristics features of the carbohydrate-recognition domain of C-type animal lectin were revealed at C-terminal sequence of hemocytin . When cDNA encoding this region was introduced into baculovirus vector, hemagglutinating activities were detected in the culture fluid of a recombinant virus-infected cells . These activities were inhibited by D-mannose, N-acetyl-D-galactosamine, and D-maltose which are haptenic saccharides of authentic hemocytin . Analysis of dot and Northern blot hybridization revealed that hemocytin gene was transcribed in hemocytes of the silkworm at larval-pupal metamorphosis and/or after the injection of Escherichia coli and lipopolysaccharide . After silkworm larvae were injected with C-terminal portion of hemocytin, aggregation of hemocytes was observed in the hemolymph . Hemocytin has significant homology with mammalian von Willebrand factor which involves in platelet adhesion to subendothelium . Also, hemocytin has a homologous region with coagulation factor V and VIII . These results suggest that hemocytin molecule is an adhesive protein and relates to hemostasis or encapsulation of foreign substances for self-defense.

Biochemistry, 1995 Feb 21, 34(7), 2284 - 8
Photoinduced spin-polarized radical pair formation in a DNA photolyase.substrate complex at low temperature; Rustandi RR et al.; Electron spin polarization is a phenomenon characterized by anomalous line intensities (emission or enhanced absorption) in the EPR spectrum . It is highly diagnostic of radical pairs, such as those formed in photoinduced electron transfer reactions . Electron spin polarization behavior (E/A pattern) is observed in light-modulated EPR spectra obtained at 4 K with fully reduced DNA photolyase.substrate complexes . Similar results are obtained with complexes formed with native enzyme or reconstituted enzyme containing fully reduced flavin as its only chromophore . No signal is observed for fully reduced enzyme or substrate alone . The results suggest that the electron spin polarization signal is due to photoinduced formation of a flavin/substrate radical pair (FADH./T < > T.-); splitting of T < > T.- does not occur at 4 K, and the radical pair can only undergo back-electron-transfer reactions . The data are consistent with the proposal that electron transfer initiates DNA repair in the photolyase reaction.

Biochemistry, 1995 Feb 21, 34(7), 2251 - 9
The major, N2-Gua adduct of the (+)-anti-benzo{a}pyrene diol epoxide can be unstable in double-stranded DNA; Drouin EE et al.; The mechanisms of mutagenesis by the (+)-anti diol epoxide of benzo{a}pyrene {(+)-anti-B{a}PDE} was investigated in supF of the Escherichia coli plasmid pUB3 {Rodriguez & Loechler (1993) Biochemistry 32, 1759} . pUB3 was reacted with (+)-anti-B{a}PDE, then either (1) transformed immediately into E . coli or (2) heated at 80 degrees C for 10 min prior to transformation--the latter to probe mechanism . Qualitatively, heating did not have a statistically significant effect on the mutagenic pattern, except at the major base substitution hot spot, G115, in supF; principally, G115-->T mutations were obtained prior to heating, while after heating, G115-->A and G115-->C mutations became more prevalent . Quantitatively, heating caused an approximately 2-fold decrease in mutation frequency . Heating released a small fraction of adducts (approximately 5%), and the chemistry and implications of this reaction are investigated herein . Although the major adduct of (+)-anti-B{a}PDE (formed at N2-Gua) is generally regarded to be heat stable, it can be quite unstable in double-stranded (but not single-stranded) DNA at low {Mg2+} . Heating releases the corresponding tetraols from (+)-anti-B{a}P-N2-Gua in approximately the same ratio as for simple hydrolysis of (+)-anti-B{a}PDE itself . This and other results suggest that guanine remains in DNA when (+)-anti-B{a}P-N2-Gua adducts are hydrolyzed . {No evidence for any other chemical change in (+)-anti-B{a}PDE adducts was found.} While no general acid/base or nucleophilic catalysis was observed, adduct hydrolysis was specific acid catalyzed down to pH approximately 5.6, where the pH-rate profile showed a break to a slope of approximately 0.0 . This break probably indicates the pKa of (+)-anti-B{a}P-N2-Gua protonated at the N2-position, which is higher than expected.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 21, 34(7), 2148 - 52
Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin results in a change in heat capacity of the native protein; Ladbury JE et al.; Two site-directed mutants of Escherichia coli thioredoxin (L78K and L78R) were designed to study the effect of placing a charged residue in the hydrophobic core of the protein . Both mutants retain catalytic activity in the assembly of phage M13 . Thermal denaturation of both these mutant proteins at pH 7.0 shows a reduction of stability of approximately 4 kcal.mol-1 with respect to the oxidized wild-type form . The thermal denaturation of the protein fits a dimeric state model . A significant reduction in the change in heat capacity (delta Cp) on unfolding is observed compared to oxidized wild-type thioredoxin . We present data to indicate that this reduction in delta Cp is attributable to structural perturbations resulting in localized unfolding of the native protein and exposure to solvent of residues that are buried in the wild-type protein.

J Biotechnol, 1995 Feb 21, 39(1), 67 - 73
Thermo-inducible expression of a recombinant fusion protein by Escherichia coli lac repressor mutants; Yabuta M et al.; The lac I gene of Escherichia coli encodes the lactose repressor . We isolated temperature-sensitive mutants of the lac I gene by in vitro mutagenesis with hydroxylamine . The new mutation sites were determined, and replacement of a single amino acid had respectively occurred at amino acid positions 241 (Ala-->Thr), 265 (Gly-->Asp) and 300 (Ser-->Asn) . These mutation sites were located in the core region of the lac repressor protein . Temperature-dependent expression of beta-galactosidase was observed in the strains having these mutant lac I genes . By using these temperature-sensitive lac I genes, we developed a thermo-inducible expression system for a foreign gene under the control of the lac promoter . A recombinant fusion protein, consisting of a derivative of E . coli beta-galactosidase and the human calcitonin precursor peptide, was efficiently produced by using this system.

Biochemistry, 1995 Feb 21, 34(7), 2260 - 6
Mechanism of inhibition of HIV reverse transcriptase by toxiusol, a novel general inhibitor of retroviral and cellular DNA polymerases; Loya S et al.; Toxiusol, a natural product isolated from the Red Sea sponge Toxiclona toxius, has been shown to be a potent inhibitor of various viral reverse transcriptases (RT) {i.e., of human immunodeficiency virus (HIV-1), equine infectious anemia virus, and murine leukemia virus} and cellular DNA polymerases (i.e., of DNA polymerases alpha and beta and Escherichia coli DNA polymerase I) . A thorough investigation of the mode of inhibition was conducted with HIV-1 RT-associated DNA polymerase activity . The inhibition is unaffected by the nature of template-primer used . The inhibitory active site of toxiusol is attributable to the polar moieties at the benzene ring . The presence of either sulfate groups in the natural lead compound or hydroxyl groups in the corresponding hydroquinone is critical, because both compounds are equally effective at low micromolar concentrations . Conversely, the presence of acetyl groups in the same position in the derivative toxiusol diacetate lowers significantly or abolishes the inhibitory activity . Toxiusol binds the HIV-1 RT irreversibly and in a noncompetitive way with high affinity (Ki = 1.2 microM), probably through polar groups . The replacement with acetyl moieties in the analog toxiusol diacetate hampers the binding of the inhibitor to the enzyme (Ki increases to about 26 microM) . Still, the compound binds irreversibly, probably through its hydrophobic structure skeleton . Toxiusol diacetate loses its ability to inhibit the first step in the DNA polymerization process (that is, the formation of the DNA-enzyme complex as measured by a gel retardation assay), which contributes to its poor inhibitory capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 21, 34(7), 2241 - 50
Investigation of the mechanism of phosphoribosylamine transfer from glutamine phosphoribosylpyrophosphate amidotransferase to glycinamide ribonucleotide synthetase; Rudolph J et al.; Phosphoribosylamine (PRA) is a product of glutamine phosphoribosylpyrophosphate amidotransferase (PRPP-AT) and a substrate for glycinamide ribonucleotide synthetase (GAR-syn), the first two enzymes in the de novo purine biosynthetic pathway . PRA has a half-life of 5 s under physiological conditions, hydrolyzing to ribose 5-phosphate . The instability of this purine precursor brings to question how the efficiency of transfer from one active site to the next is ensured: Is PRA transferred by free diffusion, or is it transferred directly from one enzyme to the next through a process defined as substrate channeling? Kinetic investigations of reactions containing both enzymes monitoring the appearance of the intermediate PRA and/or the product GAR were performed and compared with the predicted kinetics assuming a free diffusion mechanism of transfer . A significant discrepancy exists between the free diffusion model and the experimental data when the ratios of the two enzymes are varied . To accommodate this discrepancy, a direct transfer mechanism is proposed that is facilitated by protein-protein interactions . Experiments to provide evidence for these stable protein-protein interactions including gel chromatography, fluorescence spectroscopy, chemical cross-linking, and affinity gel chromatography; however, have all been unsuccessful . These results suggest that the requisite channeling interaction between PRPP-AT and GAR-syn, which is indicated by the kinetic results, must be a transient one.

Mol Gen Genet, 1995 Feb 20, 246(4), 514 - 8
An Escherichia coli strain deficient for both exonuclease V and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation; Estevenon AM et al.; An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive . A DNA fragment from an E . coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322 . Comparison of its restriction map with the physical map of the E . coli chromosome revealed complete identity to the recBD genes . ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB . This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity.

Mol Gen Genet, 1995 Feb 20, 246(4), 401 - 10
A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs); Shibata W et al.; A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture . The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs . The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues . These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase . Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2 . The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms . Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.

FEBS Lett, 1995 Feb 20, 360(1), 93 - 6
The use of electrospray mass spectrometry to identify an essential arginine residue in type II dehydroquinases; Krell T et al.; The arginine-specific reagent phenylglyoxal has been used to identify a hyper-reactive arginine residue which is essential for activity in the type II dehydroquinases of Streptomyces coelicolor and Aspergillus nidulans . Electrospray mass spectrometry was used both to characterise the phenylglyoxal modified protein, and to identify the phenylglyoxal modified peptides following enzymatic digestion . The advantages of using electrospray mass spectrometry for monitoring arginine modication aimed at identifying functional residues in proteins are discussed.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 62 - 8
Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme; Ohsugi I et al.; To study the structure/function relationships of peptidylarginine deiminase (PAD), we constructed an Escherichia coli expression plasmid for mouse uterine PAD . First, segments of a cDNA encoding murine PAD were subcloned into a single plasmid, and the resulting plasmid, pKSPAD1, was inserted into an expression vector, pKK223-3, at the EcoRI and HindIII restriction sites . Since no detectable amount or activity of the PAD was produced by E . coli carrying that plasmid, the 5'-untranslated sequence of the cDNA was replaced with several synthetic DNAs . One of the constructed plasmids, pKKPAD4, which had a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron inserted into the adjacent 5'-region of the coding region, produced a large quantity of mouse PAD as an unfused protein in E . coli . The purified recombinant PAD was indistinguishable from the native enzyme with respect to some structural properties, such as molecular mass, amino- and carboxyl-terminal sequences, and circular dichroism spectra . However, the alpha-amino group of the amino-terminal methionine residue of the recombinant PAD was not acetylated as was that of the native enzyme . Comparison of the recombinant PAD with the natural enzyme did not indicate significant differences in their sensitivity to activation by Ca2+ and in their substrate specificity toward arginine derivatives . The rates of modification of soybean trypsin inhibitor (Kunitz) were also similar for the recombinant and native PADs . These results indicate that the recombinant PAD has biological activities identical to those of the native enzyme and that the N alpha-acetyl group in the native PAD does not appear to have any particular role in the enzyme's catalytic function.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 57 - 61
Superoxide dismutase protects Escherichia coli against killing by human serum; McManus DC et al.; To assess the role of superoxide dismutase in protecting Escherichia coli from killing by human serum and neutrophils, we constructed isogenic, smooth-lipopolysaccharide K-12 strains, either sod wild-type, delta sodA, or delta sodA delta sodB . The delta sodA delta sodB strain was killed by serum much more readily than either the wild-type or delta sodA strain . After allowing for this serum sensitivity difference, the delta sodA delta sodB strain also showed increased susceptibility to phagocytic killing by human neutrophils . These results indicate that superoxide dismutase protects E . coli from killing by serum (complement system) and by human neutrophils, possibly by a role in maintaining bacterial membrane structure.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 33 - 8
Intrinsic fluorescence of the chloroplast H(+)-ATPase; Kirch RD et al.; We have examined the intrinsic fluorescence properties of a highly purified chloroplast H(+)-ATPase (CF0F1) preparation {R . D . Kirch and P . Graber (1992) Acta Physiol . Scand . 746, 9-12) . Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluorescence both at 277-nm excitation, favoring tyrosine excitation, and at 295-nm excitation, favoring tryptophan excitation . A broad tryptophan fluorescence peak was observed with a maximum at around 335 nm and a broad shoulder around 350 nm . Denaturation of the enzyme complex with guanidine-HCl resulted in a significant increase (approximately 40%) in tyrosine fluorescence . The fluorescence spectrum (lambda ex = 295 nm) of the inhibitory epsilon subunit isolated from CF1 resembled that of CF1, indicating the presence of two tryptophan species located in different environments . Fluorescence quenching by potassium iodide indicated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of epsilon from CF1 . Thus, when epsilon binds to CF1, a tryptophan residue becomes partially buried, probably at an interface between epsilon and another (possibly gamma) CF1 subunit . Removal of the epsilon subunit from CF1 leads to an increase in tyrosine fluorescence of a magnitude similar to that obtained upon denaturation of the CF0F1 complex . The results suggest that the reversible association of the epsilon subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescence of the enzyme complex.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 275 - 84
Characterization of flavin-containing monooxygenase 5 (FMO5) cloned from human and guinea pig: evidence that the unique catalytic properties of FMO5 are not confined to the rabbit ortholog; Overby LH et al.; Several full-length clones encoding the human and guinea pig orthologs of flavin-containing monooxygenase 5 (FMO5) have been isolated from libraries constructed with hepatic mRNA . The clones were detected by hybridization with the cDNA encoding FMO5 expressed in rabbit . The human and guinea pig cDNAs encode for proteins of 533 amino acids that contain putative pyrophosphate binding domains characteristic of mammalian FMOs . The sequences derived for the human and guinea pig FMO5 proteins are 87% identical and are 85 and 82% identical, respectively, to the sequence of rabbit FMO5 . As is the case with other FMOs, FMO5 in human and guinea pig is encoded by multiple transcripts . Rabbit FMO5 expressed in Escherichia coli was purified and used to elicit antibodies in goat . These antibodies detected FMO5 in samples from livers of adult humans, rabbits, and guinea pigs and fetal livers of humans . The human and guinea pig forms of FMO5 were expressed in E . coli and characterized . Neither enzyme effectively catalyzed the metabolism of methimazole, a general FMO substrate; however, both were active with n-octylamine . The responses of the human FMO5 and guinea pig FMO5 to detergent, ions and elevated temperature are all similar to the responses described for rabbit FMO5 . These results indicate that the unique properties of FMO5 from rabbit are species-independent and that this form of the flavin-containing monooxygenase is not readily classified as a drug-metabolizing enzyme.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 253 - 8
Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1; Ito-Maki M et al.; A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25-->Ala and His132-->Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli . We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase . His132-->Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity . In contrast, His25-->Ala mutation completely abolished the enzyme's catalytic activity . A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex . Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 244 - 52
Properties of fructose-1,6-bisphosphate aldolase from Escherichia coli: an NMR analysis; Szwergold BS et al.; A class II Zn(2+)-dependent fructose-1,6-bisphosphate (FBP)- aldolase was purified from an overproducer strain of Escherichia coli and characterized by standard biochemical techniques and 13C NMR spectroscopy . The principal finding of these studies was identification, by 13C NMR spectroscopy, of an enzyme-bound reaction intermediate, the enediol(ate) form of dihydroxyacetone phosphate (DHAP) . Formation of this intermediate requires the presence of Zn2+ and is pH dependent, with increasing amounts of this tautomer appearing at alkaline pH's . This pH dependence closely parallels the pH activity profile of the enzyme, suggesting an involvement of the enediol-DHAP form in the reaction pathway . In addition to these results the following observations were made on this enzyme: (a) E . coli FBP aldolase binds and utilizes only the carbonyl forms of FBP and DHAP; (b) the function of Zn2+ in this metalloaldolase appears to be polarization of the C = O bond of DHAP; (c) activity of this enzyme is unaffected by glycolytic intermediates or nucleotide phosphates such as ATP . Although these studies provide some information about the catalytic mechanism of E . coli FBP aldolase, they do not provide an explanation for the apparent regulation of this enzyme reported in previous in vivo NMR studies . While the possibility that the enzyme is allosterically regulated cannot be excluded at this time, an interesting possibility suggested by this and other studies is that in E . coli glycolytic substrates may be channeled through a multienzyme complex.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 222 - 34
Cloning and expression of hydrolase C, a member of the rat carboxylesterase family; Yan B et al.; Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase . This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney . Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence . Both enzymes have an 18-amino-acid signal peptide at the N-terminus . The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum . As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum . Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61 . The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228 . Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver . When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol . Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced . In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution . Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al . (Arch . Biochem . Biophys . 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme . The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 215 - 21
Functional complementation of the mvrA mutation of Escherichia coli by plant ferredoxin-NADP+ oxidoreductase; Krapp AR et al.; Escherichia coli cells carrying the mvrA mutation are unable to grow aerobically in the presence of the radical propagator methyl viologen (MV) . Resistance against MV toxicity could be restored by the introduction of cloned DNA sequences encoding pea chloroplast ferredoxin-NADP+ reductase (FNR), a member of a class of flavoenzymes involved in redox pathways in bacteria, plants and animals . Complementation was strictly dependent on the accumulation of a functional transgenic FNR, since mutated reductases showing decreased enzymatic activities only partially rescued the MV-resistant phenotype . These results support recent observations suggesting that the E . coli mvrA gene encodes a ferredoxin (flavodoxin)-NADP+ reductase (V . Bianchi et al . (1993) J . Bacteriol . 175, 1590-1595) . The mvrA mutant cells showed a moderate decrease in the flavodoxin-dependent activation of enzymes essential for anaerobic growth of E . coli . This effect is prevented by expression of a functional pea FNR in the mutant cells.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 201 - 7
The inducible 9, 10-dihydrophenanthrene pathway: characterization and expression of bibenzyl synthase and S-adenosylhomocysteine hydrolase; Preisig-Muller R et al.; Tricyclic 9,10-dihydrophenanthrenes originate from phenylpropane derivatives by chain elongation and cyclization according to the polyacetate rule . Bibenzyls are bicyclic intermediates, and O-methylation is a prerequisite for their conversion into dihydrophenanthrenes . cDNA clones encoding bibenzyl synthases and S-adenosylhomocysteine hydrolase of the orchid Phalaenopsis sp . were isolated from a cDNA library representing the stage of elicitor-induced plants . The deduced amino acid sequences of two clones, pBibSy811 and pBibSy212, indicated that we obtained two full-length sequences of bibenzyl synthases characterized by their homology to stilbene synthases previously investigated . That indeed bibenzyl synthase cDNAs rather than a homologous stilbene synthase cDNA or chalcone synthase cDNA have been isolated was demonstrated by expression of two enzymatically active bibenzyl synthase proteins in Escherichia coli . These proteins showed virtually the same selectivity towards m-hydroxyphenylpropionyl-CoA as substrate as the enzyme isolated from orchid plants . In young sterile Phalaenopsis plants, the formation of both bibenzyl synthase mRNAs and S-adenosylhomocysteine hydrolase mRNAs was increased upon elicitation more than 100-fold . The time courses of gene expression exhibited transient profiles, reaching maximum mRNA levels 20 h after onset of fungal infection followed by a rapid decline to 40 h.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 185 - 90
Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase; Ohlrogge J et al.; Expression of a plant lauroyl-acyl carrier protein (ACP) thioesterase in an Escherichia coli strain deficient in beta oxidation results in the accumulation of free fatty acids in the culture . Overall fatty acid production by the cultures is increased severalfold, particularly in the late log and stationary stages of growth . In control E . coli cells, malonyl-ACP levels and rates of fatty acid synthesis are highest during rapid logarithmic growth and decline to undetectable levels in stationary stage . In contrast, in cells expressing plant acyl-ACP thioesterase, malonyl-ACP levels remain high in late log and stationary stage in association with the continued fatty acid production . In addition, the biotin carboxyl carrier protein component of acetyl-CoA carboxylase is expressed at higher levels in cultures expressing the acyl-ACP thioesterase . The data presented indicate that removal of the acyl-ACP products of fatty acid synthesis results in increased production of both malonyl-ACP and fatty acids, which may in turn result from higher activity and/or expression of acetyl-CoA carboxylase.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 161 - 9
Biochemical characterization of lauric acid omega-hydroxylation by a CYP4A1/NADPH-cytochrome P450 reductase fusion protein; Chaurasia CS et al.; The binding and hydroxylation of lauric acid by a genetically engineered and expressed fusion protein comprised of an N-truncated form of rat CYP4A1 linked to an N-truncated form of rat NADPH cytochrome P450 oxidoreductase (OR) (constructed by Fisher et al., (1992) Proc . Natl . Acad . Sci . USA 89, 10817-10822) has been characterized biochemically and compared to that shown by purified reconstituted rat CYP4A1 and liver microsomes from clofibrate-induced rats . In all systems lauric acid induced a Type I cytochrome P450 difference spectrum with Ks values in agreement with prior literature (range 10-18 microM) . When provided with NADPH and oxygen but no other proteins or lipid, the fusion protein (called f4A1) catalyzed omega-hydroxylation of lauric acid with apparent Km and Vm of 3-4 microM and 4-5 nmol product/min/nmol P450 irrespective of buffer concentration or cation (NaPi or KPi, 25-200 mM); comparable values for reconstituted CYP4A1 and microsomes from clofibrate-induced rats are 9 microM and 34 min-1 and 5 microM and 10 min-1, respectively . (omega-1)-Hydroxylation of lauric acid was barely detectable (omega/(omega-1) = 135) with f4A1 or with reconstituted CYP4A1, but it accounted for up to 50% of total products formed by microsomes from clofibrate-induced rats . When added to the f4A1 system, OR stimulated hydroxylation up to fivefold at a OR:f4A1 ratio of 5:1; additionally, (omega-1)-hydroxylation was routinely observed as a minor process (< 4% of total product) in this system . These effects were also independent of buffer concentration . In contrast addition of cytochrome b5 (b5) caused a small (25%) decrease in omega-hydroxylation, while added phospholipid had no effect . However, the combination of OR, b5, and lipid stimulated turnover approximately 10-fold compared to f4A1 alone, and 11-hydroxylauric acid was regularly formed as a minor (3-4% of total) product . These observations indicate that the fusion protein f4A1 is functionally equivalent to reconstituted CYP4A1 with respect to binding and hydroxylation of lauric acid and suggest that it can be used as an alternative to reconstituted systems for structure-function and mechanistic studies of fatty acid omega-hydroxylation.

