Escherichia coli

J Infect Dis, 1993 Feb, 167(2), 461 - 54
Endotoxin and tumor necrosis factor-alpha-induced interleukin-8 release in humans; van Deventer SJ et al.; Neutrophil recruitment and activation are thought to play an important role in tissue damage observed in septicemia . Interleukin-8 (IL-8) is a small cytokine with important neutrophil-activating and chemoattractant properties . IL-8 release was studied after injection of human volunteers with low doses of either endotoxin (2 ng/kg of body weight) or tumor necrosis factor-alpha (TNF alpha) (50 micrograms/m2) . After TNF-alpha injection, IL-8 appeared at 30 min, whereas increased levels were first observed after 90 min in endotoxin-challenged volunteers . Peak levels were measured at 120 min after both endotoxin (192 +/- 193 ng/L) and TNF alpha (500 +/- 236 ng/L) injection . These data indicate that IL-8 is released in humans after injection of endotoxin and TNF alpha and suggest that endotoxin-induced IL-8 release is mediated by TNF alpha.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1993 Feb, 13(2), 94 - 7, 69
{Herbal decoction of qingwen baidu yin in treating endotoxic fever in rabbits}; Xie T; Qingwen Baidu Yin (QBY) has good curative effects on the endotoxic fever of rabbits induced by injecting endotoxin of E . Coli . The test group was given QBY orally, while the control group was given NS orally instead . Result showed QBY could: (1) Markedly inhibit the fever, it was effective in reducing febrile curve . delta T and TRI5 of the test group were smaller (P < 0.001) . (2) Ameliorate the leukocytopenia and leukocytosis, and improve thrombocytopenia . (3) Antagonize hyperviscosity syndrome and had the actions of depolymerization and dilution . (4) In test group, the increased cAMP content in plasma was reduced, and the decreased cGMP content raised, the ratio of cAMP and cGMP was nearly normal . All these provided the clue in elucidating the essence of "Excessive Yang causes Heat" and "Predominance of Yang leads to disorder of Yin" . (5) Pathomorphological examination showed that QBY had the functions of protecting the internal organs and reducing the organic damage induced by endotoxing in rabbits.

Bioorg Khim, 1993 Feb, 19(2), 150 - 60
{Study of the function of multienzyme systems of Escherichia coli cellular structures in organic media microemulsions by (31)P-NMR and spin probing}; Nikolaev BP et al.; Dynamics of substrate pools and level of inorganic phosphate P(i) in the course of glucose-6-phosphate (G6P) utilization by Escherichia coli have been studied by 31P NMR and EPR methods on cells immobilised in a three component water-in-oil microemulsion composed from tridecane, nonionic surfactant Tween 81 and a low amount of water . The state of the electron transport chain in cell membranes has been evaluated on the basis of reduction rates of exogenous nitroxide radicals . Low water activity a(w) in the external microemulsion appears to inhibit, to some extent, glycolytic enzymes and electron transport proteins in the membrane structures without disturbing the membrane integrity and solubilization of lipoproteins in reverse micelles . Enzyme activity has been restricted by the micellar substrate diffusion and by decrease in water activity a(w) in the course of the extensive molecular interchange of bound water between the external microemulsion phase and the internal cell structures.

Protein Eng, 1993 Feb, 6(2), 189 - 93
Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases; Miyashita K et al.; Picornavirus 3C proteinases (3Cpro) are cysteine proteinases but recent sequence analyses have shown that they are related to trypsin-like serine proteinases . Two models of 3Cpro structure have been presented . Both models indicate that residues His40 and Cys147 are members of the catalytic triad but the models differ in the designation of the third member of the catalytic triad, which is assigned as either Glu71 or Asp85 . To test the importance of these four residues in the catalytic activity of 3Cpro of coxsackievirus B3, a member of the enterovirus subgroup of the picornavirus family, single amino acid substitutions were introduced at each of the four sites . All of these mutations resulted in the reduction or inactivation of autocatalytic cleavage of the 3C precursor protein expressed in Escherichia coli, suggesting that all of these residues are essential for the proteolytic reaction . The substitution of Cys147 with Ala abolished 3Cpro activity while the mutant in which Cys147 was replaced with Ser retained reduced proteolytic activity both in cis and in trans . Our results strongly support the proposal that Cys147 of 3Cpro functions as a nucleophile analogous to Ser195 of trypsin-like serine proteinases.

Mol Gen Genet, 1993 Feb, 237(1-2), 241 - 50
The Escherichia coli C homoprotocatechuate degradative operon: hpc gene order, direction of transcription and control of expression; Roper DI et al.; Homoprotocatechuate (HPC; 3,4-dihydroxyphenylacetate) is catabolized to Krebs cycle intermediates via extradiol (meta-) cleavage and the necessary enzymes are chromosomally encoded in a variety of bacteria . Based on an analysis of the cloned pathway genes, the Escherichia coli C hpc gene cluster was thought to be arranged in two gene blocks transcribed from a central, divergent, operator/promoter region, which was negatively regulated by the Hpc repressor . By a variety of techniques including expression of cloned hpc genes in pUC18/19 vectors, unidirectional deletion subcloning, hybridization studies and nucleotide sequencing it has now been shown that the hpc pathway structural genes are transcribed in one direction . These experiments have also indicated that a decarboxylase and an isomerase of the pathway are encoded by a single gene (hpcE) and have established the exact structural gene order as hpcRphpcECBDGH . The position of the putative regulatory gene, hpcR, is upstream of the first structural gene (hpcE) for the Hpc pathway enzymes . The deduced open reading frame for the Hpc repressor specifies a protein of 148 amino acids with a subunit molecular weight of 17 kDa . The region between hpcR and the first gene for the pathway enzymes has a sequence similar to that for catabolite activator protein (CAP) binding . This region is immediately upstream of a promoter for the pathway structural genes, which has been identified by transcript mapping.

Mol Gen Genet, 1993 Feb, 237(1-2), 206 - 14
Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli; Disque-Kochem C et al.; The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F . In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event . The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity . The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein . The binding site for the purified D protein is located within the mini-F NcoI-HpaI DNA fragment (192 bp) . Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E . coli . The binding site, which is a region of approximately 28 bp and is located 160 bp downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.

Am J Trop Med Hyg, 1993 Feb, 48(2), 243 - 8
Etiology of acute diarrhea among United States military personnel deployed to South America and west Africa; Bourgeois AL et al.; A study of acute diarrhea was conducted from 1985 to 1987 among U.S . military personnel participating in routine shipboard exercises in South America and West Africa and ground troops deployed to coastal Ecuador . An enteropathogen was identified in 146 (51%) of 289 acute cases of diarrhea . Enterotoxigenic Escherichia coli, found in 50 (17%) patients with diarrhea, was the most commonly identified enteropathogen . Viral enteropathogens were also found in a high percentage of acute cases of diarrhea: rotavirus was detected in 11% of the patients and Norwalk virus infection in 10% . Most enteric pathogens were acquired in equal frequencies in South America and West Africa, except for rotavirus infection which was identified more often in West Africa and enteroaggregative E . coli infection which was identified more often in South America . Bacterial enteropathogens were frequently resistant to trimethoprim/sulfamethoxazole, but no resistance to quinolone drugs was observed, indicating that quinolone drugs have become important agents for the treatment of diarrhea in South America and West Africa.

Int J Biochem, 1993 Feb, 25(2), 233 - 8
Modification of tryptophan 149 of inorganic pyrophosphatase from Escherichia coli; Kaneko S et al.; 1 . The inorganic pyrophosphatase from Escherichia coli was almost completely inactivated on chemical modification of Trp-149 with N-bromosuccinimide (NBS) . 2 . The presence of a complex of Mg2+ and a substrate analogue, iminodiphosphate (PNP), provided considerable protection against the inactivation, whereas Mg2+ or PNP alone afforded only slight protection.

PCR Methods Appl, 1993 Feb, 2(3), 204 - 9
Simplified construction of a subtracted cDNA library using asymmetric PCR; Houge G; A novel method for the direct construction of subtracted plasmid cDNA libraries in the plasmid pBluescript is presented . Two libraries in lambda-ZAP were compared starting with general phagemid excision from both libraries . Thereafter, single-stranded (ss) plasmids from one library were subtracted with biotinylated cDNA molecules generated by asymmetric PCR on ss plasmid templates from the other library . The nonsubtracted plasmids were used to transform Escherichia coli directly, thus making a subtracted plasmid library . Preliminary data suggest that the specificity of the method is around 25% . The method is sensitive enough to detect low-abundance mRNAs . In contrast to other subtractive methods based on lambda-ZAP, the bias introduced using PCR in this case only affects the method's specificity and not its sensitivity.

Clin Sci (Lond), 1993 Feb, 84(2), 119 - 28
Molecular studies on an ancient gene encoding for carbamoyl-phosphate synthetase; Schofield JP; 1 . Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution . The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication . The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae . Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS . The DNA sequence of the D . discoideum carbamoyl-phosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M . barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated . 2 . Genes with ancient duplications provide unique information on their evolution . A study of the intron/exon organization of the rat carbamoyl-phosphate synthetase I gene and the carbamoyl-phosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old . Evidence is provided that some introns must have been present in the ancestral precursor before its duplication . 3 . The human carbamoyl-phosphate synthetase I gene has been isolated and characterized . A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I . A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene . The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46kb . This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.

Carcinogenesis, 1993 Feb, 14(2), 237 - 44
Mutagenesis in Escherichia coli K-12 mutants defective in superoxide dismutase or catalase; Prieto-Alamo MJ et al.; Escherichia coli K-12 strains with diminished levels of superoxide dismutase (SOD) due to inactivation of the sodA, sodB or sodA sodB genes were constructed in order to quantify the role of O2 . in mutagenesis . Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance (AraR) . No sodA sodB mutant inability to grow in aerobic minimal medium was found, in contrast to that previously reported for a different E . coli wild-type genetic background . The role of SOD for coping with the damaging effects of superoxide became evident after the increase in intracellular O2- . flux by growing cells under hyperoxygenation, but particularly by using redox cycling compounds such as plumbagin, paraquat and menadione . Bacteria completely devoid of SOD activity showed very high levels of AraR-induced mutants at doses that were non-mutagenic for the SOD-proficient parental or the sodA or sodB single mutants . The mutagenicity of nifurtimox and quercetin were studied to further compare the responses of the SOD-deficient bacteria to those of their SOD-proficient counterparts . The relative importance of SOD and catalase for coping with the damaging effects of O2- . and H2O2 was quantified by comparing SOD-deficient bacteria with isogenic catalase-deficient cells (a katG katE double mutant) . The mutagenicities of plumbagin and menadione were much higher in SOD-deficient than in catalase-deficient bacteria, in agreement with the role of the O2- . radical in the so-called metal-catalyzed Haber-Weiss reaction . The relevance of catalase in protecting against the damaging effects of H2O2 was evident from the hypersensitivity of the katG katE double mutant to the mutagenic and lethal effects of this oxidizing agent . It is concluded that the Ara mutagenicity assay combined with depletion in specific antioxidative enzymes could be a tool in establishing the extent to which DNA damage by oxygen radicals is relevant to mutagenesis.

Biochem J, 1993 Feb 1, 289 ( Pt 3), 709 - 18
Cytochrome bo from Escherichia coli: identification of haem ligands and reaction of the reduced enzyme with carbon monoxide; Cheesman MR et al.; Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo {Au and Gennis (1987) J . Bacteriol . 169, 3237-3242} . The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells . Optical e.p.r . and low-temperature m.c.d . (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E . coli . Low-spin ferric haem b, the origin of a rhombic e.p.r . spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r . m.c.d . spectrum at 1622 nm . It is therefore concluded that haem b is co-ordinated by two histidine residues . The low-temperature m.c.d . spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o . The bands arising from haem o show a direct correspondence with those in the m.c.d . spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine . This assignment was confirmed by measuring the e.p.r . spectrum of the nitric oxide derivative of fully reduced cytochrome bo . This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine . The hyperfine splittings observed were 1.95 +/- 0.05 mT for the 14N of the NO ligand and 0.75 +/- 0.05 mT for the 14N of the proximal histidine . The e.p.r . spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6 . These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-CuB, suggesting that the binuclear site may display heterogeneity . Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1, and the dissociation constant for this reaction was determined to be 1.7 x 10(-6)M . The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 {Gibson and Greenwood (1963) Biochem . J . 86, 541-554}.

Arch Biochem Biophys, 1993 Feb 1, 300(2), 751 - 5
Expression, purification, and binding properties of human cellular retinoic acid-binding protein type I and type II; Fogh K et al.; Human cellular retinoic acid-binding protein (CRABP) type I and type II were expressed in Escherichia coli from cloned cDNAs . Expressed proteins were purified by gel filtration and ion-exchange chromatography, resulting in highly pure proteins . The yield after gel filtration was approximately 50 mg/liter bacterial culture . In binding studies the equilibrium dissociation constant, Kd, of retinoic acid (RA) for E . coli-derived CRABP-I and CRABP-II was 6.8 and 39 nM, respectively . The Kd of the synthetic retinoid analog CD 367 was 2.2 nM for CRABP-I and 3.0 nM for CRABP-II . RA competed with the binding of CD 367 to CRABP-I and CRABP-II with IC50 values of 20.0 and 90.0 nM, respectively . Retinoid analogs competed with the binding of CD 367 to CRABP-I and CRABP-II in the following order: (p-{(E)-2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphtyl)-1-propenyl}-benzoic acid (TTNPB) > 4-oxo-RA > 4-OH-RA > 13-cis-RA = 9-cis-RA . m-carboxy-TTNPB and CD 271 were found not to compete with the binding of CD 367 to CRABP-I or CRABP-II even at 500-fold molar excess . These data demonstrate that E . coli-derived CRABP-I has a higher affinity for RA than CRABP-II and that retinoic acid metabolites have a lower affinity for these proteins . The observed difference in affinity for RA supports the idea that CRABP-I, which is constitutively expressed, and CRABP-II, which is induced by RA, have different functions in the cell . In addition, 9-cis-RA, a natural ligand for the retinoid X receptors, is not a physiological ligand for either CRABP-I or CRABP-II.

J Clin Microbiol, 1993 Feb, 31(2), 265 - 71
Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B; Sinclair R et al.; The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli . Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections . An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses . A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA . Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site . The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.