Virology, 1995 Feb 20, 207(1), 179 - 84
Protection of coliphage lambda O initiator protein from proteolysis in the assembly of the replication complex in vivo; Wegrzyn A et al.; We have shown previously that, in contrast to the free coliphage lambda O initiator protein rapidly degraded by ClpP/ClpX protease, the lambda present in the replication complex (RC) is protected from proteolysis . Now we asked at which step of the pathway of RC assembly in vivo does the stabilization of lambda O occur . In accordance with the in vitro established order we found that lambda P and DnaB helicase functions are, but those of DnaJ and GrpE chaperones are not, required for the protection of lambda O from proteolysis . Therefore, our results suggest that the first lambda O protecting structure of the pathway of RC assembly is the lambda O-lambda P-DnaB preprimosome . The next step of the pathway, the chaperone-mediated rearrangement of the preprimosome, is not essential for lambda O stabilization . However, in contrast to other chaperones, the DnaK function was required for the protection of lambda O from proteolysis, suggesting an earlier access of DnaK to the pathway of RC assembly in vivo, in accordance with current models by which molecular chaperones facilitate protein assembly.

J Mol Biol, 1995 Feb 17, 246(2), 264 - 72
Intragenic domains of strand-specific repair in Escherichia coli; Kunala S et al.; Heterogeneity of DNA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and DNA strands . If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo . Here we measure the frequency of UV-induced cyclobutane pyrimidine dimers at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation . Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites . In lacZ, the domain of faster repair began 32 bases downstream of the transcription start site and required the mfd gene . Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo . Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (2) induction of lacZ transcription with isopropyl-beta-D-thiogalactoside overcame this reduction . Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.

J Mol Biol, 1995 Feb 17, 246(2), 248 - 53
Hypercooperativity induced by interface mutations in the phosphofructokinase from Escherichia coli; Auzat I et al.; The saturation of the allosteric phosphofructokinase from Escherichia coli by its substrate fructose-6-phosphate is highly cooperative and seems to occur in an "all-or-none" process at all active sites . This cooperativity measured by the Hill coefficient can still be markedly increased by mutation of a single residue located at a subunit interface, Arg152 . X-ray crystallography shows that Arg152 forms an ion-pair with Glu148 within an alpha-helix of one subunit . This ion-pair is close to a symmetry axis and interacts with the ion-pair Glu148-Arg152 of the neighbouring chain across the subunit interface . Mutations of Glu148 affect cooperativity much less than those of Arg152 . The substitution of Arg152 by lysine increases the Hill coefficient by two-fold to a value larger than the number of substrate binding sites, which exceeds the maximum cooperativity predicted by the two "classical" models, concerted or sequential, of allosteric regulation . This indicates that the steady-state overall hypercooperativity is (at least partly) of kinetic origin . The hypercooperative mutants of Arg152 also show an enhanced cooperativity in their allosteric inhibition by phospho-enol-pyruvate . These results suggest that the allosteric coupling between distant sites involves (1) electrostatic interactions across the subunit interface between residues Glu148 and Arg152 from two adjacent chains, and (2) a relative movement of the alpha-helices containing Glu148 and Arg152 that could propagate and amplify a conformational change between the interface and the active site within each subunit.

J Biol Chem, 1995 Feb 17, 270(7), 3179 - 85
Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli; Ladant D; Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins, defined by the photoreceptor cell-specific protein, recoverin . Recoverin, which regulates the desensitization of photo-excited rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch . Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding . In this work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin delta . I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner . Both myristoylated and unmyristoylated proteins bind three calcium ions . However, the unmyristoylated form exhibits a higher affinity for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium . These data support the model for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 11569-11573; Dizhoor, A . M., Chen, C . K., Olshevskaya, E., Sinelnikova, V . V., and Hurley, J . B . (1993) Science 259, 829-832) . Using point mutations, I have investigated the relative importance of each of the three functional EF hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin . Calcium and membrane binding properties of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein to membranes.

J Biol Chem, 1995 Feb 17, 270(7), 3123 - 31
Differential transcriptional activation in vitro by NF-kappa B/Rel proteins; Lin R et al.; Distinct NF-kappa B subunit combinations contribute to the specificity of NF-kappa B-mediated transcriptional activation and to the induction of multiple cytokine genes including interferon-beta (IFN-beta) . To evaluate the regulatory influence of different homo- and heterodimers, NF-kappa B subunits were analyzed for transcriptional activity in vitro using test templates containing two types of NF-kappa B recognition elements (the human immunodeficiency virus type 1 enhancer and the IFN-beta-positive regulatory domain-II (PRDII) as well as IFN-beta PRDIII-PRDI-PRDII linked to the -56 minimal promoter of rabbit beta-globin . Recombinant NF-kappa B subunits (p50, p65, c-Rel, p52, and I kappa B alpha) and interferon regulatory factor 1 were produced from either Escherichia coli or baculovirus expression systems . Transcriptional analysis in vitro demonstrated that 1) various dimeric complexes of NF-kappa B differentially stimulated transcription through the human immunodeficiency virus enhancer or PRDII up to 20-fold; 2) recombinant I kappa B alpha specifically inhibited NF-kappa B-dependent transcription in vitro; and 3) different NF-kappa B complexes and interferon regulatory factor 1 cooperated to stimulate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains of the IFN-beta promoter . These results demonstrate the role of NF-kappa B protein dimerization in differential transcriptional activation in vitro and emphasize the role of cooperativity between transcription factor families as an additional regulatory level to maintain transcriptional specificity.

J Biol Chem, 1995 Feb 17, 270(7), 2901 - 5
Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase; Herrmann C et al.; The Ras-binding domain (RBD) of human Raf-1 was purified from Escherichia coli, and its interaction with Ras was investigated . Its dissociation constant with p21ras.guanyl-5'-yl imidodiphosphate was found to be 18 nM, with a slight preference for H-ras over K- and N-ras . Oncogenic forms bind with slightly lower affinity . The affinity of RBD for effector region mutants or the GDP-bound form of p21ras is in the micromolar range, which means that 100-fold lower affinity is not sufficient for signal transduction . The rate of the GTPase of p21ras is not modified by RBD . Since P(i) release is found not to be rate limiting, the Ras-Raf signal of the cell may be terminated by the intrinsic GTPase of p21ras.

J Biol Chem, 1995 Feb 17, 270(7), 2881 - 4
The sequence of phosphatidylinositol-4-phosphate 5-kinase defines a novel family of lipid kinases; Boronenkov IV et al.; Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) occupies an essential position in the phosphoinositide signal transduction cascades as the precursor to second messengers and is thought to regulate many cellular proteins directly . The final step in the synthesis of PtdIns(4,5)P2 is the phosphorylation of PtdIns(4)P- by PtdIns(4)P 5-kinase (PIP5K) . Using peptide sequences from a purified PIP5K, a cDNA for a human placental PIP5K was isolated and sequenced . Expression of this cDNA in Escherichia coli produced an active PIP5K . Surprisingly, the sequence of this PIP5K has no homology to known PtdIns kinases or protein kinases . However, the PIP5K is homologous to the Saccharomyces cerevisiae proteins Fab1p and Mss4p.

Carbohydr Res, 1995 Feb 17, 267(2), 263 - 9
The structure of the O-specific polysaccharide from Escherichia coli O113 lipopolysaccharide; Parolis H et al.; The O-specific polysaccharide from Escherichia coli O113 lipopolysaccharide was separated from the core and lipid A by mild acid hydrolysis and purified by GPC . Methylation analysis and 1H and 13C NMR spectroscopic studies of the O-deacetylated polysaccharide allowed the determination of the structure of the pentasaccharide repeating unit of the polysaccharide which can be written as {equation: see text} The position of the O-acetyl groups was not determined.

J Biol Chem, 1995 Feb 17, 270(7), 3454 - 61
Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA; Desai SY et al.; 2'-5' oligoadenylate (2-5(A)) synthetase and protein kinase, RNA activated (PKR) are the only two known enzymes that bind double-stranded RNA (dsRNA) and get activated by it . We have previously identified their dsRNA binding domains, which do not have any sequence homology . Here, we report a profound difference between the two enzymes with respect to the structural features of the dsRNA that are required for their activation . The adenoviral virus-associated type I (VAI) RNA cannot activate PKR, although it binds to the protein and thereby prevents its activation by authentic dsRNA . In contrast, we observed that VAI RNA can both bind and activate 2-5(A) synthetase . Mutations in VAI RNA, which removed occasional mismatches present in its double-stranded stems, markedly enhanced its 2-5(A) synthetase-activating capacity . These mutants, however, are incapable of activating PKR . Other mutations, which disrupted the structure of the central stem-loop region of the VAI RNA, reduced its ability to activate 2-5(A) synthetase . These debilitated mutants could bind to the synthetase protein, although they fail to bind to PKR.

J Biol Chem, 1995 Feb 17, 270(7), 3385 - 91
Lck unique domain influences Lck specificity and biological function; Carrera AC et al.; Src-family tyrosine kinases share structural and amino acid sequence homology, particularly in the catalytic domain as well as in the SH2 and SH3 domains of the regulatory region . However, each src-family member also contains a unique domain which is specific to and characteristic of each individual tyrosine kinase . These unique or specific domains may contribute to the functional specificity of each src-family kinase . To address this possibility, we analyzed the kinase activities and substrate specificities of the lymphoid src-kinase, pp56lck, and a mutant of pp56lck lacking its specific domain . Our data show that both the wild type enzyme and the specific domain-deleted mutant displayed similar affinities for ATP and the non-physiological substrate denatured enolase . However, the specific domain-deleted mutant failed to phosphorylate a number of physiological substrates of pp56lck . In addition, the ability of pp56lck to mediate induction of the interleukin-2 promoter was strongly impaired upon deletion of its specific domain . Thus, the unique domain is not required for the intrinsic kinase activity of pp56lck, however, it influences substrate preference and contributes to the unique physiological function of this src-family tyrosine kinase.

Nature, 1995 Feb 16, 373(6515), 636 - 40
Crystal structure of the GreA transcript cleavage factor from Escherichia coli; Stebbins CE et al.; Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains . One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment . Here we report the crystal structure of GreA at 2.2 A resolution . The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases . A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript . The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.

Nature, 1995 Feb 16, 373(6515), 632 - 5
Interaction with RAP74 subunit of TFIIF is required for transcriptional activation by serum response factor; Joliot V et al.; A few general transcription factors, in particular TFIID and TFIIB, have been found to bind transcriptional activators . Here we show that the general transcription factor TFIIF is also a target for a transcriptional activator, namely serum response factor (SRF), which binds to the c-fos promoter . Using a yeast interaction assay, we find that SRF binds the RAP74 subunit of TFIIF and that SRF's transcriptional activation domain is the region involved in this binding . Further, RAP74's central charged cluster domain is required for binding to SRF's activation domain . Deletion of this domain impairs RAP74's ability to support SRF-activated transcription in vitro but has little effect on the protein's basal transcription activity or its ability to support SP1-activated transcription . The correlation of SRF-RAP74 binding with transcriptional activation suggests that RAP74 is a critical target for SRF-activated transcription.

Genes Dev, 1995 Feb 15, 9(4), 387 - 98
The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP; Danese PN et al.; DegP is a heat-shock inducible periplasmic protease in Escherichia coli . Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by sigma 32 . Rather, the degP gene is transcriptionally regulated by an alternate heat shock sigma factor, sigma E . Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter . To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion . Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP . Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP . These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein . Cpx-mediated transcription of degP is partially dependent on the activity of E sigma E, suggesting that the Cpx pathway functions in concert with E sigma E and perhaps other RNA polymerases to drive transcription of degP.

Eur J Biochem, 1995 Feb 15, 228(1), 74 - 8
cDNAs for S-adenosyl-L-methionine decarboxylase from Catharanthus roseus, heterologous expression, identification of the proenzyme-processing site, evidence for the presence of both subunits in the active enzyme, and a conserved region in the 5' mRNA leader; Schroder G et al.; S-Adenosyl-L-methionine decarboxylases (AdoMetDC) are pyruvoyl-dependent enzymes producing the aminopropyl group for spermidine biosynthesis, and this reaction is the rate-limiting step in polyamine biosynthesis . We characterized cDNAs from Catharanthus roseus (Madagascar periwinkle) and investigated the enzyme after heterologous expression . The largest cDNA (1842 bp) had an 5' leader of 469 bp and encoded a protein of 357 residues and 30-35% identity with mammalian AdoMetDC . The proenzyme expressed in Escherichia coli was processed into active enzyme, and the processing site was identified by site-directed mutagenesis as the second Ser in the sequence Leu-Ser-Glu-Ser-Ser . The analysis of affinity-purified proteins indicated that the active enzyme contained both subunits . The Km for S-adenosyl-L-methionine was 35-40 microM, and the enzyme activity was not stimulated by putrescine . The 5' leader of the mRNA contained start and stop codons for a polypeptide of 51 amino acids, and this region was conserved in the 5' leaders of other plant AdoMetDC mRNAs . The putative polypeptide had no similarity with the hexapeptide responsible for modulation of AdoMetDC mRNA translation in mammals . The possibility is discussed that plants evolved a different type of translational regulation.

Eur J Biochem, 1995 Feb 15, 228(1), 68 - 73
Enoyl-CoA hydratase and isomerase form a superfamily with a common active-site glutamate residue; Muller-Newen G et al.; Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily . Their substrates and reaction mechanisms are similar {Muller-Newen, G . & Stoffel, W . (1993) Biochemistry 32, 11,405-11,412} . Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities . We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction . We functionally expressed rat liver mECH wild-type and {E164Q} mutant enzymes in Escherichia coli . Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the kcat value more than 100,000-fold, whereas the Km value is only moderately affected . We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity . Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.

Eur J Biochem, 1995 Feb 15, 228(1), 184 - 90
Mutation of the conserved Gly83 and Gly94 in Escherichia coli elongation factor Tu . Indication of structural pivots; Kjaersgard IV et al.; Elongation factor Tu from Escherichia coli cycles between an active conformation where GTP is bound, and an inactive conformation where GDP is bound . Between the two conformations, elongation factor Tu undergoes major structural changes . The aim of this work has been to reveal the role of two very well conserved glycine residues, Gly83 and Gly94, in the switch mechanism . Gly83 has been mutated alone or in combination with Gly94, both glycine residues being mutated to alanine . Enzymic characterisation of the two mutants have shown that they have an altered nucleotide affinity, a decrease in aminoacyl-tRNA affinity, an increase in intrinsic GTP hydrolysis, different behaviours in effector stimulation of the intrinsic GTPase activity, and that they are completely unable to sustain poly(Phe) synthesis in an in-vitro poly(U)-directed system . Our results indicates that particularly Gly83 is an important pivot point in elongation factor-Tu.

Eur J Biochem, 1995 Feb 15, 228(1), 176 - 83
Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli . Elucidation of their structural and functional roles; Knudsen CR et al.; All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches . Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism . Gly94 and Gly126 were separately mutated to alanine residues to study their role in the switch mechanism . The mutant proteins are denoted {G94A}EF-Tu and {G126A}EF-Tu, respectively . Both mutations affect the affinities for guanine nucleotides considerably, resulting in a decrease in the characteristic preference for GDP over GTP . Furthermore the {G94A}EF-Tu mutant possesses an increased GTPase activity . The aminoacyl-tRNA affinity is much reduced for {G94A}EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40 . Surprisingly, however, both mutants in their GDP forms have a low, but significant affinity for aminoacyl-tRNA, which is not seen for the wild-type elongation factor Tu . The mutants only exhibit minor changes compared to the wild type with respect to in vitro translation of a poly(U) messenger.

Eur J Biochem, 1995 Feb 15, 228(1), 144 - 8
Immunochemical determination of human cholesterol 7 alpha-hydroxylase; Maeda YR et al.; To produce antibodies for determination of the protein mass of human cholesterol 7 alpha-hydroxylase, a fusion protein was prepared from an in-frame fusion gene containing the cDNA for human cholesterol 7 alpha-hydroxylase near the 3' terminus of the lacZ gene of Escherichia coli . The fusion protein was purified by (NH4)2SO4 fractionation and gel-filtration chromatography on a Sephacryl column . Rabbits were immunized with this fusion protein and antisera were obtained . IgG was prepared by submitting antisera to chromatography on protein-A--Sepharose . Antibodies directed against bacterial proteins including beta-galactosidase were removed by affinity chromatography on a column to which bacterial proteins of E . coli containing beta-galactosidase had been immobilized . Evidence that the antibodies are indeed reactive against human liver cholesterol 7 alpha-hydroxylase was obtained by immunoblot analysis with human cholesterol 7 alpha-hydroxylase expressed in COS cells from the coding region of the human cholesterol 7 alpha-hydroxylase cDNA . The antiserum inhibited the activity of cholesterol 7 alpha-hydroxylase in human liver microsomes by approximately 70% . On immunoblotting of solubilized human liver microsomes, a positive band was obtained at a position corresponding to the protein mass for human cholesterol 7 alpha-hydroxylase . When calibration was performed using the fusion protein, a linear relationship was observed between the density and the amount of protein . Proportionality was also observed between the density and the amount of protein for microsomes of COS cells transfected with the coding region of the human cholesterol 7 alpha-hydroxylase cDNA . Liver microsomes from patients treated with cholestyramine (n = 3) were shown to contain levels of cholesterol 7 alpha-hydroxylase protein approximately twofold higher than those of liver microsomes from untreated patients (n = 6; P < 0.02), whereas cholesterol 7 alpha-hydroxylase activity was approximately six-fold higher in liver microsomes from the cholestyramine-treated patients than in the corresponding preparations from the untreated patients (P < 0.02) . The higher activities observed in cholestyramine-treated patients, therefore, cannot be explained only by an increased amount of protein, suggesting a posttranslational mechanism to increase the activity of human cholesterol 7 alpha-hydroxylase in addition to the transcriptional control.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 515 - 20
The use of glycerol to link DNA damage from hydroxyl radicals with the activities of DNA repair enzymes; Ewing D et al.; We show that glycerol can be used to study the relationship between DNA damage caused by hydroxyl radicals and the activities of different DNA repair enzymes . We suggest that such tests with glycerol will lead to a better understanding of the mechanistic basis for radiation-induced cell death, as well as provide valuable information on the specific roles of different DNA repair enzymes.

Biochem J, 1995 Feb 15, 306 ( Pt 1), 57 - 61
Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation; Kaderbhai MA et al.; A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep . These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes.

Biochem J, 1995 Feb 15, 306 ( Pt 1), 167 - 75
Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role; Gijon MA et al.; Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c . Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL) . N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side . Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2 . The enzyme showed optimal activity on {14C}oleate-labelled autoclaved E . coli, on synthetic phosphatidylethanolamine, and on {3H}arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of {3H}arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs) . In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties . The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells . The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Structure, 1995 Feb 15, 3(2), 163 - 76
The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli; Onesti S et al.; BACKGROUND: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA . The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known . Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase . In Escherichia coli there are two isoforms of the enzyme, LysS and LysU . Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA) . These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response . RESULTS: The crystal structure of E . coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site . The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule . Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases . CONCLUSIONS: A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified . The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain . When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ . The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428 . The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

Structure, 1995 Feb 15, 3(2), 131 - 4
Recombining the structures of HIV integrase, RuvC and RNase H; Yang W et al.; The recently reported crystal structures of two recombination enzymes, the catalytic domain of HIV integrase and Escherichia coli RuvC, an endonuclease, are surprisingly similar to that of ribonuclease H suggesting the possibility that they have a common enzymatic mechanism.

FEMS Microbiol Lett, 1995 Feb 15, 126(2), 189 - 95
Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and alpha-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96; Blum G et al.; The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors {P-fimbriae (pap and prs), F1C-fimbriae (foc) and Type 1-fimbriae (fim)}, two alpha-hemolysins (hlyI and II) and the cytotoxic necrotizing factor type 1 (cnf1) . Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hlyII, prs and cnf1 genes . The three virulence associated determinants are linked on one particular region on the chromosome, which is termed 'pathogenicity island II' (Pai II).

Genes Dev, 1995 Feb 15, 9(4), 471 - 80
Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli; Liu F et al.; The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1) . Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro . This reaction can be stimulated by proteins extracted from both E . coli and HeLa cells . When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by approximately 80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 708 - 14
Monoclonal anti-FLAG antibodies react with a new isoform of rat Mg2+ dependent protein phosphatase beta; Schafer K et al.; The FLAG peptide has been widely used as a multi-purpose tag for the identification and detection of recombinant FLAG fusion proteins . The practicability of this approach depends on specific detection of FLAG fusion proteins with no or very little cross-reactivity to cellular proteins . We have isolated a rat cDNA clone coding for a new splicing isoform of Mg2+ dependent protein phosphatase beta (MPP beta) by screening a rat brain expression library with monoclonal antibody Anti-FLAG M2 . MPP beta reacts strongly both as a MPP beta-beta-galactosidase- and as a glutathione S-transferase fusion protein with anti-FLAG M2 antibodies . Sequence analysis of MPP beta revealed a sequence motif with five out of eight amino acid residues identical to the FLAG peptide hitherto believed to be mono-specific.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 485 - 91
Specific aspartic acid-rich sequences are responsible for silver staining of nucleolar proteins; Valdez BC et al.; Ag-NOR proteins are silver-stainable proteins in the nucleolar organizer regions and are used to distinguish benign from malignant tumors . B23 and C23 are the two major Ag-NOR proteins . This study shows that only one of the two acidic clusters of B23 is responsible for its silver staining property . Fusion of this region of B23 (amino acids 161-188) to glutathione S-transferase produced an Ag-NOR positive fusion protein . The same result was obtained when amino acids 233-277 of C23 was fused with glutathione S-transferase . The aspartate residues, but not the glutamate residues, were found to be primarily responsible for the silver staining of the acidic clusters.

Gene, 1995 Feb 14, 153(2), 277 - 8
A murine Thy-1.2 reporter vector containing a SV40 origin for rapid cloning and analysis of eukaryotic promoters; Kadokawa Y et al.; A new vector, pATO, was constructed for rapid cloning and analysis of eukaryotic promoters . When a recombinant pATO, carrying a promoter sequence in its multiple cloning site, was introduced into COS cells, Thy-1.2 protein was produced on the cell surface, and was easily identified by an fluorescein-conjugated anti-Thy-1.2 antibody . The intensity of the fluorescence reflected the strength of the inserted promoter . Since pATO could replicate efficiently in COS cells, the recombinant plasmids recovered from a single COS cell were sufficient to transform Escherichia coli cells . This plasmid is applicable for the rapid and labor saving cloning of promoter elements.

Gene, 1995 Feb 14, 153(2), 197 - 202
Mammalian cell/vaccinia virus expression vectors with increased stability of retroviral sequences in Escherichia coli: production of feline immunodeficiency virus envelope protein; Wang RF et al.; Many eukaryotic DNA sequences, especially lenti-retrovirus proviral genomes and their env genes, are unstable when cloned in high-copy-number plasmids in Escherichia coli . Stability can be increased by the use of low-copy-number vectors, although plasmid yields are low . Vectors are described here that contain the intermediate-copy-number P15A ori for cloning, stable propagation and higher-yield production of plasmid DNA in E . coli, and the f1 ori for propagation as single-stranded phage . These vectors also have the capacity to direct high-yield production of protein in mammalian cells, and the option of incorporation into and expression via a T7 promoter in vaccinia virus . The SR alpha promoter, encephalomyocarditis (EMC) virus untranslated leader sequence, and poly(A) signal sequence serve as a high-yield mammalian cell expression cassette without the requirement for mRNA capping . A polyhistidine sequence is available at the 3' end of the cassette to facilitate chromatographic purification of protein . neo and gpt genes were included in some vectors to serve as selectable markers, and the dhfr gene was included in one to achieve gene amplification in mammalian cells . Dicistronic mRNAs can be generated by insertion of coding sequences up and downstream from the EMC leader . The utility of these vectors was shown through expression of feline immunodeficiency virus (FIV) Env protein, in conjunction with the tissue plasminogen activator (tPA) leader sequence.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 964 - 7
Maize branching enzyme catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli; Guan H et al.; The structure of alpha-glucan, isolated from wild-type Escherichia coli B, a glycogen branching enzyme (BE)-deficient E . coli AC71 (glgB-), or from AC71 transformed with genes coding for maize BEI and BEII individually as well as with both genes, was analyzed by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection . Transformation of the maize BE gene(s) in AC71 (glgB-) showed complementation in branching activity . Analysis by HPAEC revealed different structures between glycogen of E . coli B and alpha-glucan of AC71 transformed with a different maize BE gene(s) . The individual chains of the alpha-glucan debranched with isoamylase were distributed between chain length (CL) 3 and > 30 and the chain with CL 6 was the most abundant . In comparison with the glycogen of E . coli B, the alpha-glucan of AC71 transformed with the maize BE gene(s) consisted of a lesser amount of chains with CL 7-9 and a larger amount of chains with CL > 14 . It also showed a broad peak with chains of CL 9-12 as in maize amylopectin . This study provides in vivo evidence that glycogen BE and maize BE isozymes may have different specificities in the length of chain transferred . Furthermore, this study suggests that the specificity of glycogen synthase and starch synthase and their concerted action with BE play an important role in determining the structure of the polysaccharide synthesized.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1232 - 6
Amino acid sequence of rat kidney glutathione synthetase; Huang CS et al.; Glutathione (GSH) synthetase {gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3}, an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine . Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms . The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits . The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme . The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids . The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail . The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation . The cDNA was expressed in Escherichia coli . The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E . coli and shows some similarity to those deduced for the yeast and frog enzymes . Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1227 - 31
Contribution of cotranslational folding to the rate of formation of native protein structure; Fedorov AN et al.; To compare the process of protein folding in the cell with refolding following denaturation in vitro, we have investigated and compared the kinetics of renaturation of a full-length protein upon dilution from concentrated urea with the rate of folding in the course of biosynthesis . Formation of enzymatically active bacterial luciferase, an alpha beta heterodimer, occurred 2 min after completion of beta-subunit synthesis in an Escherichia coli cell-free system . Renaturation of urea-denatured beta subunit, either in the presence of the cell-free protein synthesis system or in buffer solutions, proceeded more slowly . Cellular components present in the cell-free protein synthesis system slightly accelerated the rate of refolding of urea-unfolded beta subunit . The results indicate that the luciferase beta subunit begins the folding process cotranslationally and that cotranslational folding contributes to the rapid formation of the native structure in the cell.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1213 - 7
Identification of galectin-3 as a factor in pre-mRNA splicing; Dagher SF et al.; Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells . Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate . Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation . Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity . Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity . The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity . The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution . Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1172 - 6
A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli; Fan C et al.; Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4) . a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology . Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core . Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids . In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1117 - 21
Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements; Josaitis CA et al.; Escherichia coli uses at least two regulatory systems, stringent control and growth-rate-dependent control, to adjust rRNA output to amino acid availability and the steady-state growth rate, respectively . We examined transcription from rrnB P1 promoters containing or lacking the cis-acting UP element and FIS protein binding sites after amino acid starvation . The "core promoter" responds to amino acid starvation like the full-length wild-type promoter; thus, neither the UP element nor FIS plays a role in stringent control . To clarify the relationship between growth-rate-dependent regulation and stringent control, we measured transcription from growth-rate-independent promoters during amino acid starvation . Four rrnB P1 mutants defective for growth-rate control and two other growth-rate-independent promoters (rrnB P2 and pS10) still displayed stringent regulation . Thus, the two systems have different promoter determinants, consistent with the idea that they function by different mechanisms . Two mutations disrupted stringent control of rrnB P1: (i) a multiple base change in the "discriminator" region between the -10 hexamer and the transcription start site and (ii) a double substitution making the promoter resemble the E sigma 70 consensus promoter . These results have important implications for the mechanisms of both stringent control and growth-rate-dependent control of rRNA transcription.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1113 - 6
Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4; Heilek GM et al.; Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit . Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA . {Fe-Cys31}S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins . Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain . One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies . A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome . These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1053 - 6
Contributions made by individual methylation sites of the Escherichia coli aspartate receptor to chemotactic behavior; Shapiro MJ et al.; To determine the extent to which chemotactic behavior depends on methylation at multiple sites, chemotaxis assays were performed on bacteria that expressed mutant aspartate receptors in which methylation site residues were mutated from glutamate to aspartate . It was found that chemotaxis was impaired when methylation sites were mutated and that the effect on chemotaxis of mutating a rapidly methylated site was more severe than the effect of mutating a less-rapidly methylated site . Expression of mutant receptors in a wild-type strain interfered with chemotaxis to only a minor extent . In vivo methylation assays showed that the chemotactic defects of most mutants could be explained by the decreased rates at which methylation levels increased in response to aspartate.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1043 - 7
Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach; Cui K et al.; NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH . To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli . The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH . This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions . The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis . The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates . With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer . With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer . These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor . This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.