J Gen Virol, 1993 Feb, 74 ( Pt 2), 229 - 37
Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus; Stoll R et al.; The isolation and characterization of two avian polyomaviruses, from chicken (BFDV-2) and a parrot (BFDV-3), is reported . Both isolates are closely related to the non-mammalian polyomavirus budgerigar fledgling disease virus (BFDV) isolated from budgerigars (now called BFDV-1), and all three viral genomes are shown to have the same basic size of 4981 bp . A 151 bp insertion was, however, observed in the non-coding region of BFDV-2 which represented an exact duplication of the left half of the non-coding region, including the putative early promoter and amino terminus of the large T antigen . With a further 15 base pairs exchanged elsewhere throughout the three genomes, these viruses have distinct degrees of tropism for various avian species . The production of antibodies directed against a beta-galactosidase-large T antigen fusion protein of BFDV-1 is described . These antibodies detected the large T antigen, with an M(r) of approximately 80K, and the small t antigen, with an M(r) of approximately 24K, in cells infected with BFDV isolates . Whereas these antibodies bind with low affinity to the large T antigen of simian virus 40 (SV40), SV40- or mouse polyomavirus-specific antibodies will not bind to the BFDV large T antigen . Antibodies directed against BFDV structural polypeptides exhibit broad, reciprocal cross-reactivities with all three structural proteins of mammalian polyomaviruses . The significance of polyomavirus infections in various avian species is discussed . Based on unique structural and biological properties we propose that these viruses should be placed in a distinct subgenus (Avipolyomavirus) within the polyomaviruses.

Mol Cell Biol, 1993 Feb, 13(2), 1232 - 7
Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro; Clark ME et al.; Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection . This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID . To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP) . Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited . Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C . Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein . The cleavage of TBP by protease 3C occurs directly . Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro . These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

Infect Immun, 1993 Feb, 61(2), 448 - 56
Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells; Dytoc M et al.; Adhesion of Helicobacter pylori was reported previously to be morphologically identical to "attaching and effacing" Escherichia coli . Therefore, the aim of the present study was to define the adhesion phenotype of H . pylori LC-11 to HEp-2, KATO-III, HEL, and CHO tissue culture cells . By using both staining of F-actin with fluorescein-labeled phalloidin and ultrastructural analysis, diffuse bacterial adhesion to discrete microvillus-denuded regions of the plasma membrane was observed in each of the infected cell lines . However, strain LC-11 did not induce formation of F-actin adhesion pedestals on the eukaryotic cells . H . pylori was negative by colony blot hybridization with an E . coli attaching and effacing gene probe . Elevations in inositol triphosphates followed infection of HEp-2 cells with H . pylori (405% of control values +/- 147%; P < 0.05) . To correlate the observed histopathology with expression of the H . pylori phosphatidylethanolamine receptor, a thin-layer chromatography overlay-binding assay was used to identify receptors in each of the cell lines . H . pylori adhered to eukaryotic cells regardless of the presence (HEp-2, KATO-III, and CHO cells) or absence (HEL cells) of the lipid receptor as detected under the assay conditions . However, in comparison to cell lines that possess the phosphatidylethanolamine receptor, HEL cells demonstrated less quantitative H . pylori binding . These findings suggest that mechanisms distinct from E . coli enteropathogens underlie the adhesion of H . pylori to mucosal surfaces . In addition to the phosphatidylethanolamine H . pylori receptor, another host factor(s) likely mediates the attachment of H . pylori to human eukaryotic cells.

Biol Pharm Bull, 1993 Feb, 16(2), 207 - 9
S-methyl methane thiosulfonate, a new antimutagenic compound isolated from Brassica oleracea L . var . botrytis; Nakamura Y et al.; Though various antimutagens with desmutagenic activities have been found in our daily foods of plant origin, the numbers of antimutagens with bio-antimutagenic activities found so far are limited . In the present study, a compound with potential bio-antimutagenic activity to Escherichia coli B/r WP2 was newly isolated from cauliflower, Brassica oleracea L . var . botrytis, and its chemical structure was identified to be S-methyl methane thiosulfonate by NMR and MS analysis.

Genet Anal Tech Appl, 1993 Feb, 10(1), 16 - 23
Open reading frame analysis by selective PCR-mediated deletion mutagenesis; Verhasselt P et al.; Recently, we demonstrated that a nested set of DNA fragments can be obtained by using one specific primer and one semirandom primer in a polymerase chain reaction (PCR) . We now describe a strategy for selective deletion mutagenesis that is based on this observation . The gene of interest is cloned as a fusion construct with a selectable marker in a small vector, allowing for PCR amplification of the entire recombinant plasmid . The specific primer is complementary to the vector sequence beyond the gene of interest and is oriented downstream . The 3' end of the semirandom primer is complementary to a triplet (GAT) that is scattered over the entire open reading frame (ORF) . It is shown by nucleotide sequence analysis that deletion mutants result exclusively from annealing of the semirandom primer at different GAT triplets . PCR products resulting from annealing to GAT triplets elsewhere in the plasmid are counterselected by the need for replication functions and for the expression of the selectable marker . This technique is demonstrated on the Saccharomyces cerevisiae ORF YCL56C.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1993 Feb, 15(1), 12 - 6
{Experimental studies on the elimination of minimal residual leukemia in vivo by alternate half body irradiation}; Chu J; The purpose of this study is to develop a new way for leukemia patients to tolerate an ablative chemoradiotherapy without BMT . Previous studies had demonstrated that only a few leukemia cells remained in the body during remission phase following chemotherapy and did not migrate to distant organs via the circulation until they had given rise to 3-4 x 10(5) new cells . The period required for such growth was about 20 days . However, hematopoietic stem cells migrate earlier than do leukemia cells . Therefore, alternate half body irradiation (HBI) within this period would kill the residual leukemia cells, but hematopoietic stem cells would migrate from shielded marrow to irradiated marrow and reconstruct hematopoiesis . BN rats were injected i.v . with 10 BNML subline LT12nl #15 cells, which had been transfected with the E . coli Lac-Z and Neo genes by retrovirus-mediated gene transfer . Cytochemical X-gal staining was used to identify the leukemia cells in agar culture . At day 9 after inoculation of leukemia cells, the rats were treated with cyclophosphamide (Cy 120 mg/kg), and 3 days later irradiated with 10 Gy (2 Gy/min, 6 MV, X-rays) on the upper half body, with the lower body shielded by a lead brick . They were then irradiated with the same dosage in the lower body with the upper half body shielded a week later . The preliminary results were as follow: At day 9 after inoculation, the cellularity of bone marrow (humerus, sternum, femur and tibia) was in the normal range, and the number of L-CFU ranged from 3.6 x 10(-5) to 9.0 x 10(-5) in agar culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant J, 1993 Feb, 3(2), 261 - 71
Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development; Allen RL et al.; A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize . A 2.87 kbp section of the promoter fused to E . coli beta-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants . However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes . The maize polygalacturonase gene is one member of a highly conserved gene family . The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.

Mol Microbiol, 1993 Feb, 7(4), 545 - 53
Regulation of pyelonephritis-associated pili phase-variation in Escherichia coli: binding of the PapI and the Lrp regulatory proteins is controlled by DNA methylation; Nou X et al.; Expression of pyelonephritis-associated pili (Pap) in Escherichia coli is under a phase-variation control mechanism in which individual cells alternate between pili+ (ON) and pili- (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam) . Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells . The GATC1028 site of phase ON cells is non-methylated and the GATC1130 site is fully methylated . Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non-methylated . Two transcriptional activators, PapI and Lrp (leucine-responsive regulatory protein), are required for this specific methylation inhibition . DNA footprint analysis using non-methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas PapI does not appear to bind to pap regulatory DNA . However, addition of Lrp and PapI together results in an additional DNaseI footprint around the GATC1028 site . Moreover, Dam methylation inhibits binding of Lrp/PapI near the GATC1028 site and alters binding of Lrp at the GATC1130 site . Our results support a model in which Dam and Lrp/PapI compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.

Mol Gen Genet, 1993 Feb, 237(1-2), 26 - 32
Floral organ-specific and constitutive expression of an Arabidopsis thaliana heat-shock HSP18.2::GUS fusion gene is retained even after homeotic conversion of flowers by mutation; Tsukaya H et al.; Organ-specific and constitutive expression of the Arabidopsis HSP18.2 gene under normal growth conditions (22 degrees C) was observed in transgenic A . thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for beta-glucuronidase (GUS) from Escherichia coli . In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9 . The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.

FEMS Microbiol Lett, 1993 Feb 1, 106(3), 301 - 8
A structural model for the GroEL chaperonin; Marco S et al.; Individual particle analysis of end views from negatively stained specimens of purified GroEL from Escherichia coli showed the presence of two different particle populations, those with a six-fold symmetry and those with a seven-fold symmetry, when studied at pH 7.7 and 5.0 . Image processing of particles from frozen-hydrated specimens revealed at both pH values a homogeneous population of particles with a strong seven-fold symmetry component and an average image with seven asymmetric units . Biochemical analysis of purified GroEL showed unequivocally the presence of a single polypeptide with the N-terminal sequence identical to that of GroEL . These results are compatible with a structural model of GroEL as an asymmetric aggregate built up by two rings of seven-fold and six-fold symmetries, respectively.

J Clin Microbiol, 1993 Feb, 31(2), 311 - 4
Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs; Westerman RB et al.; Monoclonal antibodies directed against the a determinant of K88 pili from porcine enterotoxigenic Escherichia coli which react with all three K88 variants have been produced . These antibodies have been used for diagnosis of porcine enterotoxigenic E . coli in a direct enzyme-linked immunosorbent assay with sensitivity to 50 ng of pilus protein per ml.

Am Rev Respir Dis, 1993 Feb, 147(2), 442 - 7
Inhibition of lipopolysaccharide-induced pulmonary emphysema by intratracheally instilled recombinant secretory leukocyte proteinase inhibitor; Rudolphus A et al.; Experiments were performed to test whether recombinant secretory leukocyte proteinase inhibitor (rSLPI) was able to prevent the development of lipopolysaccharide (LPS)-mediated pulmonary emphysema, hemorrhage, and secretory cell metaplasia (SCM) in hamsters . Several groups of eight animals were intratracheally treated for four weeks, twice a week with 0.5 mg Escherichia coli LPS or with saline . In the first experiment, an additional group of eight hamsters was treated with 0.5 mg LPS mixed with 0.5 mg rSLPI, and the animals received another instillation of 0.5 mg rSLPI 7 h later . In the second experiment, 0.5 mg LPS, mixed with 1 mg rSLPI, was given while additional instillations of 1 mg rSLPI were performed 7 h and 31 h after the first dosage . In the third experiment, 0.5 mg LPS, mixed with 0.5, 1.5, or 3.0 mg rSLPI, was given while additional instillations of 0.5, 1.5, and 3.0 mg rSLPI, respectively, were performed 24 h and 48 h after the first dosage . Hamster lungs were examined for emphysema, hemorrhage, and SCM . In all three series of experiments, we observed a significant inhibition of LPS-mediated emphysema by rSLPI . This inhibition tended to be dose related . Inconclusive results were obtained on the inhibition of LPS-mediated hemorrhage . The development of LPS-mediated SCM was not affected by rSLPI . The LPS-mediated polymorphonuclear leukocyte (PMN) influx did not change when administrations of rSLPI were given additionally . We conclude that rSLPI is able to diminish significantly the development of LPS-mediated pulmonary emphysema in hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1993 Feb 1, 211(3), 891 - 8
Developmental and hormonal regulation of the Xenopus liver-type arginase gene; Xu Q et al.; Liver-type L-arginase is a major urea-cycle enzyme which is strongly induced during amphibian metamorphosis, but little is known about the molecular mechanisms underlying this induction . As a first step towards elucidating the possible mechanisms, we have isolated a cDNA clone for L-arginase from an adult Xenopus laevis liver cDNA library . Sequence comparison of Xenopus liver-type L-arginase cDNA shows a strong conservation at the amino acid level with those of human, rat and yeast . Using a Xenopus arginase cDNA fragment as a hybridization probe, we have shown by Northern blotting that the gene is highly expressed in the liver, and very slightly in kidney and spleen, of adult Xenopus . The expression is developmentally regulated . Only traces of arginase mRNA can be detected in pre-metamorphic tadpoles, but its accumulation increases very markedly at the onset of natural metamorphosis, being maintained at a high concentration constitutively upon completion of this developmental process . Amphibian metamorphosis is under the strict control of thyroid hormones . It is therefore significant that exposure of pre-metamorphic tadpoles (at stages before endogenous thyroid hormone secretion) to exogenous hormone (1 nM triiodothyronine) precociously activated the L-arginase gene . The time course of this precocious hormonal induction paralleled that of serum albumin gene in the liver . Polyclonal antibodies were raised against recombinant Xenopus L-arginase expressed in Escherichia coli as a fusion protein with glutathione S-transferase in the plasmid expression vector pGEX . Western blotting using this antibody showed that, although arginase mRNA is present in high concentration in Xenopus tadpole liver at the onset of natural metamorphosis, the protein is detected only upon its completion . Our results show a complex transcriptional and post-transcriptional regulation of the Xenopus liver-type L-arginase gene during post-embryonic development . They also demonstrate that this gene can be exploited as a target for thyroid hormones in further studies to analyze the mechanisms underlying the establishment of the adult phenotype during amphibian metamorphosis.

Biochem J, 1993 Feb 1, 289 ( Pt 3), 735 - 41
Mouse synexin (annexin VII) polymorphisms and a phylogenetic comparison with other synexins; Zhang-Keck ZY et al.; Two sets of cDNAs encoding mouse synexin were isolated from a liver cDNA library and sequenced . The coding regions of synexin clones show 99% identity . By contrast, the two mouse synexin cDNAs differ in a number of ways in both 5' and 3' non-coding regions . The two sets of cDNA encode a polypeptide of 463 amino acid residues which has a deduced molecular mass of 50 kDa . The amino acid sequence of mouse synexin shows a high degree of similarity to both the unique N-terminal domain and the highly conserved C-terminal domain of previously cloned human synexin . Northern-blot analysis using mouse liver polyadenylated RNA revealed two transcripts of 1.8 kb and 2.6 kb, corresponding to group I and group II respectively . Further hybridization analysis using specific sequences from each set of clones showed that the two sizes of mRNAs differ in the length of the 3' non-coding region which corresponded to the cDNAs . Both mouse liver synexin and recombinant mouse synexin expressed in Escherichia coli reacted after Western-blot analysis with a goat antibody against bovine synexin . Only in the larger group-II cDNAs do we find point mutations leading to amino acid replacements of Ser to Ala at residue 145 in the unique N-terminal domain, and of Ala to Gly at residue 304 in the transition zone between repeats II and III . We conclude from a comparison of mouse, human and Dictyostelium synexins that changes occur predominantly in the hydrophobic N-terminal domain, or, in the C-terminal region at the ends of some predicted alpha-helices, on the hydrophobic face of the amphipathic C-helices, and within a lengthy non-helical domain connecting major repeats II and III.