Biochemistry, 1995 Feb 14, 34(6), 1959 - 67
Influence of alpha-subunits on the high-pressure stability of apo and holo beta 2-subunits in the bienzyme complex tryptophan synthase from Escherichia coli; Sindern S et al.; At a hydrostatic pressure of up to 2 kbar, the isolated alpha-subunit of tryptophan synthase from Escherichia coli proved to be a stable enzyme by virtue of specific activity as well as UV absorption and fluorescence emission spectra . The protein can therefore be regarded as a suitable effector for the investigation of structure-function relationships in the dimeric beta 2-subunit under the influence of high hydrostatic pressure . Complete deactivation of the beta 2-component in the alpha 2 beta 2 bienzyme complex occurs above 1300 bar (midpoint of transition for alpha apo beta 2, 790 bar; for alpha 2 holo beta 2, 1057 bar) . Sucrose (13%) shifts both midpoints of transition to values higher by about 300 bar . As shown by sucrose gradient centrifugation and limited trypsinolysis, deactivation of the beta 2-dimer is paralleled by dissociation into denatured beta-chains . At 10 degrees C, the corresponding dissociation constants K at 1 bar as well as the reaction volumes of dissociation delta V are calculated as 4.2 x 10(-9) M and -196 mL/mol for the apo-beta 2-component and as 9.8 x 10(-19) M and -632 mL/mol for the holo-beta 2-component in the bienzyme complex . Furthermore, large negative activation volumes are determined, reflecting the rate increase with increasing pressure: -89 mL/mol for the apo-beta 2-dimer and -195 mL/mol for the holo-beta 2-dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 14, 34(6), 1921 - 9
Characterization of two membrane-bound forms of OmpA; Rodionova NA et al.; The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy . In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands . For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC) . Limited proteolysis revealed two membrane-bound forms of OmpA, namely an "adsorbed" (35 kDa) and an "inserted" (30 kDa) form {Surrey, T., & Jahnig, F . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 7457-7461} . Which form was found after membrane binding and refolding depended on the lipids used and on the temperature . Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms . The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form) . Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal . The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 14, 34(6), 1867 - 77
Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy; Jones BE et al.; Time-resolved fluorescence techniques were utilized to monitor the kinetic refolding reaction of Escherichia coli dihydrofolate reductase (DHFR) . Measurements of emission and anisotropy decay lifetimes of both the five intrinsic tryptophan residues and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) during the folding reaction were used to characterize the compactness and development of tertiary structure in transient intermediates formed during the folding of DHFR . Experiments monitoring bound ANS show that a rapidly-formed intermediate (< 20 ms) has a rotational time of approximately 10 ns and, therefore, a compactness similar to that for the native conformation . All of the tryptophan residues in this burst phase species rotate as freely as in the unfolded state . In the set of four intermediates which then appear over the next few hundred milliseconds, the apparent rotational time measured by ANS fluorescence increases to a maximum rotational time of approximately 20 ns . An increase in the average tryptophan lifetime for these intermediates suggests these side chains become excluded from solvent and associated dynamic quenching mechanisms . As the folding reaction proceeds to a set of four native conformers the bound ANS rotational time then decreases to approach that for the native protein, 10.5 ns, and the average tryptophan rotational time increases to the same value . During these rate-limiting, final steps in folding, the static quenching effects which reflect the formation of specific tertiary contacts involving tryptophans also appear.

Biochemistry, 1995 Feb 14, 34(6), 1845 - 50
Enhanced binding of enterotoxigenic Escherichia coli K99 to amide derivatives of the receptor ganglioside NeuGc-GM3; Lanne B et al.; A natural receptor in pig small intestine {Teneberg, S., Willemsen, P., de Graaf, F . K., & Karlsson, K.-A . (1990) FEBS Lett . 263, 10-14} for the enterotoxigenic bacteria Escherichia coli K99 is the ganglioside NeuGc-GM3 (NeuGc alpha 3Gal beta 4Glc beta Cer) {e.g., H . Smit, W . Gaastra, J . P . Kamerling, J . F . G . Vliegenthart, & F . K . de Graaf (1984) Infect . Immun . 46, 578-584} . Chemical modifications of the carboxyl group of this ganglioside were performed, giving five different amides, the methyl ester, and the primary alcohol . The products were purified, and their structures were investigated by negative FAB mass spectrometry . Binding of E . coli K99 was tested by incubating 35S-labeled bacteria with derivatized compounds separated on thin-layer chromatograms . Modification of the carboxyl group to a primary amide strengthened the binding at least 5-fold, as estimated from autoradiography of dilutions on thin-layer plates . Some strengthening of the binding was also obtained with the methylamide as well as with the carboxyl group reduced to the alcohol . The ethylamide bound equally well as the underivatized NeuGc-GM3 . Amide substituents as large as propyl amide and benzyl amide were still recognized by the bacteria, although they bound weaker . The methyl ester was not stable in the chromatogram-binding assay with silica gel and water present, and it reverted to the acid.

Eur J Pharmacol, 1995 Feb 14, 274(1-3), 225 - 8
A hypotensive response induced by des-Arg9-bradykinin in young brown/Norway rats pretreated with endotoxin; Tokumasu T et al.; A hypotensive effect of intravenously injected des-Arg9-bradykinin was found in Brown/Norway strain young male rats which were pretreated with a small amount of endotoxin 24 h before the experiment, whereas the hypotensive effect of bradykinin was unaffected by the endotoxin . The potency of des-Arg9-bradykinin for the hypotensive effect was comparable to that of bradykinin . On the other hand, in endotoxin-pretreated aged rats, this effect of des-Arg9-bradykinin was not observed . Only during the intravenous infusion of des-Arg9{Leu8}bradykinin, a bradykinin B1 receptor antagonist, was the hypotensive effect of des-Arg9-bradykinin inhibited, whereas that of bradykinin was potentiated . After the end of infusion of des-Arg9{Leu8}bradykinin, the response to des-Arg9-bradykinin rapidly recovered . The results suggest the possibility that des-Arg9-bradykinin might play a role in inflammatory diseases.

FEBS Lett, 1995 Feb 13, 359(2-3), 123 - 5
GroES and the chaperonin-assisted protein folding cycle: GroES has no affinity for nucleotides; Todd MJ et al.; The E . coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins . GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold . On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested {Martin, et al., Nature, 366 (1993) 279-282} . We confirm the photolabeling of GroES with 8-azido-ATP . However, other proteins not known to contain nucleotide binding sites also became photolabeled suggesting that labeling is non-specific . Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected . We conclude that GroES has no nucleotide binding site.

Am J Physiol, 1995 Feb, 268(2 Pt 1), L239 - 44
Response of cultured human pulmonary artery endothelial cells to endotoxin; Meyrick B et al.; Endotoxemia is the leading cause of the adult respiratory distress syndrome . The effects of endotoxin on pulmonary endothelium, both in vivo and in culture, are diverse and complicated, and vary between species and cellular origin . Species such as sheep and cows are particularly sensitive to endotoxin, whereas rats and mice are more resistant . Studies using cultured pulmonary endothelial cells confirm these findings . Such species variations lead us to question whether human pulmonary artery endothelial cells (HPAEC) are directly affected by endotoxin . The present study examined the effects of endotoxin on HPAEC . Cells were exposed to endotoxin (0.001-10 micrograms/ml) for 24 h and were examined by phase-contrast microscopy, and measurements were made of lactate dehydrogenase, prostacyclin, and prostaglandin E2 release in the cell-free supernatant . In the presence of serum, endotoxin doses as small as 0.01 microgram/ml resulted in endothelial retraction and pyknosis compared with controls (P < 0.05) . Exposure to 10 micrograms/ml of endotoxin resulted in a significant increase in the number of pyknotic cells (P < 0.05), and lactate dehydrogenase release paralleled this finding . Endotoxin also resulted in a gradual increase in prostaglandin E2 release, reaching significance at 1 and 10 micrograms/ml of endotoxin (P < 0.05) . A similar trend was noted for prostacyclin release . We conclude that the direct cytotoxic effects elicited by endotoxin on HPAEC may contribute to the onset of pulmonary edema in patients with adult respiratory distress syndrome.

Nucleic Acids Res, 1995 Feb 11, 23(3), 370 - 6
Rho-independent terminators without 3' poly-U tails from the early region of actinophage øC31; Ingham CJ et al.; Previous work has identified three intergenic regions from the early region of actinophage oC31 where transcription was either terminated or the mRNA was processed . Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated eta, etb and etc . Transcripts through eta-c would be expected to form stable RNA stem-loops but would lack poly-U tails . Eta-c contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT . A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by eta-c from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphII) . In pUGT1 etb was at best a minor terminator in vivo whilst eta and etc exhibited strong termination activity . In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing eta-c inserted downstream . All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies eta > etc > etb . As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated . Termination at etb was unaffected by the presence or absence on the transcription template of CIR3 . CIR4 forms the central 17 bp of etc and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro . Eta was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of eta inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination . Taken together these data show that eta-c are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

J Mol Biol, 1995 Feb 10, 246(1), 8 - 13
Refined structures of two insertion/deletion mutants probe function of the maltodextrin binding protein; Sharff AJ et al.; The X-ray structures of the maltose bound forms of two insertion/deletion mutants of the Escherichia coli maltodextrin binding protein, MalE322 and MalE178, have been determined and refined . MalE322 involves a one residue deletion, two residue insertion in a hinge segment connecting the two (N and C) domains of the protein, an area already identified as being critical for the correct functioning of the protein . MalE178 involves a nine residue deletion and two residue insertion in a helix at the periphery of the C-domain . The function of both mutant proteins is similar to the wild-type, although MalE322 increases the ability to transport maltose and maltodextrin whilst inhibiting the ability of the cell to grow on dextrins . Both proteins exhibit very localized and conservative conformational changes due to their mutations . The structure of MalE322 shows some deformation of the third hinge strand, indicating the likely cause of change in its biochemistry . MalE178 is stable and its activity virtually unchanged from the wild-type . This is most likely due to the long distance of the mutation from the binding site and conservation of the number of interactions between the area around the deletion site and the main body of the protein.

J Mol Biol, 1995 Feb 10, 246(1), 54 - 62
Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388; Llosa M et al.; TrwC is required for conjugal DNA transfer of the broad host range plasmid R388 . The purified protein shows in vitro DNA helicase activity . Here we report that it also has in vitro oriT-endonuclease activity . TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS . Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence . Both the 5' and the 3' ends of the nick were mapped . The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC . Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction . Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites . During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.

J Mol Biol, 1995 Feb 10, 246(1), 35 - 42
A sequence-induced superhelical DNA segment serves as transcriptional enhancer; Brahms G et al.; The initiation of transcription at the sigma 54-dependent promoter glnAp2 of Escherichia coli is activated by the protein NR1(NTRC)-phosphate, which binds to two sites located upstream of the promoter that together constitute an enhancer . The cooperative binding facilitates the oligomerization of NR1-phosphate endowing it with the ATPase activity required for its ability to serve as transcriptional activator . We show here that these sites can be replaced by sequence-dependent superhelical inserts, lacking any homology to the nucleotide sequence of the enhancers . These superhelical inserts, irrespective of their chirality, are as effective as the paired sites in binding NR1-phosphate and in stimulating its oligomerization . We conclude that a specific sequence of nucleotides and the three-dimensional structure of DNA can determine its affinity for the NR1 activator protein capable of binding to DNA.

J Mol Biol, 1995 Feb 10, 246(1), 28 - 34
Tetravalent miniantibodies with high avidity assembling in Escherichia coli; Pack P et al.; We have designed tetravelent miniantibodies assembling in the periplasm of Escherichia coli . They are based on single-chain Fv fragments, connected via a flexible hinge to an amphipathic helix which tetramerizes the molecule . The amphipathic helix is derived from the coiled coil helix of the transcription factor GCN4, in which all hydrophobic a positions of every heptad repeat have been exchanged to leucine and all d positions to isoleucine . Gel filtration shows tetramer assembly of the miniantibody even at low concentrations . As expected, the functional affinity (avidity) of the tetravalent miniantibody is higher in ELISA and BIAcore measurements than that of the bivalent construct and the gain is dependent on surface epitope density.

J Mol Biol, 1995 Feb 10, 246(1), 227 - 39
The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution; Mirwaldt C et al.; The crystal structure of dihydrodipicolinate synthase from E . coli was determined by multiple isomorphous replacement methods . The structure was refined at a resolution of 2.5 A and the final R-factor is 19.6% for 32,190 reflections between 10.0 A and 2.5 A and F > 2 sigma (F) . The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry . A tetramer with approximate 222 symmetry is built up by crystallographic symmetry . The tetramer is almost planar with no contacts between the subunits related by the non-crystallographic dyad . The active sites are accessible from a wide water-filled channel in the center of the tetramer . The dihydrodipicolinate synthase monomer is composed of two domains . Each polypeptide chain is folded into an 8-fold alpha/beta barrel and a C-terminal alpha-helical domain comprising residues 224 to 292 . The fold is similar to that of N-acetylneuraminate lyase . The active site lysine 161 is located in the alpha/beta barrel and has access via two entrances from the C-terminal side of the barrel.

J Mol Biol, 1995 Feb 10, 246(1), 132 - 43
Intramolecular transmission of the ATP regulatory signal in Escherichia coli aspartate transcarbamylase: specific involvement of a clustered set of amino acid interactions at an interface between regulatory and catalytic subunits; De Staercke C et al.; Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP . Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites . The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP . These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain . Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding . These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model . This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.

J Mol Biol, 1995 Feb 10, 246(1), 1 - 7
Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex; Lazzaroni JC et al.; TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope . TolQ and TolR span the inner membrane with three and with one alpha-helical segments, respectively . The tolQ925 mutation (A177V), located in the third putative transmembrane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts and tolerance towards colicin A but not colicin E1, unlike a null tolQ mutation, which induces tolerance to all group A colicins . Since TolQ is required for colicin A and E1 uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutation might affect an interaction between TolQ and TolR . We therefore searched for suppressor mutations in TolR that would restore cell envelope integrity and colicin A sensitivity to the tolQ925 mutant . Five different tolR alleles were isolated and characterized . Four of these suppressor mutations were found to be clustered in the single putative transmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein . In addition, we isolated a spontaneous intragenic suppressor localized in the first transmembrane helix of TolQ (TolQ-I) . These observations strongly suggest that TolR and TolQ interact via their transmembrane segments . Sequence analysis indicates that Ala177 lies on the alpha-helix face of TolQ-III that, according to its composition and evolutionary conservation, is the most likely to be involved in protein/protein interaction . Energy minimization of atomic models of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a direct steric hindrance of this residue with neighboring transmembrane segments, and that suppressor mutations may alleviate this effect either directly or indirectly, e.g . by affecting the stability of conformational equilibrium of the transmembrane region of the complex.

J Biol Chem, 1995 Feb 10, 270(6), 2815 - 7
Assembly of F0 sector of Escherichia coli H+ ATP synthase . Interdependence of subunit insertion into the membrane; Hermolin J et al.; The F0 sector of the Escherichia coli H+ transporting ATP synthase is composed of a complex of three subunits, each of which traverses the inner membrane . We have studied the interdependence of subunit insertion into the membrane in a series of chromosomal mutants in which the primary mutation prevented insertion of one of the F0 subunits . Subunit insertion was assessed using Western blots of mutant membrane preparations . Subunit b and subunit c were found to insert into the membrane independently of the other two F0 subunits . On the other hand, subunit a was not inserted into membranes that lacked either subunit b or subunit c . The conclusion that subunit a insertion is dependent upon the co-insertion of subunits b and c differs from the conclusion of several studies, where subunits were expressed from multicopy plasmids.

J Biol Chem, 1995 Feb 10, 270(6), 2607 - 13
Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning; Swoboda I et al.; Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity . Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques . This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants . However, until now, only limited structural data for Bet v 1 have been published . Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F . D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W . R., Valent, P., Kungl, A . J., Breitenbach, M., Kraft, D., and Scheiner, O . (1993) J . Biol . Chem . 268, 19574-19580) . Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry . Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5'-terminal primer and a nonspecific 3'-terminal primer or by immunological screening of a birch pollen cDNA library . These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M . (1989) EMBO J . 8, 1935-1938) is now referred to as Bet v 1a . High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen . Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.

J Biol Chem, 1995 Feb 10, 270(6), 2563 - 70
C-terminal post-translational proteolysis of plant lectins and their recombinant forms expressed in Escherichia coli . Characterization of "ragged ends" by mass spectrometry; Young NM et al.; Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli . The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to "ragged" ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains . Recombinant forms of the lectins from E . corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms . Discrepancies between the protein and cDNA sequences of the E . corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated . Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment . This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.

J Biol Chem, 1995 Feb 10, 270(6), 2489 - 96
Effect of the FruR regulator on transcription of the pts operon in Escherichia coli; Ryu S et al.; The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRP.cAMP) . In addition, glucose stimulates pts operon expression in vivo . The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRP.cAMP-binding sites . The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR . In vitro, FruR can repress P1b transcription, which is activated by CRP.cAMP, and restore P1a transcription, which is repressed by CRP.cAMP . FruR can displace CRP.cAMP from its binding site in the presence of RNA polymerase even though FruR and CRP.cAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase . FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose . Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR- mutant cells as in cells grown in the presence of glucose . These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.

J Biol Chem, 1995 Feb 10, 270(6), 2447 - 50
Heat-inducible DNA binding of purified heat shock transcription factor 1; Goodson ML et al.; The heat-induced expression of heat shock proteins, called the cellular stress response, is mediated by heat shock transcription factor 1 (HSF1) . HSF1 exists in unstressed cells in an inactive form, which is converted to the DNA binding from upon exposure of cells to elevated temperature . We have developed a protocol for isolation of the non-DNA binding form of recombinant mouse HSF1, involving expression and affinity purification of HSF1 as a fusion with the glutathione S-transferase protein in Escherichia coli, followed by specific protease cleavage to release pure HSF1 protein . We report here that the purified inactive HSF1 can be converted to the DNA binding form by heat treatment in vitro . Chemical cross-linking analysis demonstrates that this conversion is accompanied by oligomerization of HSF1 from a monomeric to a trimeric native structure, similar to that observed for HSF1 in heat-shocked cells . These results indicate that elements residing in the HSF1 polypeptide are sufficient both for maintenance of this factor in the non-DNA binding from and for its heat-induced conversion to the DNA binding form and support a role for HSF1 as the "molecular thermostat" in eukaryotic cells, which senses adverse environmental conditions and activates the cellular stress response.

J Biol Chem, 1995 Feb 10, 270(6), 2443 - 6
Generation of the glycyl radical of the anaerobic Escherichia coli ribonucleotide reductase requires a specific activating enzyme; Sun X et al.; The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure . The radical is generated by an enzyme system present in E . coli . The reductase is coded for by the nrdD gene located at 96 min . Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase . The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase . The "activase" contains iron, required for activity . The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase . For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.