Planta, 1993 Feb, 189(2), 174 - 81
Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli; Sonnewald U et al.; Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylene glycol and chromatography over diethylaminoethylcellulose, omega-aminohexyl-agarose, Mono Q and Blue Affinity columns . The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1 . On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa . A new activity stain was developed to allow visualization of SPS in gels . The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively . A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize . Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone . Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al . 1991, Plant Cell 3, 1121-1130) . Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.

Biotechnology (N Y), 1993 Feb, 11(2), 201 - 6
A streptavidin mutant containing a cysteine stretch that facilitates production of a variety of specific streptavidin conjugates; Sano T et al.; The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites . A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity . Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer . Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry . In this manner, S-{14C}carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared . These conjugates contain almost twenty {14C}carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive . More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.

Biol Pharm Bull, 1993 Feb, 16(2), 120 - 4
Effects of synthetic trypsin inhibitors on the growth of Escherichia coli; Kato M et al.; The inhibitory effects of various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), potent trypsin inhibitors, on the growth of Escherichia coli were examined and the effects were compared with those of well known synthetic trypsin inhibitors . Various GMCHA esters strongly inhibited the growth of E . coli and their effects were markedly affected by the species and position of substitution on the phenyl nucleus of the GMCHA phenyl esters . No correlation was observed between the effects on the growth of E . coli and Ki's for trypsin . Inhibitory effects of benzamidine, phenylmethane sulfonylfluoride and diisopropyl fluorophosphate were less than 10% at 200 microM . 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPhtBu), a representative of various GMCHA esters, dose-dependently inhibited the growth of E . coli and the growth inhibition was preceded by a dose- and time-dependent inhibition of DNA synthesis . Tosyl-L-lysine chloromethyl ketone (TLCK) and pentamidine isethionate, potent trypsin inhibitors, also dose-dependently inhibited the growth of E . coli and the DNA synthesis . However, their effects were transient and disappeared after a short while . These results indicate that the effects of TLCK and pentamidine isethionate differ from those of GMCHA esters . GMCHA-OPhtBu and pentamidine isethionate also inhibited RNA and protein synthesis.

Acta Paediatr, 1993 Feb, 82(2), 132 - 6
The chemoluminescence response of human polymorphonuclear leukocytes to Escherichia coli O and K antigens; Miyata H et al.; The interactions between Escherichia coli O or K antigens and polymorphonuclear leukocyte function were studied . Five types of O antigen and three types of K antigen were extracted from E . coli . These included O1, O6, O75 and K1 antigens from pyelonephritopathogenic strains, O44 and K74 antigens from an enteropathogenic strain and O14 and K7 antigen from a standard strain . The antigens all reacted specifically to their specific antisera and no cross-reactions were observed . The O1 or O44 antigen stimulated a significantly greater chemoluminescence response in polymorphonuclear leukocytes obtained from normal volunteers than O75, O6 or O14 antigen . In addition, the K1 or K74 antigen stimulated polymorphonuclear leukocytes significantly more than K7 antigen . These results suggest that pyelonephritopathogenic or enteropathogenic E . coli may produce severe tissue damage as a result of the response to their O or K antigens, as well as via adhesive agents such as pyelonephritopathogenic P-pili or the enteroadhesive factor, and exotoxins such as hemolysin or verotoxin.

Nippon Rinsho, 1993 Feb, 51(2), 338 - 43
{Antigenicities of groups I and II hepatitis C virus}; Kohara M; HCV genomes are considerable heterogeneities in nucleotide and amino acid sequences among individual isolates . The primary structure of the putative core and NS3 protein regions are relatively conserved among HCV isolates, while those of envelope proteins (E1 and E2) and NS4 are variable . On the basis of nucleotide sequence homology of parts of HCV genomes, several research groups have reported the possible existence of multiple subtypes of HCV isolates . From comparative sequence studies on HCV cDNA clones corresponding to NS3 and NS4 regions followed by diagnostic studies using these clones, have shown that there are at least two groups of HCV, group I and group II . The peptide produced in E . coli, carrying group I and II cDNA clones (A.A . positions 1676-1736 of NS4 region) are recognized by circulating antibodies specific to group I and II HCV . These group specific antigens of NS4 region are highly reliable in detecting (over 90%) circulating antibodies against either groups of HCV . In this study, most of the HCV isolates used were shown to be classified into group I or II HCV based on their amino acid differences and phylogenic analysis within the putative 5'UTR, core and NS3-4 regions . Biological significance of these two groups of HCV is suggested by the observation that the group I HCV was resistance to the IFN therapy (10-20% showing a good response), on the other hand the group II HCV showed a good response (70-90%).

Lab Anim Sci, 1993 Feb, 43(1), 48 - 57
Evaluation of a nude mouse tumor model using beta-galactosidase-expressing melanoma cells; Dooley TP et al.; We developed and evaluated an in vivo athymic nude mouse model for tumor growth, angiogenesis, metastasis, and antineoplastic drug development . Melanoma cell lines expressing beta-galactosidase encoded by the Escherichia coli lac Z gene have been created by infecting an immortal murine melanocyte cell line with a recombinant retrovirus expressing the v-Ha-ras oncogene and lac Z to generate the MRB (melanoma, ras, beta-galactosidase) cell lines . The amelanotic, phorbol ester-independent, transformed melanoma cell lines developed tumors rapidly when injected subcutaneously into nude mice, as well as experimental lung metastases when injected i.v . into the tail vein . beta-galactosidase-expressing subcutaneous tumors and lung metastases stained blue with X-gal . The melanomas produced in nude mice have been characterized by using various histochemical and immunohistochemical staining methods to detect melanoma- and endothelial-cell-specific markers to determine the extent of neovascularization in MRB nude mouse tumors . Optimal staining of endothelial cells involved in tumor angiogenesis was observed by using ADPase activity and antiangiotensin-converting enzyme antibody staining . Attempts at indirect quantification of metastatic tumor cell number within the lung by either beta-galactosidase enzymatic activity or ELISA immunoreactivity were unsuccessful . However, the MRB cell lines should be useful in screening for and studying the mechanisms of action of antineoplastic, antimetastatic, and angiostatic drugs in vivo in athymic nude mice.

Biotechnol Appl Biochem, 1993 Feb, 17 ( Pt 1), 91 - 102
Inhibition of the RNA-directed DNA polymerase activity of a recombinant HIV-1 p51 reverse transcriptase by a p15 ribonuclease H domain; Evans DB et al.; The polymerase domain of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, called the p51 reverse transcriptase (p51 RT), was expressed in Escherichia coli . The recombinant protein also contained an N-terminal affinity tag designed to facilitate its purification by immobilized metal affinity chromatography . The purified p51 RT is a predominantly monomeric protein and it catalyses RNA-dependent DNA polymerization with poly(rA).oligo(dT) as the template.primer . Recently we have also reported the isolation of the recombinant RNAase H domain of HIV-1 RT that is enzymically active (Evans, Brawn, Deibel, Tarpley and Sharma {1991} J . Biol . Chem . 266, 20583-20585) . The latter directly inhibits the RNA-dependent DNA polymerase activity of p51 RT . Kinetic experiments show that the p15 RNAase H-mediated inhibition of p51 RT is competitive with respect to the poly(rA).oligo(dT) template.primer (Ki = 320 +/- 50 nM), and it does not interfere directly with the binding of dTTP to the enzyme . Thus the kinetic behaviour is consistent with the binding of p15 RNAase H at or near the template.primer-binding site in this replicase . If the binding of the p15 RNAase H involves only a small segment of this protein, then identification of that segment may open up new opportunities towards the design of novel inhibitors of RNA-dependent DNA polymerase activity.

Biochem J, 1993 Feb 1, 289 ( Pt 3), 875 - 81
Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon; Schaap D et al.; In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid . A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments . To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli . Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase . No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases . Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase . For PKC-epsilon, DG kinase is the first in vivo substrate identified . Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine . Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.

J Clin Microbiol, 1993 Feb, 31(2), 386 - 9
Evaluation of the fluorescence actin staining test for detection of enteropathogenic Escherichia coli; Shariff M et al.; Enteropathogenic Escherichia coli (EPEC) strains designated on the basis of their serotypes are epidemiologically associated with diarrhea . They adhere to the intestinal mucosa, producing the characteristic attaching and effacing (AE) lesion in an in vitro organ culture system . EPEC manifest localized adherence (LA) in the HEp-2 cell assay, and this is commonly used for clinical diagnosis . Recently, the fluorescence actin staining (FAS) test was proposed for the identification of E . coli causing the AE lesion . We therefore compared the FAS test with the HEp-2 cell assay and the EPEC adherence factor (EAF) probe assay for the detection of EPEC strains . Among 240 stool samples from children with diarrhea examined, 176 yielded E . coli and 14 of these strains showed the LA pattern in the HEp-2 cell assay; 11 of these were positive by both the EAF and the FAS tests . By using the HEp-2 cell assay as the "gold standard," the FAS test gave a sensitivity of 78.5% and a specificity of 100% . The three localized adherent FAS-negative strains tested subsequently were positive by the enteroaggregative E . coli DNA Probe and failed to produce the AE lesions characteristic of EPEC . When these strains were not considered, the sensitivity of the FAS test for detecting isolates that manifest LA was 100% . Against the EAF probe, the sensitivity and specificity of the FAS test were 91.6 and 100%, respectively . The FAS test avoids infrequent but nevertheless important phenotypic misclassifications in the HEp-2 cell assay, and it may therefore serve as a confirmatory test for EPEC.

J Bacteriol, 1993 Feb, 175(4), 1043 - 52
Identification of endonucleolytic cleavage sites involved in decay of Escherichia coli trxA mRNA; Arraiano C et al.; The degradation of individual mRNAs in Escherichia coli has been studied through the use of a multiple mutant carrying the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and rne-1 (RNase E) alleles . In this triple mutant, discrete mRNA breakdown products are stabilized in vivo at the nonpermissive temperature (Arraiano, C . M., S . D . Yancey, and S . R . Kushner, J . Bacteriol . 170:4625-4633, 1988) . In the case of thioredoxin (trxA) mRNA decay, degradation fragments accumulated at early times after a shift to the nonpermissive temperature . Using Northern (RNA) blots, S1 nuclease analysis, and primer extensions, we identified a series of specific endonucleolytic cleavage sites that occur throughout the transcript in both the triple mutant and a wild-type control . The implications of the complex decay patterns observed are discussed.

Curr Genet, 1993 Feb, 23(2), 181 - 3
Epitope-tagging vectors designed for yeast; Reisdorf P et al.; In order to facilitate the process of epitope-tagging of yeast proteins, we have constructed two Saccharomyces cerevisiae-Escherichia coli shuttle vectors that allow fusion of a sequence encoding an epitope of the human c-myc protein at the 3' end of any gene . An example of the use of this technique is presented.

Am J Vet Res, 1993 Feb, 54(2), 280 - 6
Resuscitation of anesthetized endotoxemic pigs by use of hypertonic saline solution containing dextran; Hellyer PW et al.; We evaluated the biochemical and hemodynamic response to hypertonic saline solution plus dextran in isoflurane-anesthetized pigs infused IV with Escherichia coli endotoxin (5 micrograms/kg of body weight for 0 to 1 hour + 2 micrograms/kg for 1 to 4 hours) . After 120 minutes of endotoxemia, pigs were treated with a bolus (4 ml/kg over 3 minutes) of either normal saline solution (NSS; 0.9% NaCl), or hypertonic saline solution plus dextran (HSSD; 7.5% NaCl + 6% dextran-70) . Administration of HSSD significantly (P < 0.05) increased serum osmolality and concentrations of sodium and chloride for approximately 2 hours during endotoxemia . Plasma total protein concentration decreased significantly (P < 0.05) for 2 hours after treatment with HSSD, indicating hemodilution and increased plasma volume . Although HSSD transiently increased cardiac index (CI) for approximately 15 minutes, this effect was not sustained; however, the endotoxin-induced decrease in CI was ameliorated from 120 to 180 minutes . In pigs of the endotoxin + NSS group from 180 to 240 minutes, CI decreased significantly (P < 0.05), compared with baseline and control values . The endotoxin-induced increases in mean pulmonary arterial pressure and pulmonary vascular resistance were not attenuated by HSSD . At 135 minutes, total peripheral vascular resistance was transiently lower (for approx 15 minutes) in pigs treated with HSSD, compared with control pigs . The endotoxin-induced increase in plasma lactate concentration was not attenuated by HSSD, indicating continued peripheral O2 debt . We conclude that, despite sustained increases in serum osmolality and concentrations of sodium and chloride, HSSD has only transiently beneficial cardiopulmonary effects during endotoxemia in pigs.

Clin Exp Immunol, 1993 Feb, 91(2), 295 - 300
Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC); Mollnes TE et al.; We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products . In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement . An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react . Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2 . The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity . Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described . In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period . These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock . The results are discussed with particular emphasis on activation of the terminal complement pathway.

Mutat Res, 1993 Feb, 291(1), 53 - 60
Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons; Mersch-Sundermann V et al.; To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E . coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs) . Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use . Initially, we used 0-5000 ng (= 0-20 nmole) benzo{a}pyrene (B{a}P) as a reference compound for the test procedure in the presence of standard S9 mix . Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol . We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0 . 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs . In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay) . Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts . The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.

Mutat Res, 1993 Feb, 285(2), 219 - 24
The concomitant detection of gene mutation and micronucleus induction by mitomycin C in vivo using lacZ transgenic mice; Suzuki T et al.; A new assay system that can simultaneously provide gene mutagenicity and clastogenicity data in vivo is described . Transgenic mice (Muta Mouse) harboring the lacZ gene as a target for mutation analysis were injected intraperitoneally with mitomycin C (MMC), either once or on 5 successive days . Micronucleus assays were performed with small amounts of peripheral blood collected from a tail vessel . The spontaneous frequency of micronucleated reticulocytes was 0.42% . For the mutation analysis, DNA was extracted from bone marrow and liver cells at several harvest times . The lacZ gene was rescued by lambda packaging and infection of E . coli C (lac-), followed by plating on agarose plates containing X-gal . The spontaneous lacZ mutant frequencies were 37 and 29 x 10(-6) in bone marrow and liver, respectively . In the micronucleus assay, single treatments with 1.0 and 2.0 mg/kg of MMC induced micronuclei in 3.6 and 5.8% of reticulocytes, respectively, peaking 48 h after treatment . Muta Mouse sensitivity to micronucleus induction was similar to nontransgenic strains used routinely for the micronucleus test . On the other hand, single treatments with MMC at 1.0 and 2.0 mg/kg did not induce any significant increases in the frequency of lacZ- mutants in bone marrow or liver . N-Ethyl-N-nitrosourea, used at 100 mg/kg as a positive control, yielded a 5-fold increase in mutant frequency above untreated animals in bone marrow only . After 5-day treatments, MMC induced approximately a 2-fold increase in mutant frequency in bone marrow only for the sublethal dose of 2 mg/kg . Therefore, this study indicated that the strong clastogenic activity of MMC in bone marrow was not accompanied by significant gene mutagenic activity.