Science, 1995 Feb 10, 267(5199), 897 - 9
Selfish behavior of restriction-modification systems; Naito T et al.; Plasmids carrying gene pairs encoding type II DNA restriction endonucleases and their cognate modification enzymes were shown to have increased stability in Escherichia coli . The descendants of cells that had lost these genes appeared unable to modify a sufficient number of recognition sites in their chromosomes to protect them from lethal attack by the remaining restriction enzyme molecules . The capacity of these genes to act as a selfish symbiont is likely to have contributed to the evolution of restriction-modification gene pairs.

Anal Biochem, 1995 Feb 10, 225(1), 94 - 100
Alternative methods of preparing whole-cell DNA from fungi for dot-blot, restriction analysis, and colony filter hybridization; Min J et al.; There is a large and increasing number of methods for preparing whole-cell DNA from fungi . Modifications have evolved for two reasons . This first is to simplify the protocol as much as possible to allow processing of large sample numbers, in some cases for very specific uses, e.g., dot-blots . The second is to increase the quality of the DNA . Most preparations are contaminated with varying amounts of polysaccharides and unknown wall contaminants that can inhibit subsequent restriction or ligation . The extent of contamination varies with the species, the individual isolate, and at least in Neurospora, with the method or extent of growth . This paper offers three new methods . The first is a simplified procedure for isolating denatured DNAs from filamentous fungi for dot-blot analysis . The second is a rapid method for isolating DNAs from large numbers of small- to medium-scale cultures of filamentous fungi . These preparations are sufficiently pure for a variety of enzymatic reactions . The third is a nonenzymatic method for yeast colony filter hybridization that is simple, inexpensive, and efficient and results in uniform signals for a variety of species.

Anal Biochem, 1995 Feb 10, 225(1), 18 - 23
Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring; Bouhss A et al.; GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase . The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes . The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N . The proton NMR spectrum of {15N}GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz . When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

Anal Biochem, 1995 Feb 10, 225(1), 81 - 8
Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence; Fadden P et al.; Reaction conditions were defined for the selective quantitative derivatization and fluorophore labeling of phosphoserine residues on peptides and proteins . Phosphoserine was derivatized with 1,2-ethanedithiol using a modification of the reaction conditions defined by R . C . Clark and J . Dijkstra (1967) Int . J . Biochem . 11, 577-585 and H . E . Meyer, E . Hoffman-Posorke, H . Korte, and M . G . Heilmeyer (1986) FEBS Lett . 204, 61-66 for stabilizing the phosphoamino acid during Edman degradation reactions . Following derivatization, the thiol-serine residues were coupled to fluorescence by iodoacetate reaction . Characterization by capillary zone electrophoresis and laser-induced fluorescence allowed quantitation of phosphoserine content of peptides and proteins at < 75 amol . In three separate experiments, the overall reaction efficiency for 1,2-ethanedithiol derivatization of phosphoserine was estimated at 89.27 +/- 2.44% (SDM) . Subsequent coupling of the derivatized serine residue with 6-iodoacetamidofluoroscein was estimated at > 98% efficiency . Fluorescent probe tagging of phosphoamino acids on proteins and peptides offers direct quantitative evaluation of cellular phosphorylation states at the attomole level in tissue samples derived from plants, animals, and humans, without the use of radioisotopes, antibodies, or mass spectrometry.

J Mol Biol, 1995 Feb 10, 246(1), 144 - 63
Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme; Mandel AM et al.; Ribonuclease H is an endonuclease that hydrolyzes the RNA moiety of RNA-DNA duplex molecules . Escherichia coli ribonuclease H is involved in DNA replication, and retroviral ribonuclease H is essential for reverse transcription of the viral genome . To characterize the intramolecular dynamical properties of E . coli ribonuclease H, spin-lattice relaxation rate constants, spin-spin relaxation rate constants and steady state nuclear Overhauser effects for the 15N nuclear spins were measured by using proton-detected heteronuclear NMR spectroscopy . The relaxation data were analyzed by using a series of dynamical models in conjunction with a statistical model selection protocol . Ribonuclease H exhibits a complex array of dynamical features, most notably in the parallel beta-strands of the principal five-stranded beta-sheet, the coiled-coil helical interface, the active site, and the loop regions surrounding the active site . The dynamical properties are correlated with local structural environments of the 15N spins and suggest possible relationships to the functional properties of ribonuclease H . Results for E . coli ribonuclease H are compared to previously reported results for the human immunodeficiency virus type 1 ribonuclease H domain of reverse transcriptase.

J Biol Chem, 1995 Feb 10, 270(6), 2716 - 21
Mutation of Leu25 and Val27 introduces CC chemokine activity into interleukin-8; Lusti-Narasimhan M et al.; Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes . The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils . Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu25 in IL-8, is always replaced by tyrosine in CC chemokines . There is also a high degree of conservation among the CXC chemokines of the adjacent Val27 residue, which points out from the same side of the beta-sheet as Leu25 . In RANTES, Val27 is also replaced by a tyrosine . In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu25-->Tyr, Val27-->Tyr and the double mutant Leu25-->Tyr, Val27--> Tyr of IL-8 . These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material . All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils . The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in {125I}IL-8 competition assays . Additionally, the Leu25-->Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8 . We therefore studied the displacement of {125I}MIP-1 alpha by IL-8 Leu25-->Tyr from the CC-CKR-1 receptor . The mutant displaces MIP-1 alpha ligand with an affinity only 12-fold less than MIP-1 alpha itself . This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.

J Biol Chem, 1995 Feb 10, 270(6), 2483 - 8
Epitope insertions define functional and topological features of the Escherichia coli ferric enterobactin receptor; Armstrong SK et al.; The outer membrane protein FepA of Escherichia coli is the receptor for the ferric enterobactin siderophore complex and colicins B and D . A foreign antigenic determinant inserted into selected FepA sites allowed mutational analysis of receptor function and in situ immunological tracking of specific protein domains with respect to the bacterial cell compartment . Immunoblot analysis of bacterial proteins using an epitope-specific antibody detected the peptide determinant in the receptor fusions . The impact of the insertions on FepA function was examined by ferric enterobactin-mediated iron uptake experiments and colicin sensitivity tests . In all cases, FepA retained biological activity despite introduction of the foreign sequence . To further develop the topological model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their isolated membranes . One epitope resided in a region on the exterior of the cell, at the surface of the FepA protein, while other epitopes appeared to be localized to the periplasm or within the outer membrane.

Nature, 1995 Feb 9, 373(6514), 539 - 44
Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution; Jones EY et al.; The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref . 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref . 3) . VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily . In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents . The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains . Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup . We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1 . The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1 . This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules . These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.

Biochemistry, 1995 Feb 7, 34(5), 1787 - 97
Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains . Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies; Williams G et al.; The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor {De Vos, A . M., Ultsch, M., & Kossiakoff, A . (1992) Science 255, 306-312}, a distant relative in the cytokine receptor superfamily . In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold . The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli . Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding . Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells . Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure . The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way . The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.

Biochemistry, 1995 Feb 7, 34(5), 1686 - 94
Analysis of nuclear pore protein p62 glycosylation; Lubas WA et al.; Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation . The function and control of these post-translational modifications are poorly understood . Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate . Rat p62 was expressed in Escherichia coli and purified to near homogeneity . Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (Km = 0.30 microM) for the transfer of GlcNAc from UDP-GlcNAc (Km = 1.8 microM) . Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of {14C}GlcNAc-labeled proteolytic fragments . The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys22 and Lys97; the carboxyl terminus has one known glycosylation site at Ser471 . The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser270 to Thr294 . A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (Km = 30 microM) and a potent competitive inhibitor of p62 glycosylation (Ki = 15 microM) . It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains . O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.

Biochemistry, 1995 Feb 7, 34(5), 1678 - 85
Aminolevulinate synthase: functionally important residues at a glycine loop, a putative pyridoxal phosphate cofactor-binding site; Gong J et al.; 5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes . The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as its cofactor . Lysine-313 in murine erythroid aminolevulinate synthase has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate {Ferreira, G . C., Neame, P . J., & Dailey, H . A . (1993) Protein Sci . 2, 1959-1965} . However, other residues involved in binding and orienting the cofactor remain unknown . We studied the informational content of each residue within an 11 amino acid glycine-rich region, which we propose to be part of the phosphate-binding motif, based on amino acid sequence comparison with other pyridoxal 5'-phosphate-dependent enzymes and nucleotide-binding proteins . Partial random mutagenesis of this region in murine erythroid aminolevulinate synthase gene was followed by an efficient biological selection, using a hemA- Escherichia coli strain to recover functional unnatural enzymes . Among the total of 5444 variants produced, 283 were found to be functional . DNA sequencing results of 226 functional mutants indicated that most residues in this region contained a low informational content, being able to tolerate several other amino acid substitutions . However, three residues, namely, Arg-149, Gly-142, and Gly-144, were found to contain high informational content; Arg-149 was conserved in all of the functional mutants sequenced, while Gly-142 and Gly-144 could only tolerate alanine replacement . Two codon-specific random libraries of Arg-149, and Gly-142 and -144, respectively, were constructed to test further the stringency of these three positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 7, 34(5), 1661 - 8
X-ray absorption spectroscopy of the iron site in Escherichia coli Fe(III) superoxide dismutase; Tierney DL et al.; The local structure of the iron site in ferric superoxide dismutase from Escherichia coli has been characterized by X-ray absorption spectroscopy . In the resting state of the enzyme at pH 7.0, the iron is five-coordinate with an average metal-ligand bond length of 1.98 A . Binding of azide causes a reduction in the intensity of the bound state 1s-->3d transition and an increase of 0.08 A in average bond length . Both are indicative of an increase in the iron coordination number . Raising the pH from 7.0 to 10.5 causes a similar 0.08 A increase in the average bond length, again suggesting an increase in the iron coordination number . At intermediate pH (9.4), the average bond length is 2.03 A, consistent with an approximately 50:50 mixture of the limiting high and low pH forms . Similarly, the absorption edge structure varies continuously from pH 7 to 10.5 . These spectra can be fit to a titration curve with a pKa of approximately 9.8 . These data suggest that the pH-dependent transition, previously identified by UV-vis, EPR, and activity measurements, may be the conversion of the iron from five- to six-coordinate, presumably through coordination by hydroxide . The 1s-->3d transition for ferric superoxide dismutase at high pH is broader but not significantly less intense than that at pH 7 . This suggests that the high pH form may be significantly distorted from octahedral symmetry . At pH 7, the ferric and ferric + azide samples undergo slow X-ray induced photoreduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 7, 34(5), 1635 - 45
Determination of local protein structure by spin label difference 2D NMR: the region neighboring Asp61 of subunit c of the F1F0 ATP synthase; Girvin ME et al.; Purified subunit c from the H(+)-transporting F1F0 ATP synthase of Escherichia coli folds as an antiparallel pair of extended helices in a solution of chloroform-methanol-water . A similar hairpin-like folding is predicted for the native protein in the multisubunit transmembrane Fo sector of the ATP synthase . A single Cys variant (A67C) of subunit c was created and modified with a maleimido-PROXYL {{3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyl}oxy} spin label . Pairs of 1H 2D correlation and NOE spectra were collected with the nitroxide oxidized (paramagnetic) and reduced (diamagnetic) . The pairs of spectra were subtracted, yielding difference spectra containing only cross-peaks from 1H within 15 A of the spin label . These greatly simplified spectra were easily analyzed to provide complete assignments for residues 10-25 and 52-79 of the protein, 150 NOE distance restraints, and 27 hydrogen-bonding restraints . The chemical shifts and NOE patterns observed in the derivatized mutant were virtually identical to those which were resolved in the unmodified wild-type protein, strongly suggesting that the spin label was not perturbing the protein structure . The restaints enabled us to calculate a detailed structure for this region of subunit c . The structure consisted of two gently curved helices, crossing at a slight (30 degrees) angle . The C-terminal helix was disrupted from Val60 to Ala62 near the essential Pro64 . Asp61, the residue thought to undergo protonation--deprotonation with each H+ transported across the membrane, was in ver der Waals contact with Ala24 . The proximity of these residues had been predicted from mutant analyses, where H+ translocation was retained on moving the Asp from position 61 to 24.

Biochemistry, 1995 Feb 7, 34(5), 1583 - 8
Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites; Pope B et al.; Gelsolin is composed of six repeating segments of sequence (G1-6) and contains three distinct actin binding sites, two that bind to G-actin and one that binds to filaments . The calcium-dependent actin monomer binding site present in the carboxyl-terminal half of the protein (G4-6) plays a critical role both in the cooperative binding of actin by gelsolin and in its nucleating activity . Here we have localized this actin binding site to segment 4 (G4) by expressing the segments G4, G4-5, G5, and G5-6 in Escherichia coli and analyzing their actin binding properties . In addition we have measured their calcium binding . G4-5 and G5-6 each bind a single calcium ion, but there is no binding by G4 or G5 . The affinity of binding by G5-6 is 10 times higher than that of G4-5, and calcium binding by G4-6 shows two sites of different affinity . Thus each actin binding site of gelsolin is restricted to a single segment (G1, G2, and G4), but the nonbinding segments G5 and G6 play an important role in the calcium regulation of actin binding and other activities of gelsolin.

Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 48 - 54
Channeling behavior and activity models for Escherichia coli K-12 acetohydroxy acid synthases at physiological substrate levels; Herring PA et al.; The channeling behavior of acetohydroxy acid synthases I and III (EC 4.1.3.18; AHAS) was studied by computer simulation of activities over a wide range of concentrations for the substrates pyruvate and 2-ketobutyrate . The ratios of reaction rates for both channels and three-dimensional plots of single-channel reaction rates versus substrate concentrations were introduced to compare the substrate channeling properties of the isozymes . Substrate ranges were identified in which AHAS I and III operated both channels, and in which they used only one . Kinetic constants were varied to simulate whether and how AHAS might be made channel-specific . Our study suggests that AHAS might be made channel-specific for acetolactate but not for acetohydroxybutyrate . We postulate specific physiological roles for AHAS I and III to support cell growth under conditions that vary the levels and balance of substrates.

Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 444 - 51
Determination of the photoaffinity-labeled site on the ligand-binding domain of retinoic acid receptor alpha; Sasaki T et al.; The ligand-binding domain of human retinoic acid receptor alpha (hRAR alpha) was photoaffinity-labeled with a fluorescent retinoid, ADAM-3, by the use of a recombinant fused protein constructed from a maltose-binding protein and the E/F-domain of hRAR alpha (MBP-RAR alpha/E), which was expressed in E . coli . The labeled site was identified as Arg-589 (this corresponds to amino acid residue 385 of hRAR alpha) or a residue in its vicinity.

Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 417 - 23
Putative hydrogen bond network in the heme distal site of horseradish peroxidase; Nagano S et al.; The N delta 1 atom of the distal His of several peroxidases is known to make a hydrogen bond with the side chain oxygen of Asn . Thus, a mutant horseradish peroxidase, in which Asn70 is replaced by Val, has been expressed in Esherichia coli to disrupt the putative hydrogen bond . Substitution of Asn70 to Val reduces the rate constant for the compound I formation from 1.6 x 10(7) (native) to 6 x 10(5) M-1s-1 . The rate constant for reduction of compound I of N70V by guaiacol has been also reduced from 7.8 x 10(6) (native) to 1.2 x 10(5) M-1s-1 . While compound I of N70V is stable and reduced to the resting state of the mutant without apparent formation of compound II at neutral pH, compound II of N70V is obtained as a stable intermediate at alkaline pH . Similar alteration of the reactivity has been observed in the reaction with guaiacol.

Mol Gen Genet, 1995 Feb 6, 246(3), 397 - 400
An unusual mutation in RepA increases the copy number of a stringently controlled plasmid (Rts1 derivative) by over one hundred fold; Yonemitsu H et al.; A copy number mutant of the Rts1 replicon (copy number 1-2 copies/cell) was obtained . A one-base substitution in the repA region results in a single amino acid change from histidine to asparagine at position 159 . This mutation increased the plasmid copy number by up to 120-fold depending upon the growth conditions . At 42.5 degrees C the plasmid with the wild type replicon was unstable while the mutated replicon was relatively stable.

FEBS Lett, 1995 Feb 6, 359(1), 89 - 92
Viral Q beta RNA as a high expression vector for mRNA translation in a cell-free system; Katanaev VL et al.; Dihydrofolate reductase (DHFR) mRNA was inserted into Q beta phage RNA instead of its coat protein cistron . Translation of this recombinant mRNA in the Escherichia coli cell-free system resulted in the synthesis of DHFR, which was two orders of magnitude higher than that in the case of translation of the control DHFR mRNA . Additionally, it resulted in a significantly enhanced synthesis of Q beta replicase as compared with its synthesis when the original Q beta RNA was used.

FEBS Lett, 1995 Feb 6, 359(1), 69 - 72
Quinacrine mustard and lipophilic cations inhibitory to both vacuolar H(+)-ATPase and F0F1-ATP synthase; Moriyama Y et al.; Various lipophilic cations, such as quinacrine mustard and dequalinium, which are known to inhibit mitochondrial F1-ATPase, strongly inhibited vacuolar H(+)-ATPase purified from bovine adrenal chromaffin granules . Quinacrine mustard bound irreversibly to vacuolar H(+)-ATPase subunit A, and the 115 kDa accessory polypeptide and dithiothreitol had no effect . The binding was competitively inhibited by chlorpromazine and quinacrine, and these compounds specifically reduced the amount of labeling of subunit A . Quinacrine mustard also prevented the binding of {alpha-32P}ATP to subunit A but had no effect on the binding of {3H}N-ethylmaleimide to either subunit A or the 115 kDa accessory polypeptide . These results suggest that the binding site of quinacrine mustard in subunit A is not related to the N-ethylmaleimide-binding site(s), which is important for activity.