Mutat Res, 1993 Feb, 285(2), 145 - 63
Comparison of the spectra of genetic damage in N4-hydroxycytidine-induced ad-3 mutations between nucleotide excision repair-proficient and -deficient heterokaryons of Neurospora crassa; de Serres FJ et al.; A comparison has been made of the mutagenic effects of N4-hydroxycytidine (HC) in the adenine-3 (ad-3) region of two-component heterokaryons of Neurospora crassa: nucleotide excision repair-proficient (uvs-2+/uvs-2+) heterokaryon 12 (H-12) and nucleotide excision repair-deficient (uvs-2/uvs-2) heterokaryon 59 (H-59) . HC was found to produce mutations predominantly, if not exclusively, by AT to GC base-pair transitions in Escherichia coli strain K12 by Janion and Glickman (1980, Mutation Res., 72, 43-47) and Sledziewska-Gojska et al . (1992, Mutagenesis, 7, 41-46) . The ad-3 forward-mutation, specific-locus assay system permits the recovery of ad-3A and/or ad-3B mutants resulting from gene/point mutation, multiple-locus mutation, and multilocus deletion mutation . Uvs-2, which is homokaryotic in H-59, results in a recovery of HC-induced ad-3 forward mutations at a frequency in H-59 that is comparable to that found in H-12 . Genetic analysis of ad-3 mutants recovered from experiments with HC treatment demonstrates that predominantly gene/point mutations were found in both strains: 99.3% (540/544) in H-12, and 97.4% (531/545) in H-59 . Genetic analysis of allelic complementation among the ad-3BR mutations demonstrated that HC induced the highest percentage of complementing mutants ever found with base analogs both in H-12 (99.7% {328/329}) and H-59 (91.2% {290/318}) . As a result of these findings, the majority of HC-induced ad-3 mutations are postulated to have resulted from missense mutations . Thus, we conclude that the results in Neurospora are consistent with the observations in E . coli strain K-12, where HC induces predominantly AT to GC base-pair transitions.

Mol Cell Biol, 1993 Feb, 13(2), 769 - 74
Antibody mimicking the action of RAS proteins on yeast adenylyl cyclase: implication for RAS-effector interaction; Suzuki N et al.; Polyclonal antisera were raised against various subregions of Saccharomyces cerevisiae adenylyl cyclase in order to examine the molecular mechanism of interaction between adenylyl cyclase and RAS proteins . One of the antisera was found to activate adenylyl cyclase to an extent comparable to that activated by saturating amounts of yeast RAS2 protein produced in Escherichia coli . The stimulatory effect of this antiserum was shown to be additive with RAS2 protein when both antisera and RAS2 protein were present at low concentrations . At saturating amounts of RAS2 protein, the antisera did not exhibit additional stimulatory effects, suggesting that the actions of RAS2 protein and the antisera are complementary with each other . The antigenic determinant for the antibody involved in the activation was mapped to a 14-amino-acid segment, 1452-NSVDNGADVANLSY-1465, located between the leucine-rich repeats and the catalytic domain of adenylyl cyclase . Certain missense mutations affecting this 14-amino acid segment significantly reduced the response of adenylyl cyclase to both activating antibody and RAS proteins . These results suggest that this segment of adenylyl cyclase is intimately involved in the mechanism by which RAS proteins activate this downstream effector.

J Bacteriol, 1993 Feb, 175(3), 674 - 83
Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori; Evans DG et al.; Gene hpaA, which codes for the receptor-binding subunit of the N-acetylneuraminyllactose-binding fibrillar hemagglutinin (NLBH) of Helicobacter pylori, was cloned and sequenced . The protein expressed by hpaA, designated HpaA, was identified as the adhesin subunit on the basis of its fetuin-binding activity and its reactivity with a polyclonal, monospecific rabbit serum prepared against NLBH purified from H . pylori . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blots (immunoblots) showed that the cloned adhesin has the same molecular weight (20,000) as that found on H . pylori . Also, HpaA contains a short sequence of amino acids (KRTIQK) which are all either identical or functionally similar to those which compose the sialic acid-binding motif of Escherichia coli SfaS, K99, and CFA/I . Affinity-purified antibody specific for a 12-residue synthetic peptide that included this sequence blocked the hemagglutinating activity of H . pylori and was shown by immuno-gold electron microscopy to react with almost transparent material on unstained H . pylori cells, which is consistent with previous observations concerning the location and morphology of the NLBH.

J Bacteriol, 1993 Feb, 175(3), 604 - 12
The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 is nonessential for ammonium assimilation; Wagner SJ et al.; The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp . strain PCC 7002) was isolated by complementing an Escherichia coli strain auxotrophic for glutamine (YMC11) with a PR-6 cosmid library . PR-6 glnA is a single-copy gene that encodes a deduced amino acid sequence that is highly homologous to the deduced glnA amino acid sequences reported for other bacteria . No homology was found between the PR-6 glnA flanking sequences and the ntrB, ntrC, or glnB genes of other bacteria . Northern (RNA) and primer extension analyses of PR-6 RNA revealed one predominant and several minor glnA transcripts of about 1.5 to 1.7 kb . The steady-state amounts of these transcripts increased three- to fivefold when the cells were starved for nitrogen . However, we found that mutant PR-6 cells lacking glnA were still able to use nitrate or ammonium as a sole nitrogen source . Although no RNA homologous to an internal fragment of the glnA gene could be detected in the mutant cells, they retained about 60% of wild-type glutamine biosynthetic activity . The mutant cells were more sensitive than the wild-type cells to methionine sulfoximine, a transition state analog of glutamate, a result that might indicate the presence of an additional glutamine synthetase; however, cell extracts of wild-type PR-6 cells and those lacking glnA were both able to use carbamyl phosphate instead of ammonium as a nitrogen donor for the synthesis of glutamine, a result that indicates the use of carbamyl phosphate synthetase to assimilate ammonium and produce glutamine.

Infect Immun, 1993 Feb, 61(2), 423 - 31
A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells; Comstock LE et al.; Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells . In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B . burgdorferi to HUVE cells by 73% . Subsequently, MAbs were generated for use in determining whether or how B . burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B . burgdorferi . Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa . Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells . None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5% . This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer) . Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence . MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 {corrected} of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells . Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA . MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B . burgdorferi.

Virology, 1993 Feb, 192(2), 430 - 7
Antigenicities of Group I and II hepatitis C virus polypeptides--molecular basis of diagnosis; Tsukiyama-Kohara K et al.; Comparative nucleotide sequence studies on the putative NS3 and NS4 regions of the genomes of hepatitis C viruses (HCV) have revealed that there are at least two groups of HCV, group I and group II . The cDNA clone E, corresponding to a boundary between the NS3 and NS4 (NS3-4) region of the group II HCV genome, encodes antigens that react to antibodies specific to group II HCV (Tsukiyama-Kohara et al . (1991) Virus Genes 5, 243-254) . To understand the molecular basis of the group-specific antigenicity of HCV peptides, the predicted amino acid sequences around the NS3-4 region of our group II HCV cDNAs were compared with those of other HCV isolates . The analysis revealed the presence of group-specific amino acids in this peptide region . Evolutionary analysis of nucleotide sequences within this region of these HCV isolates also led to the same classification . A similar result was obtained by sequence analysis of cloned cDNAs corresponding to the core region . A cDNA of the group II HCV core region was prepared by polymerase chain reaction from the cDNA synthesized with group II-specific primer complementary to the NS3-4 region . The products directed by the cDNA of the core region did not have group-specific antigenicity . The NS3 peptide region also appeared not to carry group-specific antigens . Our results indicate that most HCV isolates can be classified into either group I or II, and that the existence of two groups of HCV does not disturb HCV diagnosis as long as core and/or NS3 peptides are used to detect HCV antibodies.

J Virol, 1993 Feb, 67(2), 961 - 8
An antibody- and synthetic peptide-defined rubella virus E1 glycoprotein neutralization domain; Wolinsky JS et al.; We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets . Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J . S . Wolinsky, M . McCarthy, O . Allen-Cannady, W . T . Moore, R . Jin, S . N . Cao, A . Lovett, and D . Simmons, J . Virol . 65:3986-3994, 1991) . Both of these MAbs neutralize RV infectivity . A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain . One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA . Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239) . Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV . The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity . These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.

J Virol, 1993 Feb, 67(2), 664 - 72
B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins; Pancino G et al.; In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats . We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4) . Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific . To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed) . Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection . These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.

Mutat Res, 1993 Feb, 301(2), 99 - 105
The use of selection in recovery of transgenic targets for mutation analysis; Lundberg KS et al.; Transgenic animal mutagenesis assays using lambda shuttle vectors have recently been described for isolation and characterization of spontaneous and chemical induced DNA mutations . Extensive information on lambda and E . coli genetics provides a wealth of techniques to allow selection of mutant target genes . Here we describe the modification of an E . coli host which permits two methods for the direct selection of mutant genes . These methods reduce the number of plates needed to be screened for a comparable amount of frequency data by 20-100-fold and thus provide a significant savings of the materials and time required for the screening of mutations . In addition, mutants selected by these approaches described here may alter or broaden the spectrum of mutations that are recoverable . Ultimately, a combination of selective and nonselective techniques may prove valuable for the analysis of mutations produced in vivo in transgenic animals.

Mutat Res, 1993 Feb, 301(2), 93 - 7
Temperature-dependent antimutagenic activity of acrolein in Escherichia coli; Aikawa K et al.; The effect of temperature on the antimutagenic activity of acrolein was investigated using UV-irradiated E . coli B . When incubated at lower temperatures (30 degrees C or 37 degrees C), acrolein greatly reduced the mutation frequency in WP2 (wild-type strain), but no such effect was observed with WP2s and ZA159 (excision repair-deficient strains) . The antimutagenic activity of acrolein increased when cells were incubated at higher temperatures (40 degrees C or 42 degrees C) . Particularly in excision repair-deficient strains, the antimutagenic activity was observed only at higher temperatures . In heat shock response-deficient background, however, the antimutagenic activity was observed at 30 degrees C even in the excision repair-deficient strains.

Mutat Res, 1993 Feb, 301(2), 125 - 34
The action of 4-hydroxyaminobiphenyl in Escherichia coli: cytotoxic and mutagenic effects in DNA repair deficient strains; Suzuki M et al.; The cytotoxic and mutagenic effects of 4-hydroxyaminobiphenyl (N-OH-ABP) were studied using Escherichia coli strains with different repair capacities . N-OH-ABP was equally cytotoxic for uvrA and recA mutants as well as in wild-type cells while polA mutant strains proved particularly sensitive to its toxicity . In contrast, the mutation frequency in the uvrA strains tested was elevated to 30-400-fold the wild-type values . We suggest that aminobiphenyl-DNA adducts responsible for mutation are repaired by UVR endonuclease but different pathways exist for removal of DNA lesions responsible for bacterial killing . From the 32P-postlabeling analysis, it was concluded that ABP-DNA adducts can be relatively rapidly repaired in wild-type strains, while persisting in the uvrA strains.

Presse Med, 1993 Jan 30, 22(3), 109 - 20
{Hematopoietic growth factor (GM-CSF) after autologous bone marrow transplantation . A randomized, double-blind, multicenter study in 91 cases of non-Hodgkin's malignant lymphomas}; Gorin NC et al.; To a great extent, the risks of autologous bone marrow transplantation are related to neutropenia . Although the efficacy of the recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) on neutrophil recovery has appeared in numerous open trials, only a few randomized studies have hitherto been published . Ninety-one patients with non-Hodgkin's malignant lymphoma treated with ablative chemotherapy followed by purged or unpurged bone marrow transplantation were entered in a placebo-controlled, double-blind randomized study; 44 patients received GM-CSF (E . coli) in doses of 250 micrograms/m2/day, and 47 received a placebo . Treatment was administered daily as continuous infusion started on the day of transplantation and pursued until the absolute number of neutrophils reached 0.5 x 10(9)/l during 7 days or, if this failed, during 30 days . The median time of neutrophil recovery was 14 days in patients on rhu GM-CSF and 21 days in patients on placebo (P < 0.0001) . Patients who received a mafosfamide-purged bone marrow also had a rapid neutrophil recovery (median: 16 days versus 20.5 days; P = 0.013) . The stay in hospital was shorter in the rhu GM-CSF group (median: 23 days versus 28 days; P < 0.05) . No significant difference in the number of days with fever, infections, antibiotics administered and overall survival was detected between the two groups . The main toxicity ascribable to rhu GM-CSF was a capillary leakage syndrome found in 3 patients . Thus, after purged or unpurged autologous bone marrow transplantation rhu GM-CSF significantly reduces the duration of both neutropenia and hospital stay.

Gene, 1993 Jan 30, 123(2), 271 - 5
Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli; Davidson BL et al.; Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts . To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required . A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis . For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR . By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E . coli . The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.

Gene, 1993 Jan 30, 123(2), 259 - 62
High-level expression in Escherichia coli of the gene coding for the major structural protein (p72) of African swine fever virus; Freije JM et al.; The gene encoding the major structural protein (p72) of African swine fever virus (ASFV) has been expressed in Escherichia coli using a T7 RNA polymerase system . The use of a recombinant plasmid which contains the entire gene inserted between the T7 promoter and the transcription terminator of the expression vector allowed us to obtain a high expression level of the intact viral protein . This polypeptide, which appears in the insoluble fraction of the bacterial extracts, showed an intense reaction with the antibodies present in the sera of ASFV-infected animals, as demonstrated by Western blot and enzyme-linked immunosorbent assay . The recombinant protein was purified by size-exclusion high-performance liquid chromatography and used to develop a serological test of the disease.

Gene, 1993 Jan 30, 123(2), 203 - 10
Efficient production of biologically active human salivary cystatins in Escherichia coli; Bobek LA et al.; Different Escherichia coli expression systems were used for expression of cDNA clones encoding the human salivary cysteine proteinase (CysP) inhibitors, cystatins SN and S (CsnSN and CsnS) . These included pOTSNco12 that expresses foreign sequences as authentic (nonfusion) proteins, and pGEX-2T that directs the synthesis of foreign polypeptides as fusion proteins with glutathione S-transferase (GST) . The pOTS vector produced low levels of recombinant CsnSN (reCsnSN) that was localized in the soluble fraction, but not easily purified . The pGEX vector, on the other hand, produced much higher yields of the fusion protein, GST::CsnSN, that was localized almost entirely in the insoluble protein fraction . Solubilized and refolded GST::CsnSN inhibited the CysP, papain, more efficiently than chicken egg white Csn, indicating that the recombinant product was biologically active and that the GST carrier did not interfere with the biological activity . The pGEX-2T vector was subsequently used for the large-scale production of reCsnSN and reCsnS that were cleaved from the GST by thrombin and purified by DE-52 cellulose chromatography . ReCsnSN inhibited papain almost as efficiently as salivary CsnSN, while the reCsnS showed lower inhibitory activity as compared to both salivary CsnS and reCsnSN.

Science, 1993 Jan 29, 259(5095), 673 - 7
Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase; Hurley JH et al.; The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system . Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins . The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined . GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin . IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc . GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group . The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Cell, 1993 Jan 29, 72(2), 223 - 32
Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites; Zervos AS et al.; We used the interaction trap to isolate a novel human protein that specifically interacts with Max . This protein, Mxi1 (for Max interactor 1), contains a bHLH-Zip motif that is similar to that found in Myc family proteins . Mxi1 interacts specifically with Max to form heterodimers that efficiently bind to the Myc-Max consensus recognition site . When bound to DNA by a LexA moiety in yeast, Mxi1 does not stimulate transcription . mxi1 mRNA is expressed in many tissues, and its expression is elevated in U-937 myeloid leukemia cells that have been stimulated to differentiate . These facts are consistent with a model in which Mxi1-Max heterodimers indirectly inhibit Myc function in two ways: first, by sequestering Max, thus preventing the formation of Myc-Max heterodimers, and second, by competing with Myc-Max heterodimers for binding to target sites.

Biochem Biophys Res Commun, 1993 Jan 29, 190(2), 453 - 61
Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries; Wong P et al.; We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols . To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector . To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP) . Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones . We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.

Biochem Biophys Res Commun, 1993 Jan 29, 190(2), 340 - 6
Antibodies to a human alpha 2-C10 adrenergic receptor fusion protein confirm the cytoplasmic orientation of the V-VI loop; Vanscheeuwijck P et al.; DNA encoding the hydrophilic region between transmembrane domains V and VI of the human platelet alpha 2-adrenergic receptor (alpha 2-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST) . Expression of the recombinant plasmid in E . coli resulted in the production of a GST/alpha 2-C10 fusion protein which was purified by preparative SDS-PAGE . Chickens inoculated with the fusion protein produced antibodies that were present in their eggs . In cells expressing the alpha 2-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence . For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent . This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.

Biochem Biophys Res Commun, 1993 Jan 29, 190(2), 397 - 405
Isolation of a cDNA for chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Li L et al.; A chicken liver cDNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Lambda ZAP2 phage library . The chicken liver cDNA codes for a protein that has 89.1, 88.4 and 88.0% amino acid identity with the human, rat and bovine liver isoforms, respectively . The kinetic properties of the rat and chicken liver enzymes, purified to homogeneity after expression in E . coli, were different including negative cooperativity for ATP binding and inhibition by Mg2+ for the chicken liver 6-phosphofructo-2-kinase but not for the rat liver kinase . Differences in the beta-loop ATP signature sequences in the chicken and rat liver kinase domains may explain the kinetic differences and represent the major divergence in the evolution of the enzyme from birds to mammals.

Nature, 1993 Jan 28, 361(6410), 371 - 4
A new photoreactivating enzyme that specifically repairs ultraviolet light-induced (6-4)photoproducts; Todo T et al.; Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)photoproducts) are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by ultraviolet light irradiation of cells . The phenomenon of photoreactivation, the reduction of the lethal and mutagenic effects of ultraviolet radiation by simultaneous or subsequent irradiation with near ultraviolet or visible light, has been identified in several organisms and in some cases the enzymes that catalyse this process have been characterized in sufficient detail . CPDs are the only known substrate for the photoreactivating enzymes so far analysed and enzymatic photoreactivation of (6-4)photoproducts has not yet been reported . We report here that an enzyme that catalyses the light-dependent repair of (6-4)photoproduct exists in Drosophila melanogaster . This is, to our knowledge, the first report of such photoreactivating activity specific for (6-4)photoproducts in any organism.

Biochemistry, 1993 Jan 26, 32(3), 872 - 8
Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins; Jakoby MG et al.; Cellular retinol-binding protein II (CRBP-II) and intestinal fatty acid-binding protein (I-FABP) are both expressed in small intestinal enterocytes and exhibit 31% sequence identity . I-FABP binds a single molecule of long-chain fatty acid and forms an ion-pair electrostatic interaction between the cationic side chain of arginine-106 and the anionic fatty acid carboxyl group . In contrast, CRBP-II binds all-trans-retinol or -retinal and contains a glutamine residue in the corresponding position, residue 109 . We have characterized and compared the interactions of fatty acids and retinoids with I-FABP, CRBP-II, and two reciprocal mutant proteins . The mutants were designated CRBP-II(Q109R), where glutamine-109 was replaced by arginine, and I-FABP(R106Q), where arginine-106 was replaced by glutamine . As monitored by titration calorimetry and carbon-13 NMR spectroscopy, the fatty acid-binding properties of CRBP-II(Q109R) were found to be essentially identical to those of wild-type I-FABP . Both proteins bound 1 molecule of fatty acid with identical affinities (Kd = 0.2 microM) . The enthalpic contribution to the total free energy of binding was large for both proteins: 66% and 87%, respectively . In addition, the carboxyl groups of fatty acids bound to both proteins were solvent-inaccessible . There was little or no change in the ionization state of the bound fatty acid over a wide pH range, as monitored by the chemical shift of the fatty acid carboxyl 13C resonance . Furthermore, the binding of fatty acid to both proteins was accompanied by a selective perturbation of the guanidino 13C resonance of a single arginine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jan 26, 32(3), 812 - 8
Rat liver aromatic L-amino acid decarboxylase: spectroscopic and kinetic analysis of the coenzyme and reaction intermediates; Hayashi H et al.; The physiochemical properties of the coenzyme in rat liver aromatic L-amino acid decarboxylase (AADC) expressed in Escherichia coli have been studied by spectroscopic analysis of the enzyme, its reaction intermediates, and its complexes with substrate analogs . The enzyme, having one pyridoxal 5'-phosphate (PLP) per subunit, shows a prominent absorption maximum at 335 nm and a weaker one at 425 nm . The spectrum did not essentially change in the pH range of 6.0-8.0 . When the coenzyme was excited at 335 nm, it emitted fluorescence primarily at 520 nm . The structure for the absorption at 335 nm was ascribed to the enolimine form of the PLP-lysine Schiff base . On the reaction of AADC with L-3,4-dihydroxyphenylalanine (L-dopa), the absorption of PLP showed biphasic changes before reaching a steady-state . Results of both pre-steady-state and steady-state kinetic analyses were consistent with the model that the reaction proceeds as shown in the equation: E + S<==>X1<==>X2-->E + P . The rate constant was determined for each step, and the Km value for L-dopa was obtained as 0.086 mM . The absorption spectra of the two intermediates, X1 and X2, were postulated from the calculation of the absorption changes during the first and the second steps of the reaction in which X1 and X2 showed an absorption maximum at 425 and 380 nm, respectively, with a concomitant decrease in absorbance at 335 nm . These predicted absorption spectra of X1 and X2 showed striking resemblances to those of AADC complexed with dihydroxyphenylacetic acid (DOPAc) and L-dopa methyl ester (DopaOMe), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jan 26, 32(3), 799 - 805
Escherichia coli fumarase A catalyzes the isomerization of enol and keto oxalacetic acid; Flint DH; Fumarase A, a product of the fumA gene of Escherichia coli, has been found to catalyze the isomerization of enol to keto oxalacetic acid (OAA) in addition to catalyzing the fumarase reaction . The kcat/Km for the isomerization is almost identical to that for the fumarase reaction . The isomerization reaction apparently takes place at the same active site as the fumarase reaction since both reactions show a similar sensitivity to inactivation by O2, both reactions are strongly inhibited by 2-hydroxy-3-nitropropionate, and the isomerization reaction is inhibited by fumarate and malate . The isomerization requires the presence of a {4Fe-4S} or {3Fe-4S} cluster, perhaps for structural rather than catalytic reasons . Hydration of enol OAA to the gem diol has been ruled out as a possible mechanism of isomerization on the basis of the preservation of the oxygen on carbon 2 and the position of protonation on carbon 3 . The isomerization is not stereospecific in the position of protonation at carbon 3 but appears to be stereoselective, with protonation preferentially occurring in the 3-pro-S position . Porcine fumarase, isopropyl malate isomerase, and dihydroxyacid dehydratase do not catalyze this isomerization . Fumarase A and aconitase, two enzymes with 4Fe-4S clusters that bind a linear 4-carbon dicarboxylic acid moiety in the trans conformation during their normal hydro-lyase reaction, do catalyze this isomerization.

Biochemistry, 1993 Jan 26, 32(3), 918 - 22
Local conformational change in the B-subunit of Shiga-like toxin 1 at endosomal pH; Saleh MT et al.; Shiga and Shiga-like toxins are potent bacterial cytotoxins composed of six proteins: one A-subunit that possesses a toxic N-glycosidase activity and a pentamer of identical B-subunits that anchors the toxin to glycolipids present on mammalian cells . Following their endocytosis through coated pits, a segment of the A-subunit noncovalently associated with the B oligomer is translocated to the cytoplasm where it enzymatically inactivates the protein synthesis machinery . The fluorescence intensity of the single tryptophan residue in the B-subunit is perturbed by pH conditions typically observed in an endosomal compartment, with a sharp reversible transition in fluorescence intensity occurring at pH 4.5 . The secondary structure of the pentamer as monitored by circular dichroism is altered by pH conditions lower than 4.5 and greater than 10 . However, the conformational change observed under acidic conditions as low as pH 2 does not parallel a loss of receptor binding potential and is reversible, suggesting that the structure of the B-subunit undergoes a second conformational change between pH 4.5 and 2 without a loss of tertiary or quaternary structure . The B-subunit may thus play a role in the translocation of the A chain to the cytoplasm, an event potentially mediated by a conformational change in its structure at pH levels occurring in the endosomal or lysosomal compartments.

Nucleic Acids Res, 1993 Jan 25, 21(2), 295 - 301
The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding; Wyszynski MW et al.; All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine . It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position . We show here that substitutions of this cysteine in the E . coli methylase M . EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA . Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA . These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor . Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate . However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding . We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine . Further, we show that a DNA substrate for M . EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme . As expected, this modified substrate does not form irreversible complexes with the mutants.

Nucleic Acids Res, 1993 Jan 25, 21(2), 259 - 65
Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus; Uemori T et al.; We cloned the gene encoding the thermostable DNA polymerase from the archaeon Pyrococcus furiosus . The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA polymerase was sequenced . DNA polymerase (Pfu polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA polymerases (family B) represented by human DNA polymerase alpha and Escherichia coli DNA polymerase II . An unrooted phylogenetic tree of the alpha-like DNA polymerases based on the amino acid sequence alignment was constructed . Pfu polymerase, with two other archaeon polymerases, constitutes a group with some animal viruses . The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P . furiosus and E . coli, and the promoters were assigned upstream of the pol coding region . A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P . furiosus.

J Biol Chem, 1993 Jan 25, 268(3), 2232 - 8
Dissection of the biotinyl subunit of transcarboxylase into regions essential for activity and assembly; Shenoy BC et al.; Transcarboxylase, a multisubunit enzyme containing 12 S, 5 S, and 1.3 S subunits, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate (overall reaction) via two partial reactions . In the first partial reaction, a carboxyl group from methylmalonyl-CoA bound to the 12 S subunit is transferred to the biotin of the 1.3 S subunit, and, in the second partial reaction, the carboxylated biotin transfers its carboxyl group from biotin to pyruvate, bound to the 5 S subunit . Previously we have shown that the region around the biotinyl lysine of the 1.3 S subunit is critical for catalysis, that peptides in the amino-terminal region of 1.3 S are capable of forming complexes with 12 S and 5 S, and that amino acids in the carboxyl terminus of the 1.3 S subunit form part of the recognition site for holocarboxylase synthetase . In order to further examine the role of the sequences in this subunit, we generated 8 shortened forms of the 1.3 S biotinyl subunits missing either one or both termini . Truncated 1.3 S subunits were active in both partial reactions until deletion reached amino acid 59 . None of the truncated subunits was able to support stable complex formation with the 12 S and 5 S subunits or catalyze the overall reaction . The results suggest that the region between 59 and 78 is required for activity and the sequence 1-18 is required for enzyme assembly . Activity in the partial reactions correlated with intrinsic fluorescence enhancement of tryptophan residues in either the 12 S or 5 S subunit . Fluorescence enhancement was observed with the shortened 1.3 S subunits until truncation reached amino acid 59 implying either 1) that the internal sequence, 59-78, transiently associates with the other subunits to properly orient the biotin for catalysis or 2) that the sequence 59-78 contributes to the folded conformation of the 1.3 S subunit so that subunit interactions can take place.

J Biol Chem, 1993 Jan 25, 268(3), 2195 - 202
Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation; Sagitov V et al.; The segment Asp1064-Lys1073 in the beta subunit of Escherichia coli RNA polymerase is evolutionarily conserved and is located near the "5' face" of the nucleotide binding pocket as was shown by affinity labeling with priming substrates (Grachev, M . A., Lukhtamov, E . A., Mustaev, A . A., Zaychikov, E . F., Abdukayumov, M . N., Rabinov, I . V., Richter, V . I., Skoblov, Y . S., and Chistyakov, P . G . (1989) Eur . J . Biochem . 180, 577-585) . We engineered single Xaa-->Ala or Ala-->Ser substitutions of eight evolutionarily conserved amino acids in this segment as well as a multiple alanine (KRNK) substitution of four of these residues . The KRNK mutation as well as four of the single substitutions were dominant lethal, two of the single mutations were recessive lethal, and two were viable . RNA polymerase bearing the dominant mutations was prepared for biochemical study by in vitro reconstitution from subunits . All of the mutant enzymes formed stable, specific promoter complexes, capable of initiating RNA synthesis . However, the KRNK polymerase was totally blocked in initiation-to-elongation transition, whereas the four point mutants displayed allele-specific changes in promoter clearance rate . Each of the four mutations changed, in a specific way, both the pattern of short oligomers generated in abortive initiation and the pattern of RNA polymerase pausing during elongation . Thus, the mutations appear to distort but not destroy the active center and to alter, in allele-specific manner, the coupling between the catalytic reaction and RNA polymerase propagation along the template.