FEBS Lett, 1995 Feb 6, 359(1), 20 - 2
Cold lability of the mutant forms of Escherichia coli inorganic pyrophosphatase; Velichko IS et al.; The variants of Escherichia coli pyrophosphatase carrying the substitutions Glu20-->Asp, His136-->Gln or His140-->Gln are inactivated, in contrast to the wild-type enzyme, at temperatures below 25 degrees C: their activity measured at 25 degrees C decreases with decreasing the temperature of the stock enzyme solution . The inactivation is completely reversible and is explained by cold-induced dissociation of these hexameric enzymes into less active trimers.

Eur J Pharmacol, 1995 Feb 6, 273(3), 281 - 4
Protective effect of DS-4574, a peptidoleukotriene receptor antagonist, against endotoxin-induced intestinal injury in rats; Tabuchi Y et al.; We evaluated the protective effect of DS-4574 (6-(2-cyclohexylethyl)-{1,3,4}thiadiazolo{3,2-a}-1,2,3- triazolo{4,5-d}pyrimidin-9(3H)-one), a peptidoleukotriene receptor antagonist, against intestinal mucosal injury evoked by endotoxin in rats by exploring changes in hematocrit and plasma leakage along with morphological features . Treatment with Escherichia coli endotoxin (5 mg/kg i.v.) alone elicited hemoconcentration, vasocongestion and a marked mucosal necrosis . DS-4574 (10-50 mg/kg) effectively prevented these changes on either oral or intraduodenal administration . These results demonstrate that peptidoleukotrienes may be key mediators in the intestinal injury induced by endotoxin in rats.

Gene, 1995 Feb 3, 153(1), 99 - 104
Cloning, sequencing and expression of serine/threonine kinase-encoding genes from Streptomyces coelicolor A3(2); Urabe H et al.; A 6.3-kb DNA fragment encoding two eukaryotic-type serine/threonine protein kinases (Ser/Thr PK) was cloned from Streptomyces coelicolor A3(2) by using a PCR product obtained with primers based on highly conserved regions of eukaryotic Ser/Thr PK . The nucleotide (nt) sequence of the essential 4.4-kb fragment contained two possible ORFs . One ORF (PkaA) contained 543 amino acids (aa), while another (PkaB) consisted of 417 aa . The N-terminal half of both proteins showed significant similarity with the catalytic domain of eukaryotic Ser/Thr PK . On the other hand, the C-terminal region of PkaA, but not of PkaB, is rich in Pro and Gln residues, indicating that PkaA works as a PK as well as a transcription factor . The pkaB gene was overexpressed in Escherichia coli, and the gene product (PkaB) was found to be phosphorylated mainly at Thr . The pkaA gene was also overexpressed in E . coli, and the gene product (PkaA) was found to be phosphorylated mainly at Thr and slightly at Ser . In the case of PkaA, at least 100 aa residues from the C terminus were not essential for the PK activity . When the PCR product was used as a probe, it hybridized to DNA fragments from all the Streptomyces species tested, indicating that these types of Ser/Thr PK are distributed ubiquitously and play significant physiological roles in the various species of Streptomyces.

Gene, 1995 Feb 3, 153(1), 9 - 15
Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12; Scarabel M et al.; A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase . The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin . After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis . Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.

Gene, 1995 Feb 3, 153(1), 67 - 70
Cloning and sequencing of a previously unidentified gene that is involved in the biosynthesis of heme in Escherichia coli; Nakayashiki T et al.; We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell . Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA . This new gene, designated hemK, was located at 27 min on the linkage map of the E . coli chromosome . By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases . The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA . An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities . H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.

Gene, 1995 Feb 3, 153(1), 49 - 55
A group-I intron in the mitochondrial large-subunit ribosomal RNA-encoding gene of Dictyostelium discoideum: same site localization in alga and in vitro self-splicing; Angata K et al.; A 547-bp group-I intron belonging to subgroup IA1 was found near the 3' end of the large subunit ribosomal RNA-encoding gene (LSUrRNA) in the mitochondrial (mt) DNA of the cellular slime mold Dictyostelium discoideum . This intron was inserted in a highly conserved stretch within the sequence that encodes the peptidyl transferase center domain V in the corresponding region of the Escherichia coli LSUrRNA . Interestingly, the insertion site of the intron is the same as that of the So.LSU.2 intron of the green alga, Scenedesmus obliquus, mt DNA and the Pw.LSU.2 intron of the colorless alga, Prototheca wickerhamii, mt DNA . The intron could self-splice in vitro at a concentration higher than 20 mM MgCl2 . Polymerase chain reaction analysis showed the possible existence of an intron similar to that of D . discoideum LSUrRNA in another cellular slime mold, Polysphondylium pallidum (CK-8), but not in D . mucoroides (Dm7 and Dm11).

Gene, 1995 Feb 3, 153(1), 41 - 8
Three genes hrdB, hrdD and hrdT of Streptomyces griseus IMRU 3570, encoding sigma factor-like proteins, are differentially expressed under specific nutritional conditions; Marcos AT et al.; Three genes (hrd) homologous to the rpoD gene of Escherichia coli, that encode sigma factor-like proteins, have been cloned from DNA of the candicidin-producing strain Streptomyces griseus IMRU 3570 . They are located in different regions of the chromosome . Sequence analysis showed that the first one is analogous to the hrdB gene of S . coelicolor . The second showed high similarity to the hrdD gene of S . coelicolor and S . aureofaciens and is linked, as in S . coelicolor, to a N-acetyltransferase-encoding gene (nat) distantly related to the pat and bar genes that encode resistance to bialafos . The third showed no close homology with other known hrd genes from actinomycetes and has been named hrdT . Functional domains in the three S . griseus Hrd proteins are highly conserved in relation to those of the sigma 70 protein family . Northern analysis showed that hrdB is expressed as a 1.9-kb transcript during active growth in phosphate-rich medium, but it is less efficiently transcribed under sporulation conditions (phosphate-starved) or after a heat-shock treatment . Two other shorter transcripts of 1.2 and 0.7 kb were also detected with the same probe . The hrdD gene is transcribed as a single 1.1-kb transcript under sporulation conditions following nutritional shiftdown and, to a lower extent, during growth conditions in phosphate-rich medium . The hrdT gene is weakly transcribed (1.5-kb RNA) under all conditions tested . The hrd-encoded sigma factors probably recognize actinomycetes promoters (SEP type) with E . coli-like consensus sequences.

Gene, 1995 Feb 3, 153(1), 141 - 2
Cloning and sequencing of the putative Azospirillum brasilense gene encoding GTP cyclohydrolase II; Van Bastelaere E et al.; Sequence analysis of a fragment of Azospirillum brasilense DNA revealed the presence of a ribA homologue, of which the 3' portion encodes a putative GTP cyclohydrolase II . The 5' portion (approx . half of the ORF) does not show homology to any other sequence from the databases.

J Mol Biol, 1995 Feb 3, 245(5), 549 - 58
Characterization of the 5 S RNA binding activity of Xenopus zinc finger protein p43; Zang WQ et al.; One major component of the Xenopus 42 S ribonucleoprotein (RNP) storage particle is the p43 protein . The 5 S RNA binding protein is structurally similar to TFIIIA, containing nine zinc finger domains . The RNA binding properties of recombinant p43 were characterized using a nitrocellulose filter binding assay . The experimental conditions necessary for in vitro p43-5 S RNA complex formation include: pH 7.5, 0.1 M KCl and incubation at 22 degrees C . Under these conditions, the protein binds to Xenopus oocyte 5 S RNA with an apparent association constant of 1.61(+/- 0.12) x 10(9) M-1 . A series of mutations in 5 S RNA were used to determine which sequence and structural features of the 5 S RNA are required for high affinity binding of p43 . The primary contact points for p43 include the sequences and structures of stems II, V and loop D of the 5 S RNA . Although p43 and TFIIIA are structurally similar and are both relatively insensitive to mutations in the 5 S RNA, they do require different features of the 5 S RNA molecule for high affinity binding.

J Mol Biol, 1995 Feb 3, 245(5), 474 - 85
Role of NusA in L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli; Sha Y et al.; The transcription of the 11 gene S10 operon of Escherichia coli is autogenously regulated by one of the operon's products, ribosomal protein L4 . This protein stimulates termination of transcription in vivo at a specific site within the S10 leader . The in vivo effect can be reproduced in a purified transcription system but requires an additional factor, NusA . Our earlier in vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused complexes are further stabilized by L4, which presumably accounts for L4's stimulation of termination in vivo . Here we show that NusA is not absolutely required for RNA polymerase to recognize the attenuation site: at low (5 microM) UTP concentration, RNA polymerase pauses at the site, although the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein . Furthermore, RNA polymerase pausing at the attenuation site is not sufficient for the L4 effect, since L4 cannot stabilize a transcription complex paused at the attenuation site in the absence of NusA . We have been able to isolate paused complexes formed without NusA and/or L4; such complexes are active upon re-addition of NTPs, and respond as expected to the addition of L4 or NusA . Our experiments are consistent with the notion that L4 is a stable component of a paused transcription complex.

J Mol Biol, 1995 Feb 3, 245(5), 467 - 73
Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli; Goldman E et al.; Insertion of nine consecutive low-usage CUA leucine codons after codon 13 of a 313-codon test mRNA strongly inhibited its translation without apparent effect on translation of other mRNAs containing CUA codons . In contrast, nine consecutive high-usage CUG leucine codons at the same position had no apparent effect, and neither low- nor high-usage codons affected translation when inserted after codon 223 or 307 . Additional experiments indicated that the strong positional effect of the low-usage codons could not be accounted for by differences in stability of the mRNAs or in stringency of selection of the correct tRNA . The positional effect could be explained if translation complexes are less stable near the beginning of a message: slow translation through low-usage codons early in the message may allow most translation complexes to dissociate before they read through.

J Biol Chem, 1995 Feb 3, 270(5), 2190 - 7
Substructure of the amidotransferase domain of mammalian carbamyl phosphate synthetase; Guy HI et al.; The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases . The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified . The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active . The kcat is 347-fold higher and the KGlnm is 40-fold lower than the complete GLNase domain . Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E . coli CPSase synthetase subunit . Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity . The 19-kDa amino half, the interaction subdomain, binds tightly to the E . coli CPSase large subunit . Thus, the GLNase domain consists of two subdomains which can autonomously fold and function . The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis . The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.

J Biol Chem, 1995 Feb 3, 270(5), 2183 - 9
The DnaK chaperone system of Escherichia coli: quaternary structures and interactions of the DnaK and GrpE components; Schonfeld HJ et al.; The DnaK (Hsp70), DnaJ, and GrpE heat shock proteins of Escherichia coli constitute a cellular chaperone system for protein folding . Substrate interactions are controlled by the ATPase activity of DnaK which itself is regulated by the nucleotide exchange factor GrpE . To understand the structure-function relationship of this chaperone system, the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes were analyzed by gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and native gel electrophoresis . GrpE formed dimers in solution . DnaK formed monomers, dimers, and higher mole mass oligomers, the equilibrium between these forms being dependent on the DnaK concentration . The behavior of DnaK and GrpE in gel filtration and dynamic light scattering suggested elongated shapes of both molecules . In the absence of added nucleotides, DnaK and GrpE formed stable complexes containing one molecule of DnaK and two molecules of GrpE . A 44-kDa N-terminal ATPase fragment of DnaK also formed complexes with GrpE with the same 1:2 stoichiometry . DnaK-GrpE complex formation was unaffected by elimination of DnaK-bound nucleotides or addition of saturating concentrations of a DnaK peptide substrate . These findings allow the correlation of DnaK-GrpE interactions with a role for GrpE in the functional cycle of the DnaK chaperone system.

J Biol Chem, 1995 Feb 3, 270(5), 2024 - 31
Unwinding of nucleosomal DNA by a DNA helicase; Eggleston AK et al.; We have asked whether a DNA helicase can unwind DNA contained within both isolated native chromatin and reconstituted chromatin containing regularly spaced arrays of nucleosome cores on a linear tandem repeat sequence . We find that Escherichia coli recBCD enzyme is capable of unwinding these DNA substrates and displacing the nucleosomes, although both the rate and the processivity of enzymatic unwinding are inhibited (a maximum of 3- and > 25-fold, respectively) as the nucleosome density on the template is increased . The observed rate of unwinding is not affected if the histone octamer is chemically cross-linked; thus, dissociation, or splitting, of the histone octamer is not required for unwinding to occur . The unwinding of native chromatin isolated from HeLa cell nuclei occurs both in the absence and in the presence of linker histone H1 . These results suggest that as helicases unwind DNA, they facilitate nuclear processes by acting to clear DNA of histones or DNA-binding proteins in general.

J Mol Biol, 1995 Feb 3, 245(5), 486 - 98
RNA determinants required for L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli; Sha Y et al.; We have probed regions of the S10 leader RNA to determine their role in L4-mediated, NusA-dependent attenuation control of the S10 ribosomal protein operon . Using genetic and "antisense" oligonucleotide competition approaches, we were able to distinguish between the determinants necessary for intrinsic (NusA-independent) pausing by RNA polymerase at the S10 attenuation site, for NusA-dependent enhancement of pausing, and for L4 stabilization of the paused ternary complex . The upper stem-loop structure in the attenuator hairpin is the major determinant for the NusA-dependent pause, while the sequence at the site of pausing is important for RNA polymerase to pause in the absence of NusA . The determinants for L4 stabilization of the paused complex include the hairpin immediately upstream of the attenuator hairpin as well as the ascending side of the attenuator structure . In conclusion, our results suggest that there are three distinct pausing activities by RNA polymerase during its transcription of the S10 leader, with three corresponding signals in the S10 leader.

Rev Saude Publica, 1995 Feb, 29(1), 75 - 9
{Hydrogen peroxide as despyrogenation agent for medical and hospital product components}; Pinto TJ; The current search for alternative solvents to fluorinated products, in face of the depletion of ozone in the stratosphere calls for the development of alternative processes applicable to the manufacture of medical devices . In view of the challenge and as water is serving particularly as the choice for other industrial segments, the fast macrobial proliferation in it is of concern, as it is a potential source of endotoxins . Such a risk is inconsistent with the production of items designed for example, for use in surgical procedures in the cardio-vascular field . Thus research was undertaken for the purpose of investigating the possibility of using water as a cleansing agent for components for such products, providing that hydrogen peroxide is added . The work was carried out by inoculating water and peroxide at levels of 0.1, 0.25, 0.5 e 1.0 UE/ml . The confirmation was obtained as to the active effectiveness at a 5% concentration of hydrogen peroxide by means of analytical determination using the "in vitro" method . The effectiveness of the use of peroxide was investigated on polycarbonate injected parts designed for the manufacture of oxigenators and blood reservoirs and contained with endotoxins . The findings permit one to draw a favorable conclusion regarding the adequacy of the proposed process both biologically as well as regards the removal of impurities.

Photochem Photobiol, 1995 Feb, 61(2), 171 - 4
Photorepair of nonadjacent pyrimidine dimers by DNA photolyase; Kim ST et al.; Photolyases reverse the harmful effects of UV light on cells by converting pyrimidine dimers (Pyr{}Pyr) into two pyrimidine monomers by utilizing near-UV and visible light . Previous work has shown that photolyase repairs T{c,s}T and T{t,s}T in DNA as well as U{}U in RNA, all of which are formed by joining the two adjacent pyrimidines in a light-dependent reaction . In this report, we show that Pyr{}Pyr formed in nonadjacent pyrimidines are also substrates for DNA photolyase.

Immunity, 1995 Feb, 2(2), 149 - 54
Direct binding of the Mtv7 superantigen (Mls-1) to soluble MHC class II molecules; Mottershead DG et al.; The superantigen encoded by the mouse mammary tumor virus (MMTV) is a potent stimulator of T cells when bound to MHC class II molecules . Recent data from this laboratory have shown that the Mtv7 superantigen, Mls-1, elicits a strong T cell response when presented by HLA-DR . To expand these observations further, we have produced the 28 kDa extracellular domain and the 18 kDa carboxy-terminal subfragment of the Mls-1 protein in E . coli and studied their interaction with human MHC class II molecules in vitro . In this report, we demonstrate direct binding of these recombinant forms of the Mls-1 protein to soluble HLA-DR1 and HLA-DR4, but not to HLA-A2 . Our data imply a unique class II interaction site of retroviral superantigens that is not shared with bacterial superantigens.

Plant Mol Biol, 1995 Feb, 27(3), 629 - 33
A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase; Verwoert II et al.; In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated . Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.

Plant Mol Biol, 1995 Feb, 27(3), 607 - 17
Structure and expression of the Chlamydomonas reinhardtii alad gene encoding the chlorophyll biosynthetic enzyme, delta-aminolevulinic acid dehydratase (porphobilinogen synthase); Matters GL et al.; cDNA clones for the alad gene encoding the chlorophyll biosynthetic enzyme ALA dehydratase (ALAD) from Chlamydomonas reinhardtii were isolated by complementation of an Escherichia coli ALAD mutant (hemB) . The C . reinhardtii alad gene encodes a protein that has 50 to 60% sequence identity with higher plant ALADs, and includes a putative Mg(2+)-binding domain characteristic of plant ALADs . Multiple classes of ALAD cDNAs were identified which varied in the length of their 3'-untranslated region . Genomic Southern analysis, using an ALAD cDNA as a probe, indicates that it is a single-copy gene . This suggests that the differently sized ALAD cDNAS are not the products of separate genes, but that a primary ALAD transcript is polyadenylated at multiple sites . A time course determination of ALAD mRNA levels in 12-h light:12-h dark synchronized cultures shows a 7-fold increase in ALAD mRNA at 2 h into the light phase . The ALAD mRNA level gradually declines but continues to be detectable up to the beginning of the dark phase . ALAD enzyme activity increases 3-fold by 6 h into the light phase and remains high through 10 h . Thus, there is an increase in both ALAD mRNA level and ALAD enzyme activity during the light phase, corresponding to the previously observed increase in the rate of chlorophyll accumulation.

Int J Radiat Biol, 1995 Feb, 67(2), 187 - 91
Occupational exposure to radiation induces an adaptive response in human lymphocytes; Barquinero JF et al.; The in vitro pretreatment of phytohaemagglutinin stimulated human lymphocytes with tritiated thymidine or with low doses of X-rays induces a response that makes these cells less susceptible to further genetic damage induced by subsequent high doses of radiation . This phenomenon has been called 'Adaptive Response' because it is similar to the one described in E . coli . In the present study, we describe that lymphocytes irradiated in vitro at 2 Gy, from individuals occupationally exposed to X and gamma rays, show lower frequencies of dicentrics than those from non-occupationally exposed individuals irradiated in the same conditions . Our results could indicate that an adaptive response can also be induced in lymphocytes in vivo by very low occupational doses of radiation, and that for biological dosimetry purposes it is necessary to know the individual's history of exposure to mutagens.

Biophys Chem, 1995 Feb, 53(3), 259 - 66
Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli; Igarashi Y et al.; The molecular architecture of native GroEL has been studied by solution X-ray scattering . The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C . The maximum dimension was estimated to be 170 A, based on the pair distance distribution function . A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A . The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A . Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.

Epidemiol Infect, 1995 Feb, 114(1), 113 - 21
Clonal diffusion of EPEC-like Escherichia coli from rabbits as detected by ribotyping and random amplified polymorphic DNA assays; Leroy-Setrin S et al.; The genetic diversity and clonal relationships among 77 Escherichia coli strains isolated in France from diarrhoeic rabbits and that belonged to seven O serogroups including the predominant O103 serogroup, were estimated by ribotyping and random amplified polymorphic DNA (RAPD) assays . Fifteen ribotypes were defined . Most of the highly pathogenic O103 strains could be assigned to two major groups . Non-pathogenic strains were clearly distinguished . RAPD assays generally matched ribotyping, or gave more precision for subdividing strains from the two main O103 groups . The results on strains isolated from different areas and over a 9-year period showed the relevance of the association of these two methods for the survey of the spread of strains in breeding flocks and illustrated clonal diffusion in rabbit production structures.

Eur J Biochem, 1995 Feb 1, 227(3), 903 - 8
Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo; Ingledew WJ et al.; The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site . The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone . We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone . The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10 . The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5 . The two-electron couple has an Em7 of approximately 70 mV . Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction . The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion . In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals . Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).

Eur J Biochem, 1995 Feb 1, 227(3), 816 - 22
Substitution of Arg230 and Arg233 in Escherichia coli elongation factor Tu strongly enhances its pulvomycin resistance; Boon K et al.; Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP) . Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334 . In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis . We have purified two mutants species, {R233S}EF-TuHis and {R230V, R233F}EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography . In this study, we describe the in vitro characterization of these mutant proteins . The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance . Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin . Moreover, the mutant {R230V, R233F}EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Crit Care Med, 1995 Feb, 23(2), 308 - 15
Inhibitory effect of Escherichia coli endotoxin on skeletal muscle contractility; Gutierrez G et al.; OBJECTIVE: Endotoxemia in rabbits is associated with decreases in oxygen transport, tissue hypoxia, metabolic acidosis, and impaired oxygen extraction . In this study, we tested the hypothesis that endotoxin also inhibits skeletal muscle contractility directly . DESIGN: Randomized animal study . SETTING: Accredited animal research facility . SUBJECTS: New Zealand white rabbits of either sex, weighing 2.55 +/- 0.20 kg . INTERVENTIONS: We compared two groups of rabbits (n = 10 each) undergoing continuous electrical stimulation of the left hindlimb (maximal isometric twitch contraction at 0.25 Hz) . One group (septic) was given an intravenous infusion of Escherichia coli endotoxin . The control group was subjected to decreases in cardiac output by inflating a balloon placed in the right ventricle . MEASUREMENTS AND MAIN RESULTS: Endotoxin or balloon inflation resulted in comparable decreases in cardiac output (49% and 53%, respectively) . Hindlimb oxygen transport decreased to similar values for both groups (4.9 +/- 0.3 and 4.2 +/- 0.5 mL/min/kg, respectively) . Systemic oxygen extraction ratio was greater in the control group (0.72 +/- 0.03) than in the septic group (0.55 +/- 0.04; p < .05) . There were no differences in hindlimb oxygen extraction ratio . Decreases in hindlimb forces were greater in the septic group (42 +/- 4%) than in the control group (18 +/- 3%, p < .01) . Force frequency curves obtained at the beginning and the end of the experiment showed greater fatigue in the septic group . CONCLUSIONS: The intravenous infusion of Escherichia coli endotoxin produces a direct inhibitory effect on skeletal muscle contractility in rabbits . This phenomenon is independent of decreases in oxygen transport and blood pH . Our data support the notion of a direct cellular effect of endotoxin, or of an associated cytokine, on skeletal muscle contractility . The mechanism responsible for this phenomenon is unknown.

Am J Respir Cell Mol Biol, 1995 Feb, 12(2), 142 - 8
ICAM-1 expression on bronchial epithelium after recombinant adenovirus infection; Pilewski JM et al.; Early experience with recombinant adenoviruses for gene transfer to airway epithelium suggests that these vectors are associated with the development of inflammation . The mechanisms for this are unclear, but previous work has shown that respiratory viruses can cause increased expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells . We therefore hypothesized that recombinant adenoviruses may induce ICAM-1 expression and thereby facilitate the development of airway inflammation . To address this, primary cultures of human bronchial epithelial cells were examined for ICAM-1 expression by flow cytometry after infection with a serotype 5, E1/E3-deleted recombinant adenovirus containing the Escherichia coli LacZ reporter gene driven by the cytomegalovirus promoter (Ad.CMVlacZ) . Compared with control cells, ICAM-1 expression was unchanged after infection with Ad.CMVlacZ, but increased after infection with wild-type adenovirus . Treatment of Ad.CMVlacZ-infected cells with interferon-gamma (IFN) resulted in increased ICAM-1 expression, but to a lower level than that seen in cells treated with IFN alone, indicating that recombinant adenovirus infection blunted IFN-induced up-regulation of ICAM-1 . Adhesion of human leukocytes to human bronchial epithelial cells was not increased after Ad.CMVlacZ infection, thereby excluding an ICAM-1-independent increase in leukocyte-epithelial adhesion . The results for ICAM-1 expression were confirmed in vivo, as immunostaining of human bronchial xenografts infected with Ad.CMVlacZ revealed basilar epithelial staining with ICAM-1, but no increased expression on cells expressing beta-galactosidase . This study demonstrates that unlike other respiratory viruses, recombinant E1/E3-deleted adenovirus does not cause increased ICAM-1 expression on human bronchial epithelium in vitro or in vivo nor increased leukocyte adhesion in vitro.

Arch Biochem Biophys, 1995 Feb 1, 316(2), 917 - 25
Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis; Okamoto K et al.; Argingipain, so termed due to its peptide cleavage specificity at arginine residue, is a unique extracellular cysteine proteinase produced by the anaerobic rod Porphyromonas gingivalis, which is known as a major pathogenic factor of the progressive periodontal disease (T . Kadowaki, M . Yoneda, K . Okamoto, K . Maeda, and K . Yamamoto (1994) J . Biol . Chem . 269, 21371-21378) . The catalytic specificity and functional importance of this enzyme prompted us to elucidate its structural features . A DNA fragment for argingipain was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the NH2-terminal amino acid sequence of the purified enzyme . Although the extracellular mature enzyme was shown to have an apparent molecular mass of 44 kDa in gels, the nucleotide sequence of the isolated gene revealed a single gene coding for a 109-kDa precursor of argingipain . The deduced amino acid sequence exhibited no significant similarity to the sequences of representative members of the cysteine protease family . The precursor contained four functional domains: the NH2-terminal signal peptide required for the inner membrane transport; the NH2-terminal prosequence, which is assumed to stabilize the precursor structure; the proteinase domain; and the COOH-terminal hemagglutinin domain, which appears to be essential for extracellular secretion of the proteinase domain . Experiments involving the addition of the argingipain inhibitors to the culture medium of P . gingivalis suggested that the maturation of argingipain occurs intracellularly via an autocatalytic cleavage of the pro-argingipain propeptide.

Am J Physiol, 1995 Feb, 268(2 Pt 2), R514 - 9
Endotoxin tolerance alters thermal response of guinea pigs to systemic infusions of tumor necrosis factor-alpha; Roth J et al.; In guinea pigs, intra-arterial infusions of 5 micrograms/kg tumor necrosis factor-alpha (TNF; specific activity 20,000 U/micrograms; duration of infusion 45-50 min) evoked a biphasic elevation of abdominal temperature lasting approximately 6 h . One week after systemic infusion of TNF, the animals started to receive either five intramuscular injections of bacterial lipopolysaccharide (LPS from Escherichia coli; 20 micrograms/kg) or equivalent volumes of solvent (0.9% NaCl) in intervals of 3 days . Fever in response to repeated injections of LPS was progressively attenuated; the animals developed endotoxin tolerance . Repeated injections with solvent did not cause any measurable changes in abdominal temperature . Three days after the fifth injection of LPS or solvent, all animals again received an intra-arterial infusion of 5 micrograms/kg TNF . In guinea pigs injected five times with solvent, the biphasic elevation of abdominal temperature could again be observed in response to systemic infusions of TNF . In endotoxin-tolerant animals, however, only the first peak of the biphasic thermal response lasting approximately 60 min could be monitored after infusions with TNF . The second phase of the thermal response to administration of TNF, which normally lasts > 5 h, was abrogated almost completely . The significant suppression of the febrile response to TNF infusions did not seem to be caused by a more rapid elimination of TNF from the circulation of endotoxin-tolerant guinea pigs . Circulating levels of TNF, measured 60 and 180 min after the start of TNF infusions, were not different before and after development of endotoxin tolerance . In conclusion, the development of endotoxin tolerance in guinea pigs is accompanied by a reduced responsiveness to TNF administered exogenously.

Am J Physiol, 1995 Feb, 268(2 Pt 1), G374 - 9
Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function; Spitz J et al.; The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive . Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins . The consequences of these intracellular perturbations on host cell function, however, have not been determined . The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function . T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative . Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain . Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity . Dual 22Na+ and {3H}mannitol flux studies localized the permeability defect to tight junctions . In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC . Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment . These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

Am J Physiol, 1995 Feb, 268(2 Pt 1), G361 - 73
Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers; Salzman AL et al.; We tested the hypothesis that nitric oxide (NO) modulates the permeability of tight junctions in a model intestinal epithelium (Caco-2BBe monolayers) . Incubation with sodium nitroprusside (SNP) resulted in time- and concentration-dependent decreases in transepithelial resistance . Permeability to fluorescein sulfonic acid increased during incubation for 24 h in the presence of 1.25 mM SNP, 5 mM S-nitroso-N-acetylpenicillamine (SNAP), or 1% NO gas . SNP-induced hyperpermeability was not due to loss of cell viability, as confirmed by intact ultrastructure, unaltered lactate dehydrogenase release, and ability to recover baseline permeability . Incubation with SNP increased permeability but only minimally increased intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) . Incubation with Escherichia coli heat-stable enterotoxin greatly increased cGMP levels with only a minimal effect on permeability . Cellular ATP levels decreased after incubation with SNP, SNAP, or gaseous NO . Incubation with SNP led to diminished fluoresceinphalloidin staining of junctional actin (confocal microscopy) and widened tight junctions (electron microscopy) . We conclude that NO reduces ATP levels and reversibly increases the permeability of tight junctions in cultured Caco-2BBe cells.

J Clin Invest, 1995 Feb, 95(2), 905 - 12
Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene; Xu W et al.; To investigate the molecular basis of the phenotypic heterogeneity in congenital erythropoietic porphyria, the mutations in the uroporphyrinogen III synthase gene from unrelated patients were determined . Six missense (L4F, Y19C, V82F, V99A, A104V, and G225S), a nonsense (Q249X), a frameshift (633insA), and two splicing mutations (IVS2+1 and IVS9 delta A + 4) were identified . When L4F, Y19C, V82F, V99A, A104V, 633insA, G225S, and Q249X were expressed in Escherichia coli, only the V82F, V99A, and A104V alleles expressed residual enzymatic activity . Of note, the V82F mutation, which occurs adjacent to the 5' donor site of intron 4, resulted in approximately 54% aberrantly spliced transcripts with exon 4 deleted . Thus, this novel exonic single-base substitution caused two lesions, a missense mutation and an aberrantly spliced transcript . Of the splicing mutations, the IVS2+1 allele produced a single transcript with exon 2 deleted, whereas the IVS9 delta A+4 allele was alternatively spliced, approximately 26% being normal transcripts and the remainder with exon 9 deleted . The amount of residual activity expressed by each allele provided a basis to correlate genotype with disease severity, thereby permitting genotype/phenotype predictions in this clinically heterogeneous disease.

J Bacteriol, 1995 Feb, 177(4), 998 - 1007
Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli; Thanassi DG et al.; Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance . Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form . Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed . In E . coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small . This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier . When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm . In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes . Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion . Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E . coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin . These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm . A quantitative model of tetracycline fluxes in E . coli cells of various types is presented.

J Bacteriol, 1995 Feb, 177(4), 953 - 63
Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli; Snyder WB et al.; The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming . This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming . We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein . This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C . Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure . To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct . We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery . This suggests that jamming is caused by LacZ folding . We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel . Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm . Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.

J Bacteriol, 1995 Feb, 177(4), 938 - 47
Identification of genes negatively regulated by Fis: Fis and RpoS comodulate growth-phase-dependent gene expression in Escherichia coli; Xu J et al.; Fis is a nucleoid-associated protein in Escherichia coli that has been shown to regulate recombination, replication, and transcription reactions . It is expressed in a transient manner under batch culturing conditions such that high levels are present during early exponential phase and low levels are present during late exponential phase and stationary phase . We have screened a random collection of transposon-induced lac fusions for those which give decreased expression in the presence of Fis . Thirteen different Fis-repressed genes were identified, including glnQ (glutamine high-affinity transport), mglA (methyl-galactoside transport), xylF (D-xylose-binding protein), sdhA (succinate dehydrogenase flavoprotein subunit), and a newly identified aldehyde dehydrogenase, aldB . The LacZ expression patterns revealed that many of the fusions were maximally expressed at different stages of growth, including early log phase, mid- to late log phase, and stationary phase . The expression of some of the late-exponential- and stationary-phase genes was dependent on the RpoS sigma factor, whereas that of others was affected negatively by RpoS . We conclude that Fis negatively regulates a diverse set of genes and that RpoS can function to both activate and inhibit the expression of specific genes.

J Bacteriol, 1995 Feb, 177(4), 926 - 31
Activation of the dephosphorylation of nitrogen regulator I-phosphate of Escherichia coli; Liu J et al.; The transcription of sigma 54 RNA polymerase-dependent nitrogen-regulated genes is activated by nitrogen regulator I (NRI)-phosphate . The kinase NRII is responsible for the phosphorylation of NRI . It has been shown that NRII also has the ability to dephosphorylate NRI-phosphate but only when PII is present at a concentration greatly in excess of that of NRII . We have now shown that glutamate enables PII to stimulate the dephosphorylation of NRI-phosphate when present in equimolar concentration with NRII . This effect of glutamate appears to be a backup control that becomes effective when the normal regulation of PII activity is disabled.

J Bacteriol, 1995 Feb, 177(4), 883 - 91
Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12; Zhao G et al.; We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP) . The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography . The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN) . In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (Km = 2 and 105 microM and kcat = 0.76 and 1.72 s-1 for PNP and PMP, respectively) and produces hydrogen peroxide . Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low kcat turnover number . Comparison of kcat/Km values suggests that PNP rather than PMP is the in vivo substrate of E . coli PdxH oxidase . In contrast, the eukaryotic enzyme has similar kcat/Km values for PNP and PMP and seems to act as a scavenger . E . coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 microM, respectively . Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence . Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E . coli growing exponentially in minimal medium plus glucose at 37 degrees C . Thus, E . coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis.

J Bacteriol, 1995 Feb, 177(4), 1119 - 20
A test of the directed mutation hypothesis in Escherichia coli MCS2 using replica plating; Sniegowski PD; Excision of phage Mu from Escherichia coli MCS2 was originally put forth as a clear example of directed mutation . This claim was considerably weakened, however, by subsequent evidence that Mu excision occurs during starvation regardless of fitness consequences . Here, I use the classical replica-plating technique to examine Mu excision during starvation of MCS2 . The results support the conclusion that Mu excision is not directed but is, instead, nonspecifically induced by starvation.

J Bacteriol, 1995 Feb, 177(4), 1086 - 9
Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus; Conley DL et al.; Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene . While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability . Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding . Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions . They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.

J Bacteriol, 1995 Feb, 177(4), 1023 - 9
Regulation of the molybdate transport operon, modABCD, of Escherichia coli in response to molybdate availability; Rech S et al.; The mod (chlD) locus at 17 min on the Escherichia coli chromosome encodes a high-affinity molybdate uptake system . To further investigate the structure and regulation of these genes, the DNA region upstream of the previously identified modBC (chlJD) genes was cloned and sequenced . A single open reading frame, designated modA, was identified and appears to encode a periplasmic binding protein for the molybdate uptake system . To determine how the mod genes are regulated in response to molybdate, nitrate, and oxygen, we constructed a series of mod-lacZ operon fusions to the upstream region and introduced them in single copy onto the E . coli chromosome . Whereas molybdate limitation resulted in elevated mod-lacZ expression, neither oxygen nor nitrate had any significant effect on gene expression . A regulatory motif, CATAA, located at the modA promoter was identified and shown to be required for molybdate-dependent control of the modABCD operon . Mutations within this sequence resulted in nearly complete derepression of gene expression and suggest that transcription of the operon is mediated by a molybdenum-responsive regulatory protein.