J Biol Chem, 1993 Jan 25, 268(3), 2189 - 94
A pathogen-responsive gene of parsley encodes tyrosine decarboxylase; Kawalleck P et al.; A group of recently isolated parsley (Petroselinum crispum) cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment have been demonstrated to encode tyrosine decarboxylase (TyrDC) . The deduced TyrDC protein sequence shares extensive similarity with two functionally related enzymes, tryptophan decarboxylase from periwinkle and dopa decarboxylase from Drosophila melanogaster . Expression of TyrDC cDNA in Escherichia coli yielded catalytically active protein with high substrate specificity for tyrosine . All four identified parsley TyrDC genes have been cloned and encode at least three TyrDC isozymes . Treatment of cultured parsley cells with fungal elicitor caused very rapid and transient increases in TyrDC mRNA and enzyme activity levels.

J Biol Chem, 1993 Jan 25, 268(3), 2063 - 8
Aspartyl residue 10 is essential for ATPase activity of rat hsc70; Huang SP et al.; Three mutants of rat hsc70 were constructed, overexpressed in Escherichia coli, purified, and characterized . First, site-directed mutation was utilized to substitute Asn for Asp-10 . The recombinant protein, hsc70(D10N), loses not only its peptide-stimulated ATPase activity but also its basal ATPase activity . The measured dissociation constants of ATP (0.3 microM) and S-peptide (5 microM) for hsc70(D10N), however, are virtually identical to those of hsc70 . The intrinsic fluorescence spectra of hsc70(D10N) also remain largely unchanged . Therefore, the overall structure of the hsc70 protein is most likely intact after mutation . Second, the entire C-terminal peptide-binding domain was deleted and the resultant mutant contains only the N-terminal ATPase domain of hsc70 . This recombinant protein, Nt-hsc70, is a peptide-independent ATPase . The ATPase activity at 37 degrees C of the Nt-hsc70, 270 pmol/h/micrograms of protein, is comparable to that of maximally peptide-activated hsc70 . Third, the Asp-10 of Nt-hsc70 was replaced by Asn . Despite that this mutant, Nt-hsc70(D10N), is capable of binding ATP and it loses the capability to hydrolyze ATP . Taken together, these results indicate that aspartyl residue 10 of hsc70 is essential for ATP hydrolysis . Purified hsc70 and its mutants autophosphorylate in vitro at a substoichiometric level . On average, less than 1% of the hsc70 and Nt-hsc70 proteins are phosphorylated . Although the amount of phosphate incorporated into hsc70(D10N) and Nt-hsc70(D10) is reduced, a significant level of phosphorylation can still be achieved in these two site-directed mutants . Hence, autophosphorylation of hsc70 and its mutants is not correlated with their ability to hydrolyze ATP.

J Biol Chem, 1993 Jan 25, 268(3), 2013 - 20
Evidence for an extended structure of the T-cell co-receptor CD8 alpha as deduced from the hydrodynamic properties of soluble forms of the extracellular region; Boursier JP et al.; We expressed soluble forms of the human T-cell coreceptor CD8 alpha extracellular region, CD8 alpha 161, and the amino-terminal immunoglobulin-like domain, CD8 alpha 114, in Chinese hamster ovary cells and Escherichia coli, respectively . Both molecules were readily purified to homogeneity in milligram amounts and were recognized by a large panel of monoclonal antibodies . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that approximately 70% of CD8 alpha 161 was secreted as a disulfide-linked dimer, but CD8 alpha 114 was not disulfide-linked . To investigate the structural features of CD8 alpha 161 and CD8 alpha 114 under native conditions, we performed gel filtration and sucrose gradient sedimentation analysis . In spite of being partially or totally noncovalently bound, both recombinant molecules were stably associated homodimers, as no monomers could be detected at a fairly low protein concentration (approximately 1 microM) . This suggests that the CD8 alpha amino-terminal domain alone strongly contributes to chain association . Determination of the Stokes radius (Rs) and sedimentation coefficient (s20,w) gave results consistent with CD8 alpha 114 having a globular shape and CD8 alpha 161 being an asymmetric molecule . Taking into account the contribution of hydration to the frictional coefficient, we obtained for CD8 alpha 161 an axial ratio of approximately 5, when modeled as a prolate ellipsoid . These results indicate that the elongated structure of CD8 alpha 161 is essentially contributed by the hinge region and help to explain how the CD8 alpha is able to bridge the distance between the T-cell surface and its binding site in the alpha 3 domain of major histocompatibility complex class I molecules on the target cell.

J Biol Chem, 1993 Jan 25, 268(3), 1965 - 75
Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis . Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities; Kong H et al.; We have isolated, cloned, and characterized a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis, the Tli DNA polymerase (also referred to as Vent DNA polymerase) . The enzyme is extremely thermostable, having a half-life of 8 h at 95 degrees C and about 2 h at 100 degrees C . Pseudo-first-order kinetics at 70 degrees C reveal an extremely low Km for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high Km for dNTPs (50 microM) . Accompanying extension rates are on the order of 1000 nucleotides/min . Synthesis by the polymerase is largely distributive, adding an average of 7 nucleotides/initiation event . This distributive synthesis can generate products of at least 10,000 bases . Tli DNA polymerase contains a 3'-->5' exonuclease activity that enhances the fidelity of replication by the enzyme (Mattila, P., Korpela, J., Tenkanen, T . and Pitkanen, K . (1991) Nucleic Acids Res . 19, 4967-4973) . A 2-amino acid substitution within the conserved exonuclease domain abolishes both double and single strand-dependent exonuclease activity, without altering kinetic parameters for polymerization on a primed single-stranded template . Strand displacement activity by the mutated and unmutated forms increases with increasing temperature and is enhanced in the exonuclease-deficient form of the enzyme.

J Biol Chem, 1993 Jan 25, 268(3), 1931 - 6
DNA repair by eukaryotic nucleotide excision nuclease . Removal of thymine dimer and psoralen monoadduct by HeLa cell-free extract and of thymine dimer by Xenopus laevis oocytes; Svoboda DL et al.; Using a human cell-free extract, we have recently shown that thymine dimers are removed from DNA in oligonucleotides 27-29 nucleotides in length (Huang, J . C., Svoboda, D . L., Reardon, J . T., and Sancar, A . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 3664-3668) . In this study we find that the excision reaction is dependent on ATP, the excised fragments range in length from 27-32 nucleotides, and have 5'-P and 3'-OH termini . We also found that a thymine-psoralen furan side monoadduct is excised from the DNA with a similar incision pattern, indicating that in humans bulky adducts are removed from DNA by the same enzyme system which hydrolyzes mainly the 22-24th and the 5th phosphodiester bonds 5' and 3', respectively, to the lesion . This incision pattern might be common to eukaryotic excision nucleases as thymine dimers were removed from DNA by the same dual incision pattern by Xenopus laevis oocytes.

J Biol Chem, 1993 Jan 25, 268(3), 1805 - 10
Binding of DNA quenches tyrosine fluorescence of RecA without energy transfer to DNA bases; Eriksson S et al.; The binding of single- as well as double-stranded DNA to RecA, in the presence of the cofactor analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), leads to about 20% quenching of the tyrosine fluorescence of the protein but to no essential change of the tryptophan fluorescence . The excitation spectrum of the fluorescent DNA analog poly(d epsilon A), complexed with RecA, shows no sign of energy transfer from the tyrosine residues of RecA to the etheno-modified adenine bases of the polynucleotide . From this observation we reject stacking interaction between tyrosine residues and DNA bases . The RecA filament may bind up to three molecules of single-stranded DNA; however, the observed fluorescence change occurs only upon the binding of the first DNA strand, indicating that the binding mode of this first strand is different from those of the others . The fluorescence change is interpreted in terms of a conformational change of the RecA protein promoted by cooperative binding to DNA . A larger quenching (40%) upon the binding of single-stranded DNA is observed in the absence of cofactor . At high salt condition, which induces ATPase activity in RecA just as DNA binding does, the tyrosine fluorescence is more pronounced than at low salt conditions, indicating that the effect induced by high salt is different from the conformational change induced by DNA binding.

J Biol Chem, 1993 Jan 25, 268(3), 1677 - 83
Isozyme-specific modules on human aldolase A molecule . Isozyme group-specific sequences 1 and 4 are required for showing characteristics as aldolase A; Motoki K et al.; Vertebrate aldolase molecules bear at least four stretches of isozyme group-specific sequences (referred to as IGS) . The IGSs of the type A isozyme are known to endow the aldolase molecules with some characteristics typical of A . In order to locate the type A regions, 4 chimeric enzymes were constructed between human aldolases A and B and 5 mutant enzymes with single or double mutations in the IGS-1 region . Among engineered proteins, the chimeric enzymes bearing the type A IGS-1 to -4 (BABA34-108:306-363) and the IGS-1 and -4 (BABA34-55:306-363) exhibited similarities to isozyme A in many respects . On the other hand, neither chimeric enzyme bearing the type A IGS-1 to -3 (BAB34-108) nor that bearing the IGS-1 alone (BAB34-55) exhibited properties as isozyme A . Four mutant aldolases A (carrying single mutation in the IGS-1 region) maintained the original activity as A . Similarly, the BA306 chimera with the type B-->A substitution at positions 41 and 45 (BA306 N41K:R45S) failed to exhibit the A-like properties although the activities toward Fru-1,6-P2 and Fru-1-P significantly increased . Conclusively, the type A IGS-1, together with the IGS-4, act as indispensable modules in determining the characteristic properties of human aldolase A.

J Biol Chem, 1993 Jan 25, 268(3), 1590 - 5
Mutations of the molecular chaperone protein SecB which alter the interaction between SecB and maltose-binding protein; Gannon PM et al.; SecB is a 16-kDa cytosolic chaperone protein that is required for efficient export of particular proteins in Escherichia coli . To identify regions of SecB that contribute to efficient protein export, we isolated secB point mutants that are defective for protein export in vivo . We obtained missense mutations at residues Leu75 (SecBL75Q), Cys76 (SecBC76Y), and Glu77 (SecBE77K) in the center of the secB gene . In vivo, mutant SecBL75Q and SecBE77K proteins are capable of binding to precursor maltose-binding protein (MBP) and preventing the formation of export-incompetent precursor MBP; however, export of MBP is still defective . In vitro, purified SecBL75Q and SecBE77K proteins bound to unfolded MBP and blocked its refolding . SecBL75Q and SecBE77K were more effective than wild-type SecB at blocking the refolding of unfolded MBP, suggesting that SecBL75Q and SecBE77K have a higher affinity for unfolded MBP.

J Biol Chem, 1993 Jan 25, 268(3), 1575 - 9
Genetic and immunological analyses of the cyanobacterium Synechocystis sp . PCC 6803 show that the protein encoded by the psbJ gene regulates the number of photosystem II centers in thylakoid membranes; Lind LK et al.; The psbJ gene is a member of the psbEFLJ gene cluster in the cyanobacterium Synechocystis sp . PCC 6803 as well as in the chloroplasts of green plants . The putative product of the psbJ gene is a 4-kDa protein with one membrane-spanning domain . We have raised rabbit antibodies against a T7 gene 10-psbJ fusion protein, overexpressed in Escherichia coli . These antibodies recognized a polypeptide of expected size in the thylakoid membrane from wild type Synechocystis cells . We have also created a targeted mutant of Synechocystis 6803 in which the fourth codon of the psbJ open reading frame was modified to a translational stop codon . Thylakoid membranes from these mutant cells lacked the protein recognized by the antibodies . In the mutant cells, the partial electron transfer reaction mediated by the photosystem I complex was unaffected, whereas the rate of the photosystem II (PSII)-mediated reaction was 46% of that in wild type cells . Herbicide binding assays indicated that the PSII to chlorophyll ratio in the mutant cells was 49% of that in wild type cells . These results indicate that while the PsbJ protein is not essential for the photochemical activity it controls the amount of functionally assembled PSII complex in the thylakoid membrane.

J Biol Chem, 1993 Jan 25, 268(3), 2048 - 51
Identification of the herpes simplex virus-1 protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products; DiIanni CL et al.; Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35) . The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease . The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus . The fusion protease underwent autoproteolytic cleavage at two distinct sites . The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data . The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis . Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease . The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.

J Biol Chem, 1993 Jan 25, 268(3), 1960 - 4
Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C; Nemenoff RA et al.; Phospholipase A2 (PLA2) is the enzyme regulating the release of arachidonic acid in most cell types . A high molecular mass, 85-kDa soluble form of PLA2 (cPLA2) has recently been identified, the activity of which is stably increased by stimulation of cells with hormones and growth factors . Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA2 on serine residues, but the kinases mediating this effect have not been identified . We report here that human cPLA2 is phosphorylated in vitro by two growth factor-stimulated serine/threonine-specific kinases, p42 MAP kinase and protein kinase C (PKC) . Phosphorylation of the cPLA2 enzyme by either kinase results in an increase in catalytic cPLA2-specific activity . Domains of the cPLA2 molecule have been expressed in Escherichia coli, and the fusion proteins purified . PKC and p42 MAP kinase give different patterns of phosphorylation of the recombinantly expressed cPLA2 fragments . p42 MAP kinase selectively phosphorylates the domain of cPLA2 containing a MAP kinase consensus sequence, whereas PKC phosphorylates sites in all three recombinantly expressed domains of the enzyme . Peptide mapping indicates that the site phosphorylated by p42 MAP kinase is different from those phosphorylated by PKC . The combined action of both of these kinases is likely to mediate the effects of growth factor stimulation on arachidonic acid release through the activation of cPLA2.

J Biol Chem, 1993 Jan 25, 268(3), 1780 - 5
Isolation and properties of adenovirus type 2 proteinase; Tihanyi K et al.; We have cloned and expressed the human adenovirus type 2 proteinase gene in Escherichia coli . The expressed proteinase was isolated by a four-step chromatographic procedure . Purity and identity of the recombinant protein was established by two-dimensional gel electrophoresis, N-terminal sequencing, and specific antisera . The pure enzyme did not contain disulfide bridges, and it consisted of one subunit with a pI of 10.2 . It did not show any sign of autocleavage . Labeled iodoacetate bound the pure enzyme while labeled diisopropyl fluorophosphate did not . The protease readily cleaved the viral pVII protein, ovalbumin, fibrin, and actin but had no effect on synthetic penta-, octa-, or nonapeptides carrying the consensus sequence for cleavage . The inhibitory profile of the isolated proteinase and the affinity labeling clearly indicate that the human adenovirus type 2 proteinase is a cysteine rather than a serine proteinase as previously believed . The most likely candidate for an active site residue is one of the two conserved cysteines, Cys-104 or Cys-122.