Hum Genet, 1995 Feb, 95(2), 171 - 3
The PKU mutation S349P causes complete loss of catalytic activity in the recombinant phenylalanine hydroxylase enzyme; Knappskog PM et al.; The mutation S349P in exon 10 of the phenylalanine hydroxylase (PAH) gene was identified in one Norwegian and one Polish phenylketonuria (PKU) allele on a haplotype 1.7 background . This missense mutation in PAH codon 349 is a T to C transition in cDNA position 1267 . This mutation has been reported both on haplotype 1 and 4, suggesting recurrent mutation . In two different expression systems, the pET and the pMAL systems of Escherichia coli, it was shown that the S349P mutation, introduced by site directed mutagenesis, results in complete loss of enzymatic activity . Thus, protein instability alone does not seem to be the direct cause of the lack of activity of this PKU mutation as previously reported.

EMBO J, 1995 Feb 1, 14(3), 610 - 8
The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during the rehydration phase; Bernacchia G et al.; Transketolases, key enzymes of the reductive and oxidative pentose phosphate pathways, are responsible for the synthesis of sugar phosphate intermediates . Here we report the first molecular analysis of transketolase genes from plants . Three distinct classes of transketolase-encoding cDNA clones were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum . One class represented by the transcript tkt3 is constitutively expressed in leaves and roots under all physiological conditions tested . By biochemical analysis and protein sequencing of purified transketolase, it was shown that tkt3 is expressed in three enzymatically active isoforms . An intriguing discovery was that accumulation of the two other transketolase transcripts, tkt7 and tkt10, is preferentially associated with the rehydration process of the desiccated plant; whereas tkt10 is only expressed in leaves, tkt7 was detected in leaves and roots . This observation suggests a possible role for these transketolases in the conversion of sugars, which are a major phenomenon in the rehydration process . Despite an abundant level of tkt7 and tkt10 transcripts in rehydrating leaves, proteins could not be isolated . This is due in part to a translational control mechanism acting on the loading of mRNAs to polysomes.

EMBO J, 1995 Feb 1, 14(3), 594 - 602
Quantitative control of the stationary phase-specific sigma factor, sigma S, in Escherichia coli: involvement of the nucleoid protein H-NS; Yamashino T et al.; In Escherichia coli, recent intensive studies revealed that expression of a certain subset of genes is under the control of the stationary phase-specific sigma factor, sigma S, which is encoded by the rpoS gene . Since sigma S functions predominantly under certain growth conditions, its activity and/or cellular content has accordingly to be tightly controlled, however, the underlying molecular mechanism is at present unclear . We previously demonstrated that expression of the cbpA gene, encoding an analogue of the DnaJ molecular chaperone, is largely dependent on sigma S function . Here we have found that a mutational lesion of the hns gene, which encodes one of the well-characterized nucleoid proteins, H-NS, affects the cellular content of sigma S remarkably and consequently affects the expression of cbpA . Enhanced accumulation of sigma S in hns deletion cells was particularly observed in the logarithmic growth phase and was demonstrated to result from an elevated translational efficiency of rpoS mRNA and also from an increased stability of newly synthesized sigma S . Although H-NS is known to influence the transcription of a number of apparently unlinked genes on the chromosome, in this study we provide a novel instance in which H-NS is deeply implicated in post-transcriptional regulation(s) of the expression of rpoS . As to physiological relevance, it was also demonstrated that hns deletion cells exhibit an extreme thermotolerance even in the logarithmic growth phase, presumably because of the enhanced accumulation of sigma S.

EMBO J, 1995 Feb 1, 14(3), 503 - 11
Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product; Parry D et al.; D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, regulate events in the G1 phase of the cell cycle and may contribute to the phosphorylation of the retinoblastoma gene product (Rb) . However, in cells in which the function of Rb has been compromised, either by naturally arising mutations or through binding to proteins encoded by DNA tumour viruses, Cdk4 and Cdk6 are not associated with D cyclins . Instead, both kinases form binary complexes with a stable 16 kDa protein (p16) encoded by the putative tumour suppressor gene INK4/MTS1 on human chromosome 9p21 . Here we show an inverse correlation between Rb status and the expression of p16 . Since Rb-negative cells express high levels of p16, we suggest that in these cells p16 competes with D cyclins for binding to Cdk4 and Cdk6 and prevents formation of active complexes . In line with these predictions, DNA tumour virus oncoproteins do not disrupt cyclin D1-Cdk4 complexes in cells lacking p16.

Virology, 1995 Feb 1, 206(2), 954 - 62
Readthrough protein associated with virions of barley yellow dwarf luteovirus and its potential role in regulating the efficiency of aphid transmission; Wang JY et al.; Purified particles of barley yellow dwarf luteovirus (BYDV) contain a major 22-kDa protein and a minor protein of approximately 58 kDa . The 22-kDa capsid protein is encoded by open reading frame (ORF) 3 . ORF 5 is immediately downstream and in frame with ORF 3 and a 72-kDa protein can be translated via a readthrough suppression of the ORF 3 termination codon . Antibodies were produced against two Escherichia coli expressed polypeptides that represent the amino- and carboxyl-terminal halves of a putative 50-kDa protein encoded by ORF 5 . Immunological analyses indicated that the 58-kDa protein associated with purified virions contained sequences encoded by ORF 3 and ORF 5 . The carboxyl terminal portion of the full-length (72 kDa) readthrough protein was absent from the 58-kDa protein . The full-length readthrough protein was detected in infected oat protoplasts and plant tissue, but was not associated with virus particles purified from plants . The carboxyl-terminal portion of the 72-kDa readthrough protein was not required for aphid transmission; however, virus was transmitted more efficiently from protoplast extracts containing virions and soluble 72-kDa readthrough protein than from mock-inoculated protoplast extracts to which plant purified virus was added . The full-length readthrough protein, although not required for transmission, may increase the transmission efficiency of BYDV by aphids.

Virology, 1995 Feb 1, 206(2), 787 - 95
Probing the structure of influenza B hemagglutinin using site-directed mutagenesis; Rivera K et al.; The crystal structure of the hemagglutinin (HA) of influenza virus A/Aichi/68 (H3N2) from the X-31 reassortant virus was reported in 1981, but as yet there are no X-ray diffraction structures for hemagglutinins of other types or even subtypes of influenza virus . We have used site-directed mutagenesis to probe the structure of the hemagglutinin of influenza B/Hong Kong/8/73 . We investigated a region in the globular head domain that is helical in the influenza A HA structure, targeting sidechains that in the H3 HA point toward solvent (Thr196) or into the receptor-binding pocket (Gln197) . None of the mutations affected hemagglutination activity, but mutations T196P or Q1971 eliminated binding of a monoclonal antibody . The data suggest that this region of the influenza B HA forms a surface structure different from the alpha-helix of the influenza A HA structure and that it accounts for much of the antigenic activity of influenza B HA.

Virology, 1995 Feb 1, 206(2), 1145 - 9
Identification of the cauliflower mosaic virus movement protein RNA-binding domain; Thomas CL et al.; The in vitro RNA-binding activity of the movement protein (P1) of cauliflower mosaic virus was studied after its expression in Escherichia coli, purification, and uv-crosslinking to a radioactive probe . It was found that insoluble P1 aggregates were involved in RNA-binding activity . A series of deletion mutants were used to identify a domain within P1 required for binding activity . The RNA-binding domain is located between amino acids 120 and 197 and includes the region of homology between P1 and the movement protein (P30) of tobacco mosaic virus (TMV) . The homologous region corresponds to part of RNA-binding domain "A" in TMV P30, but unlike domain A, the P1 domain is able to bind RNA out of the context of the complete protein . The P1 RNA-binding domain shows some structural similarity with RNA-binding domains of other proteins . The conservation of this domain in the caulimo- and badnaviruses provides support for the view that this activity has biological relevance.

J Leukoc Biol, 1995 Feb, 57(2), 275 - 81
Effects of bafilomycin A1 on functional capabilities of LPS-activated alveolar macrophages; Bidani A et al.; Resident alveolar macrophages (m phi) possess plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase) that plays a crucial role in regulation of intracellular pH (pHi) . To assess the importance of this V-ATPase to m phi effector functions, resident alveolar m phi from rabbits were activated with E . coli-derived lipopolysaccharide (LPS) and exposed to bafilomycin A1, a specific inhibitor of V-ATPase . Bafilomycin caused a significant cytosolic acidification in both the absence and presence of CO2-HCO3-, and in both unstimulated and activated m phi . Superoxide production and Fc receptor-mediated phagocytosis also were reduced in bafilomycin-treated m phi . Similar effects were elicited by acidifying the cytoplasm in the absence of bafilomycin, by lowering extracellular pH (pHo) from 7.4 to 6.5-6.6 . Thus, the effects of bafilomycin on phagocytosis and superoxide production probably were related to cytosolic acidification, secondary to blockade of V-ATPase-mediated H+ extrusion across the plasma membrane . Conversely, bafilomycin significantly increased TNF-alpha release . This effect cannot be explained by a bafilomycin-induced acidosis because acidic pHo significantly reduced TNF-alpha release . The results demonstrate that V-ATPase activity is an important determinant of the effector functions of LPS-activated m phi.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 999 - 1003
Glyoxalase III from Escherichia coli: a single novel enzyme for the conversion of methylglyoxal into D-lactate without reduced glutathione; Misra K et al.; A single novel enzyme, glyoxalase III, which catalyses the conversion of methylglyoxal into D-lactate without involvement of GSH, has been detected in and purified from Escherichia coli . Of several carbonyl compounds tested, only the alpha-ketoaldehydes methylglyoxal and phenylglyoxal were found to be substrates for this enzyme . Glyoxalase III is active over a wide range of pH with no sharp pH optimum . In its native form it has an M(r) of 82000 +/- 2000, and it is composed of two subunits of equal M(r) . Glutathione analogues, which are inhibitors of glyoxalase I, do not inhibit glyoxalase III . Glyoxalase III is found to be sensitive to thiol-blocking reagents . The p-hydroxymercuribenzoate-inactivated enzyme could be almost completely re-activated by dithiothreitol and other thiol-group-containing compounds, indicating the possible involvement of thiol group(s) at or near the active site of the enzyme.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 959 - 65
Conformational changes and in vitro core-formation modifications induced by site-directed mutagenesis of the specific N-terminus of pea seed ferritin; van Wuytswinkel O et al.; Plant ferritin has a three-dimensional structure predicted to be very similar to that of animal ferritin . It has, however, an additional specific sequence of 24 amino acids at its N-terminus named extension peptide (EP) . In order to determine precisely the interactions between EP and other domains of the pea seed ferritin subunit, three point mutations were performed . The mutated residues were chosen by three-dimensional computer modelling of the pea seed ferritin subunit structure {Lobreaux, Yewdall, Briat and Harrison (1992) Biochem . J . 228, 931-939} . The mutant recombinant proteins were expressed in Escherichia coli and purified to homogeneity; all the mutants were found to be assembled as 24-mers . When Ala-13 was replaced by His, as in mammalian ferritins, ferroxidase activity was significantly reduced . Moreover, in vitro iron-core formation in Pro-X-->Ala, Lys-R-->Glu and Ala-13-->His mutants was increased after denaturation by urea followed by renaturation; this was also observed with the EP deletion mutant (r delta TP/EP) . The recombinant ferritins were also analysed using tryptophan fluorescence spectra . The r delta TP/EP, Pro-X-->Ala and Lys-R-->Glu mutants were found to be more susceptible to denaturation by urea than the native r delta TP pea seed ferritin.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 791 - 8
The effect of histone H1 and DNA methylation on transcription; Johnson CA et al.; We have previously shown that DNA methylation acts as a focus for the formation of inactive chromatin in vivo . We have investigated the mechanism further by in vitro transcription of a template containing two tRNA genes and an extensive (G+C)-rich sequence characteristic of a CpG island . The extent of transcription from the unmethylated or fully methylated template was assayed in the presence of varied levels of histone H1 . The transcriptional activity of both templates was inhibited by increasing amounts of histone H1, although inhibition with the methylated template occurs at a lower H1:DNA ratio . The H1c variant shows the greatest preferential inhibition of the methylated template . We demonstrated that histone H1 complexed to DNA is one of the factors that inhibits transcription by preventing the formation of initiation complexes, particularly on methylated template, rather than the formation of disordered H1.DNA aggregates.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 767 - 75
A gelsolin-related protein from lobster muscle: cloning, sequence analysis and expression; Luck A et al.; The tail muscle of the lobster Homarus americanus contains an actin-binding protein with an apparent molecular mass of 105 kDa determined by SDS/PAGE and gelsolin-like properties . We isolated this protein and peptide sequences were obtained after limited proteolysis with chymotrypsin . A tail-muscle-specific cDNA library was constructed in a lambda expression vector and a full-length clone was obtained by screening with a polyclonal anti-(crustacean gelsolin) antibody . The cDNA insert of approx . 3.2 kb length was sequenced . The cDNA contained an open reading frame of 2.265 kb, and the deduced amino acid sequence of 754 residues (83,469 Da) identified the protein as a cytoplasmic member of the gelsolin/villin protein family . Comparison of the lobster gelsolin amino acid sequence with other members of this protein family revealed the characteristic 6-fold repeated segmental structure as well as the three conserved sequence motifs typical of each segment {Way and Weeds (1988) J . Mol . Biol . 203, 1127-1133} . Strong homologies were found with Drosophila gelsolin, human gelsolin, villin core, Dictyostelium severin and Physarum fragmin . In addition, the gelsolin-like protein from lobster muscle revealed motifs that were clearly similar to the actin-bundling region of human villin headpiece although it did not itself contain a distinct headpiece domain . The recombinant lobster gelsolin-like protein, expressed in Escherichia coli as a fusion protein, was purified from inclusion bodies and renatured as a functional protein . There were no significant differences in the biological activity tested between the recombinant and the native protein isolated from lobster muscle.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 753 - 9
Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26); Dilsiz N et al.; The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method . The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 {Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54} . Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli . A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane . A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification . Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26 . Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane . The majority of the protein was integrated into cell plasma membrane . MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column . Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation {Takemoto and Emmons (1991) Curr . Eye Res . 10, 863-869} . We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus . The mutation was confirmed by DNA sequencing . The mutant MIP26 protein was also expressed in E . coli and incorporated predominantly into the cytoplasmic membrane.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 707 - 10
Escherichia coli chorismate synthase: a deuterium kinetic-isotope effect under single-turnover and steady-state conditions shows that a flavin intermediate forms before the C-(6proR)-H bond is cleaved; Bornemann S et al.; We report the observation of a deuterium kinetic isotope effect for the conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (6proR2HV = 1.13 +/- 0.03) using recombinant chorismate synthase from Escherichia coli . Similar isotope effects were observed for the decay of a spectroscopically characterized flavin intermediate (6proR2Hk = 1.17 +/- 0.04) during single-turnover experiments . The main rate-limiting steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either before or concomitant with its decay.

J Gen Virol, 1995 Feb, 76 ( Pt 2), 431 - 5
Sequence and expression of the ns2 protein gene of human coronavirus OC43; Labonte P et al.; The complete nucleotide sequence of the ns2 gene of human coronavirus OC43 (HCV-OC43) was determined . Sequence analysis revealed an open reading frame that could encode a protein of 278 amino acids, with an estimated molecular mass of 32.2 kDa . Six potential phosphorylation sites are present but no sites of N-glycosylation were found . The amino acid sequence of the HCV-OC43 ns2 protein shows 92% identity with that of the Mebus strain of bovine coronavirus (BCV) . However, a stretch of nine consecutive amino acids near the C terminus is completely different, causing it to be very hydrophilic, which contrasts with the hydrophobic nature of this region in BCV . As shown by immunofluorescence with a monospecific antiserum, the ns2 protein was expressed in the cytoplasm of HCV-OC43-infected HRT-18 cells.

J Infect Dis, 1995 Feb, 171(2), 465 - 8
Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers; Nataro JP et al.; Enteroaggregative Escherichia coli (EAggEC) are diarrheal pathogens defined by aggregative adherence to HEp-2 cells . In an effort to identify pathogenic EAggEC isolates, four groups of 5 volunteers were fed 1 of 4 different EAggEC strains, each at a dose of 10(10) cfu . Strain 042 caused diarrhea in 3 of 5 adults; 3 other EAggEC isolates (17-2, 34b, and JM221) failed to elicit diarrhea . A gene encoding enterotoxin EAST1 was found in strains 042 and 17-2 but not 34b or JM221; a 108-kDa cytotoxin was expressed in all 4 isolates . All 4 isolates showed a modest degree of gentamicin protection in HEp-2 cells . 17-2, 34b, and JM221 expressed the fimbrial antigen AAF/I; 042 did not express this fimbria as determined by immunogold electron microscopy and genetic probe hybridization.

J Bacteriol, 1995 Feb, 177(3), 867 - 70
The oxygen affinity of cytochrome bo' in Escherichia coli determined by the deoxygenation of oxyleghemoglobin and oxymyoglobin: Km values for oxygen are in the submicromolar range; D'Mello R et al.; Apparent oxygen affinities for Escherichia coli cells and membranes containing a terminal oxidase with only one type of ligand-binding heme, cytochrome o', were measured with oxyleghemoglobin and oxymyoglobin as sensitive oxygen reporters . Two Km values (0.15 to 0.35 microM and 0.016 to 0.085 microM) were detected, well below values determined for the purified oxidase by insensitive electrode methods.

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