J Biol Chem, 1993 Jan 25, 268(3), 1729 - 34
Physiological role of nhaB, a specific Na+/H+ antiporter in Escherichia coli; Pinner E et al.; The nhaB gene which codes for Na+/H+ antiporter activity in Escherichia coli was recently cloned (Pinner, E., Padan, E., and Schuldiner, S . (1992) J . Biol . Chem . 267, 11064-11068) . In order to elucidate the role of nhaB in Na+ and H+ ions physiology and its interaction with nhaA, we generated mutants in which the chromosomal gene has been inactivated by insertion/deletion . A mutant devoid of both nhaA and nhaB is extremely sensitive to Na+ and Li+ at all pH values, and membranes prepared from this strain show no Na+/H+ antiporter activity . As opposed with the delta nhaA mutant which contains NhaB, the pH independent Na+/H+ antiporter (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S . (1989) J . Biol . Chem . 264, 20297-20302), the delta nhaB mutant, containing NhaA, shows Na+/H+ antiporter activity highly dependent on pH . nhaB, in the absence of nhaA, confers a certain tolerance to Na+ which decreases with increasing pH . In the absence of NhaB, NhaA alone confers complete halotolerance under all conditions tested . However, when grown on agar in minimal medium on substrates which are symported with Na+ (proline, serine, and glutamate) at pH 6 and at low Na+ concentrations (< 10 mM), delta nhaB grows slower than the wild type and its Na+ dependent transport of glutamate and proline is markedly inhibited . Since both of these defects of the delta nhaB strain are alleviated upon transformation of the mutant with multicopy plasmid bearing nhaA, we conclude that nhaB is crucial when the level of NhaA activity is growth limiting, when nhaA is not sufficiently induced, and/or when NhaA is not activated.

J Biol Chem, 1993 Jan 25, 268(3), 2075 - 82
Expression of Drosophila Rrp1 protein in Escherichia coli . Enzymatic and physical characterization of the intact protein and a carboxyl-terminally deleted exonuclease-deficient mutant; Sander M et al.; Drosophila Rrp1 protein purified from embryos has four tightly associated enzymatic activities: DNA strand transfer, single-strand DNA renaturation, 3'-exonuclease, and apurinic endonuclease . Copurifying with these activities is a single polypeptide that has an apparent M(r) of 105,000 when estimated by SDS-polyacrylamide gel electrophoresis . To determine if this polypeptide is sufficient for these activities, it has been overexpressed in Escherichia coli . In crude extracts of E . coli cells, an ATP-independent Mg(2+)-dependent strand transfer activity is observed upon activation of the promoter that drives expression of Rrp1 . Rrp1 protein purified from induced E . coli cells has electrophoretic, chromatographic, and enzymatic properties similar to those of Drosophila Rrp1 protein . The carboxyl-terminal region of Rrp1 (amino acids 428-679) is homologous to E . coli exonuclease III . Rrp1 deleted for this region cannot carry out DNA strand transfer, but can renature complementary single-strand DNA . The strand transfer activity of this truncated protein can be restored if DNA 3'-exonuclease is provided in trans by pretreating the double-strand DNA substrate with E . coli exonuclease III . This demonstrates a likely role of the exonuclease in the in vitro DNA strand transfer reaction carried out by Rrp1 protein . Such a role is also suggested by an analysis of the polarity of the strand transfer reaction.

J Biol Chem, 1993 Jan 25, 268(3), 1553 - 7
pH-dependent stability and membrane interaction of the pore-forming domain of colicin A; Muga A et al.; Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy . In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M . At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation . Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved . The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A . Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7 . Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state . These findings are discussed in relation to the recent model (van der Goot, F . G., Gonzalez-Manas, J . M., Lakey, J . H., Pattus, F . (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A.

Science, 1993 Jan 22, 259(5094), 525 - 8
Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling; Duchesne M et al.; Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways . GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals . A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling . A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes . The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP . Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone . These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.

J Mol Biol, 1993 Jan 20, 229(2), 494 - 501
Domain closure in adenylate kinase . Joints on either side of two helices close like neighboring fingers; Gerstein M et al.; In large variants of adenylate kinase the AMP and ATP substrates are buried by a domain rotating by 90 degrees . Here conformational changes responsible for this domain closure are determined by an analysis of the open state of beef heart mitochondrial adenylate kinase and the closed state of Escherichia coli adenylate kinase . Although these two proteins have sequence differences, the principal structural changes responsible for the domain movements are large, and can clearly be distinguished from the effects of evolution . The mobile domain is linked to the rest of the protein by two helices packed together in an antiparallel fashion . During the closure, deformations take place in four localized regions, called joints, near the N and C termini of these helices . Three of these joints have simple motions that can be well approximated by rotations of three torsion angles, but the joint that makes contact with the ligand involves motion throughout an extended loop: i.e . two torsions on either side of a reverse turn change significantly . The main chain atoms of the joints have few packing constraints . The first pair of joints is responsible for approximately 30 degrees of the total rotation and the second pair for the remaining approximately 60 degrees . These movements carries along the regions between the joints, the two helices and the rest of the mobile domain, to a first approximation, as rigid bodies . This jointed domain closure mechanism is contrasted with the shear mechanisms found in other enzymes.

J Mol Biol, 1993 Jan 20, 229(2), 306 - 18
Lrp, a global regulatory protein of Escherichia coli, binds co-operatively to multiple sites and activates transcription of ilvIH; Wang Q et al.; Lrp (Leucine-responsive regulatory protein) has recently been recognized as a major regulatory protein that controls the expression of many operons in Escherichia coli . Footprinting and gel retardation experiments with DNA from ilvIH, one of the operons controlled positively by Lrp, indicate that Lrp binds to six sites over a 200 base-pair region upstream from the promoter . Binding of Lrp to some of these sites is highly co-operative . We suggest a consensus sequence for Lrp binding based upon a comparison of six binding sites . An analysis of mutants indicates that five out of six binding sites are important for transcription activation and that two or three adjacent Lrp binding sites act synergistically in vivo . The observed synergistic effects in vivo may result from co-operative binding of Lrp to adjacent sites . We propose a model in which multiple binding sites contribute to the formation of a nucleoprotein complex, but only a particular proximal site positions Lrp properly so that it interacts with RNA polymerase.

J Mol Biol, 1993 Jan 20, 229(2), 328 - 43
Homologous recognition and triplex formation promoted by RecA protein between duplex oligonucleotides and single-stranded DNA; Rao BJ et al.; RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5' end of its own plus strand . An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none . The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3' end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA . In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I . The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect . These results are discussed in relation to possible models of triplex DNA.

Biochemistry, 1993 Jan 19, 32(2), 472 - 81
In vivo evidence that UV-induced C-->T mutations at dipyrimidine sites could result from the replicative bypass of cis-syn cyclobutane dimers or their deamination products; Jiang N et al.; The major mutations induced by UV light are C-->T transitions at dipyrimidines and arise from the incorporation of A opposite the C of dipyrimidine photoproducts . The incorporation of A has most often been explained by the known preference of a polymerase to do so opposite noninstructional DNA damage such as an abasic site (A rule) . There are also mechanisms that suppose, however, that cis-syn dipyrimidine photodimers are instructional . In one such mechanism (tautomer bypass), the incorporation of A is directed by the tautomer of a C of a dimer that is equivalent in base-pairing properties to U {Person et al . (1974) Genetics 78, 1035-1049} . In another mechanism (deamination bypass), the incorporation of A is directed by a U of a dimer that results from the deamination of the C of a dimer {Taylor & O'Day (1990) Biochemistry 29, 1624-1632} . The viability of these mechanisms was tested by obtaining the mutation spectrum of a TU dimer in Escherichia coli by application of a standard method for site-directed mutagenesis . To this end, a 41-mer containing a site-specific TU dimer was constructed via ligation of a dimer-containing decamer that was produced by triplet-sensitized irradiation and used to prime DNA synthesis on a uracil-containing (+) strand of an M13 clone containing a double mismatch opposite the dimer . The reaction mixture was used to transfect a uracil glycosylase proficient, photoproduct repair deficient E . coli host, and all progeny phage weakly hybridizing to the parental (+) or (-) strands were sequenced . Under non-SOS conditions the TU dimer almost completely blocked replication, while under SOS conditions it directed the incorporation of two As with much higher specificity (96%) than would an abasic site . The implications of these results to the mechanism of the UV-induced TC-->TT mutation, and by extension to the CT-->TT, CC-->TC, CC-->CT, and the tandem CC-->TT mutations, are discussed.

Biochemistry, 1993 Jan 19, 32(2), 426 - 35
Comparison of backbone and tryptophan side-chain dynamics of reduced and oxidized Escherichia coli thioredoxin using 15N NMR relaxation measurements; Stone MJ et al.; The backbone and tryptophan side-chain dynamics of both the reduced and oxidized forms of uniformly 15N-labeled Escherichia coli thioredoxin have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy . Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured for more than 90% of the protonated backbone nitrogen atoms and for the protonated indole nitrogen atoms of the two tryptophan residues . These data were analyzed by using a model free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m) . The reduced and oxidized forms exhibit almost identical dynamic behavior on the picosecond to nanosecond time scale . The W31 side chain is significantly more mobile than the W28 side chain, consistent with the positions of W31 on the protein surface and W28 buried in the hydrophobic core . Backbone regions which are significantly more mobile than the average include the N-terminus, which is constrained in the crystal structure of oxidized thioredoxin by specific contacts with a Cu2+ ion, the C-terminus, residues 20-22, which constitute a linker region between the first alpha-helix and the second beta-strand, and residues 73-75 and 93-94, which are located adjacent to the active site . In contrast, on the microsecond to millisecond time scale, reduced thioredoxin exhibits considerable dynamic mobility in the residue 73-75 region, while oxidized thioredoxin exhibits no significant mobility in this region . The possible functional implications of the dynamics results are discussed.

Biochemistry, 1993 Jan 19, 32(2), 395 - 400
Selective isotopic enrichment of synthetic RNA: application to the HIV-1 TAR element; Michnicka MJ et al.; The introduction of isotopically enriched nucleotides into NMR quantities of a synthetic 29-mer RNA derived from the HIV-1 TAR element is described . RNA enriched in 13C and/or 15N is produced by a procedure which involves isolation of whole cellular RNA from Escherichia coli, nucleolysis, separation of mononucleotides, chemical or enzymatic pyrophosphorylation, and in vitro transcription by T7 RNA polymerase . Spectral characteristics of each residue type are examined in isolation . 13C chemical shifts provide an alternative method to determine ribose puckers for larger RNAs . Nonprotonated sites such as purine N7 groups can now be monitored through the use of multiple-bond 1H-15N coupling . When applied conservatively, coordinate analysis of chemical shift values should prove valuable for NMR studies of RNA structure and recognition . 1H, 13C, and 15N chemical shift data suggest that TAR residue A35 has an unusual local environment, consistent with extrusion of its base from the terminal loop.

Biochemistry, 1993 Jan 19, 32(2), 628 - 36
Assembly of the Rieske iron-sulfur subunit of the cytochrome bc1 complex in the Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunits; Van Doren SR et al.; The Rieske iron-sulfur subunit of the cytochrome bc1 complex from Rhodobacter sphaeroides has been expressed in Escherichia coli and also in a strain of Rb . sphaeroides lacking the other subunits of the bc1 complex . PCR products encoding the full-length subunit were introduced into expression vectors to produce the subunit alone or the subunit fused behind the mature portion of the E . coli maltose binding protein (MBP), but lacking the MBP signal sequence . These proteins are both located in the cytoplasmic membrane . The unfused Rieske subunit assembles a Rieske-like iron-sulfur cluster, but with EPR characteristics which differ from the normal rhombic signal observed in the cytochrome bc1 complex . The overproduced MBP fusion protein, on the other hand, does not contain an EPR-detectable iron-sulfur cluster . Subfragments of the Rieske subunit lacking the amino-terminal hydrophobic anchor also lack the iron-sulfur cluster were expressed in E . coli . When expressed in Rb . sphaeroides in the absence of the cytochrome b and c1 subunits, the fully metalated Rieske subunit with the diagnostic gy = 1.90 EPR signal is observed in the cytoplasmic membrane . The fact that the Rieske subunit has an assembled iron-sulfur cluster and is bound to either the E . coli or the Rb . sphaeroides membrane in the absence of the other subunits of the bc1 complex demonstrates a mode of membrane attachment independent of the other components of the complex . These data are consistent with models in which the Rieske subunit is bound to the membrane via a single membrane-spanning helix located near the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jan 19, 32(2), 622 - 7
Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex; Meyer TE et al.; Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions . When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone . The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5 . At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths . The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex . The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1) . Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges . It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jan 19, 32(2), 602 - 12
Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II); Runyon GT et al.; We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein . Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E . coli cells containing the overexpression plasmid . Overproduction of helicase II protein at these levels is lethal in E . coli . The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 {20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C} . We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties . The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT) . In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions . However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important . On the basis of these observations and similarities between helicase II and the E . coli Rep helicase, which appears to function as a dimer {Chao, K., & Lohman, T . (1991) J . Mol . Biol . 221, 1165-1181}, we suggest that the active form of helicase II may also be a dimer or larger oligomer.

Biochim Biophys Acta, 1993 Jan 18, 1145(1), 119 - 23
Ionophore properties of OmpA of Escherichia coli; Saint N et al.; Both porins OmpA1, from wild-strain K12 Escherichia coli, and OmpA2, from a K12 derivative deficient in both OmpF and OmpC, are able to form ion channels in virtually solvent-free membranes . The conductance has been shown to vary in a discrete fashion with different single increment values especially with OmpA2 . This behaviour seems to indicate, beside monomers, the presence of aggregates of different sizes . The estimated small pore diameter (0.6-0.7 nm) for the monomeric would explain the weak permeability of this narrow channel toward different solutes . OmpA protein, from experiments of ion selectivity and zero-current potential, is determined weakly anion selective.

Eur J Biochem, 1993 Jan 15, 211(1-2), 373 - 6
Homology of pyridoxal-5'-phosphate-dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p-aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products; Mehta PK et al.; Bacterial deletion mutants have indicated that the gene products of cobC, nifS, pabC and malY participate in important metabolic pathways, i.e . cobalamin synthesis, nitrogen fixation, synthesis of p-aminobenzoate and the regulation of the maltose system, respectively . However, the proteins themselves and their specific functions have not yet been identified . In the course of our studies on the evolutionary relationships among aminotransferases, we have found that the above gene products are homologous to aminotransferases . Profile analysis {Gribskov, M., Luthy, R . & Eisenberg, D . (1990) Methods Enzymol . 183, 146-159} based on the amino acid sequences of certain subgroups of aminotransferases as probes attributed significant Z scores in the range 5-20 SD to the deduced amino acid sequences of the above gene products as included in the protein data base . Reciprocal profile analyses confirmed the homologies . All known aminotransferases are pyridoxal-5'-phosphate-dependent enzymes and catalyze the reversible transfer of amino groups from amino acids to oxo acids . The sequence homologies suggest that the above gene products are aminotransferases or other closely related pyridoxal-5'-phosphate-dependent enzymes probably catalyzing transformations of amino acids involving cleavage of a bond at C alpha.

Eur J Biochem, 1993 Jan 15, 211(1-2), 181 - 91
The use of lac-type promoters in control analysis; Jensen PR et al.; For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady-state fluxes or concentrations . We describe an improved method for effecting the modulation, as elaborated for Escherichia coli . The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter . The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes . The lacUV5 promoter should be used when the wild-type expression level is low, the tacI promoter when the latter is high . The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lacY7am mutation (eliminating lactose permease, the transport system for the inducer isopropyl-thio-beta-D- galactoside) is introduced . The method was demonstrated experimentally by constructing E . coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter . We measured the concentration of the c subunit of H(+)-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times the wild-type level . Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level . We show that the introduction of a lacY mutation facilitated the attainment of steady induction levels of partially induced cells . The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl-thio-beta-D-galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values . These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.

Gene, 1993 Jan 15, 123(1), 87 - 92
Cloning and characterization of a gene coding for the catechol 1,2-dioxygenase of Arthrobacter sp . mA3; Eck R et al.; The catA gene, coding for the catechol 1,2-dioxygenase (C12O) of the bacterial strain Arthrobacter sp . mA3, was cloned and expressed in Escherichia coli . One plasmid containing a 6.1-kb EcoRI insert was selected by its ability to degrade catechol and to accumulate cis-cis-muconate . The DNA insert of this plasmid was mapped with restriction enzymes . The catA gene was subcloned on a 1.3-kb PstI-EcoRI fragment by deleting the adjacent restriction fragments . The nucleotide sequence of catA was determined . The C12O is coded for by a gene spanning 849 nucleotides and the deduced M(r) of the protein is 30,560 . The polypeptide encoded by the cloned catA gene was expressed in an E . coli minicell system and detected by gel electrophoresis.

Gene, 1993 Jan 15, 123(1), 115 - 9
Cloning and characterisation of an aminopeptidase P-encoding gene from Streptomyces lividans; Butler MJ et al.; An aminopeptidase P (PepP)-encoding gene has been cloned from Streptomyces lividans 66 by screening for overexpression of activity using the chromogenic substrate Gly-Pro-beta-naphthylamide as a liquid overlayer on colonies growing on agar medium . The pepP gene was localised by deletion mapping, and the nucleotide sequence was determined . The deduced amino acid sequence was found to display significant similarity to Escherichia coli PepP . The partially purified S . lividans enzyme had a 50-kDa subunit and was present as a homodimer . Direct Edman degradation of the purified protein confirmed that pepP encoded the observed intracellular PepP.

Gene, 1993 Jan 15, 123(1), 1 - 7
Increased antibody expression from Escherichia coli through wobble-base library mutagenesis by enzymatic inverse PCR; Stemmer WP et al.; We tested the value of a new library mutagenesis approach, called library enzymatic inverse PCR (LEIPCR), for expression-level enhancement of antibody Fv fragments produced in Escherichia coli . The production level of active, metal chelate-specific antibody from our constructs is limited by a low expression level of the second, heavy-chain cistron . To increase the production level, LEIPCR was applied to the wobble bases of the second cistron leader peptide . In LEIPCR mutagenesis, the entire plasmid is amplified using mutagenic primers with class-IIS restriction endonuclease (ENase) sites at their 5' ends . The PCR product is digested with the class-IIS ENase (here, BsaI; GGTCTCN{symbol: see text}NNNN{symbol: see text}), which removes its own recognition sequence, and the ends are self-ligated . Thus, LEIPCR can be used to make plasmid mutant libraries regardless of the nucleotide sequence, and independent of available ENase sites . The resulting library of 10(7) wobble mutants was screened for active Fv by a colony filter lift . A selected mutant was shown to produce between four- and elevenfold more active Fv than the wild type (wt), and fivefold more heavy chain . Mutations outside of the leader peptide were shown not to be involved . The mutated areas of the mRNAs of two different up-mutants may have less secondary structure than the wt . Thus, the sequence of the mRNA of the second leader peptide was limiting to the expression level of heavy-chain and active Fv.

Cell, 1993 Jan 15, 72(1), 29 - 38
A nuclear pore complex protein that contains zinc finger motifs, binds DNA, and faces the nucleoplasm; Sukegawa J et al.; We have molecularly cloned and sequenced a cDNA for a rat liver nucleoporin with a molecular mass of 152.8 kd, termed nup153, that shares a repetitive degenerate pentapeptide motif with a subgroup of nucleoporins of yeast and vertebrates . However, its most striking feature is a novel 4-fold repeat of a Cys2-Cys2-type zinc finger motif . When expressed in E . coli, the zinc finger domain of nup153 binds DNA in a zinc-dependent fashion . Immunoelectron microscopy localized nup153 exclusively to the nucleoplasmic side of the nuclear pore complex . We suggest that nup153 recognizes a specific DNA sequence to organize the genome three-dimensionally and to gate transcribable genes to nuclear pore complexes.

Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 250 - 6
Rational design and expression of a heparin-targeted human superoxide dismutase; Boissinot M et al.; In order to improve the therapeutic effectiveness of human Cu,Zn superoxide dismutase (HSOD) by targeting it to cell surfaces and increasing its circulatory half-life, we have designed and expressed a heparin-binding derivative of HSOD . This design was based on the idea that structurally independent protein units, HSOD and the heparin-binding A+ helix from protein C inhibitor, could be combined with a carefully chosen linker, GlyProGly, to form a stable, bifunctional protein . The chimeric HSOD-GlyProGly-A+ protein was expressed and secreted to the periplasm of E . coli and had normal SOD activity . HSOD-GlyProGly-A+ had a significantly increased retention time relative to wild-type HSOD on a heparin affinity column, indicating that it was successfully targeted to heparin, and this binding was maintained at physiological ionic strength . When administered to mice, HSOD-GlyProGly-A+ had a half-life of approximately 15 minutes, twice that of wild-type HSOD . Our rational design approach should be generally applicable to the creation of bifunctional chimeric molecules.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 615 - 9
Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli; Laity JH et al.; Wild-type ribonuclease A and five mutants thereof have been expressed in Escherichia coli as fusion proteins by using a T7 expression system . The five mutants are C{65-72}S, C{40-95}S, C{58-110}S, C{26-84}S, and K41G . The expressed fusion protein formed inclusion bodies which were then cleaved by factor Xa . The cleaved ribonuclease A was isolated as unfolded (sulfonated), soluble protein which was subsequently folded . This expression system can be used to produce mutants of ribonuclease A in yields suitable for folding and structural studies . All four native three-disulfide mutants exhibited enzymatic activity (5-30%), although only two were thermally stable at room temperature, demonstrating that no single native disulfide bond is essential for folding . The K41G mutant was enzymatically inactive with cyclic cytidine monophosphate as substrate.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 577 - 81
A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene; Sun X et al.; During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication . The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG . The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase . The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence . RVSGY, of pyruvate formate-lyase . During activation of the anaerobically induced pyruvate formate-lyase, the glycine residue of the pentapeptide becomes an organic radical {Wagner, A . F . V., Frey, M., Neugebauer, F . A., Schafer, W . & Knappe, J . (1992) Proc . Natl . Acad . Sci . USA 89, 996-1000} . The gene for the anaerobic reductase is located at a position around 96 min on the E . coli genomic map.

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 497 - 501
In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions; Sakamoto W et al.; We have used the Escherichia coli beta-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function of the 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene . Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene . In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated . However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of beta-glucuronidase activity was detected . When approximately 70% of the petD 5' UTR was deleted from the translational fusion, uidA mRNA accumulation and beta-glucuronidase activity decreased 4- to 6-fold and 8-fold, respectively . Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates . Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability . The expression of beta-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.

Science, 1993 Jan 15, 259(5093), 365 - 8
Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase; Singh R et al.; A transfer RNA (tRNA) binding protein present in HeLa cell nuclear extracts was purified and identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . Studies with mutant tRNAs indicated that GAPDH recognizes both sequence and structural features in the RNA . GAPDH discriminated between wild-type tRNA and two tRNA mutants that are defective in nuclear export, which suggests that the protein may participate in RNA export . The cofactor nicotinamide adenine dinucleotide disrupted complex formation between tRNA and GAPDH and thus may share a common binding site with the RNA . Indirect immunofluorescence experiments showed that GAPDH is present in the nucleus as well as in the cytoplasm.

Science, 1993 Jan 15, 259(5093), 358 - 61
Two open complexes and a requirement for Mg2+ to open the lambda PR transcription start site; Suh WC et al.; Potassium permanganate (KMnO4) footprinting in the absence and presence of magnesium (Mg2+) at the lambda PR promoter identified two different open complexes with Escherichia coli E sigma 70 RNA polymerase (designated RPo1 and RPo2) . The single-stranded region in RPo1 (formed in the absence of Mg2+) was at most 12 bases long, whereas that in RPo2 (formed in the presence of Mg2+) spanned at least 14 bases . Only in RPo2 did the single-stranded region extend to the start point of transcription (+1, +2) . These results provide a structural basis for the requirement for uptake of Mg2+ in the formation of RPo2 from RPo1, as deduced from kinetic studies at this promoter.

J Biol Chem, 1993 Jan 15, 268(2), 948 - 54
Determinants of catalytic activity and stability of carbonic anhydrase II as revealed by random mutagenesis; Krebs JF et al.; The functional importance of a conserved hydrophobic face in human carbonic anhydrase II (CAII), including amino acid residues 190-210, was investigated by random mutagenesis . The catalytic activity, inhibitor binding, and level of CAII expression in Escherichia coli of 57 single amino acid variants were measured revealing that the function of amino acids correlates with their secondary structure placement . Side chains of amino acids in beta-sheet structure are required for the formation of folded, stable protein while those in the turn region determine catalytic efficiency and inhibitor specificity . The CAII active site is extremely plastic, accommodating amino acid substitutions of varied size, charge, and hydrophobicity with little effect on catalysis; only substitutions at Leu198 and Thr199 decrease the rates of CO2 hydration and ester hydrolysis more than 5-fold . These results pinpoint the hydrogen bond network, including the zinc-solvent molecule and Thr199, as crucial for high catalytic efficiency and also suggest that Leu198 forms a portion of a CO2 association site . Increased activity is observed for substitutions at Thr200 (esterase) and Leu203 (hydrase) . In addition, the pKa of the zinc-bound water molecule varies upon substitution of amino acids which alter the overall charge of the active site . Three residues interact with sulfonamide inhibitors; substitutions at Thr199 decrease binding (up to 10(3)-fold) while mutations at Thr200 and Cys206 increase binding of dansylamide (up to 80-fold) . Mutations in the beta-sheet structure (Asp190-Ser197 and Val207-Ile-210) decrease the protein expression of CAII in E . coli, causing the formation of insoluble protein aggregates in many cases . This may suggest an important role for these residues in the folding process . In addition, mutations in Trp192, cis-Pro202, and Trp209 increase thermal lability (up to 5000-fold).

J Biol Chem, 1993 Jan 15, 268(2), 917 - 22
Structural and functional domains of Escherichia coli ribosomal protein L7/L12 . The hinge region is required for activity; Oleinikov AV et al.; Variant forms of Escherichia coli ribosomal protein L7/L12 were constructed, overexpressed, and purified . These included proteins that deleted residues 35-52 (delta 35-52) and 42 to 52 (delta 42-52), others that contained single cysteine substitutions at residues 63 and 89, and combinations of the deletions and cysteine substitutions . Chemical modification of the introduced cysteine residues with {14C}iodoacetamide was used to radiolabel the protein variants in order to quantify their binding to the ribosome . Neither of the deletions in the hinge domain, delta 35-52 and delta 42-52, had any effect on L7/L12 dimer formation as detected by cross-linking by dimethyl suberimidate . Perpendicular urea gradient gel electrophoresis showed that both deletion variants retained a compact structural element attributable to the globular C-terminal domain . Reconstitution of core particles depleted of wild type L7/L12 with the deletion proteins showed that delta 42-52 bound normally in 4 copies per particle, whereas delta 35-52 bound in only 2.5 copies following isolation of the particles by high speed centrifugation or gel filtration . Ribosomes mixed with an excess of the deletion variants and assayed directly for polyphenylalanine synthesis were completely inactive . The results suggest that the flexibility conferred by the hinge is required for activity, perhaps by allowing the C-terminal domain to occupy a location near the base of the L7/L12 stalk.

J Biol Chem, 1993 Jan 15, 268(2), 909 - 16
Genetic engineering of snake toxins . Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis; Pillet L et al.; To study the site by which erabutoxin a (Ea) from Laticauda semifasciata binds to the nicotinic acetylcholine receptor, we mutated most residues that are shared with other curaremimetic toxins and studied the structural and biological consequences of introduced mutations . By site-directed mutagenesis, we changed Ser-8 into Gly (EaS8G), Lys-27 into Glu (EaK27E), Trp-29 into Phe (EaW29F) and His (EaW29H), Asp-31 into His (EaD31H), Phe-32 into Leu (EaF32L), Arg-33 into Lys (EaR33K) and Glu (EaR33E), Gly-34 into Ser (EaG34S), Glu-38 into Gln (EaE38Q) and Lys (EaE38K), Gly-49 into Val (EaG49V), and Leu-52 into Ala (EaL52A) . All mutants were homogeneous as judged by various analytical procedures . EaE38Q, EaG49V, and EaL52A bound the nicotinic acetylcholine receptor with apparent Kd values close to 10(-10) M, virtually identical to wild Ea . Therefore, Glu-38, Gly-49, and Leu-52 are not important elements in the expression of curaremimetic function in Ea . Mutations of Phe-32 and Gly-34 provoked a 7-fold affinity decrease, suggesting that these residues moderately contribute to function . The 176-fold affinity decrease due to mutation of Ser-8 may reflect some structural change that operates in the polypeptide chain of the mutant, as detected by circular dichroism . Decreases in affinity by a factor of 175, 67, 46, and 318 were seen upon mutations of Lys-27 into Glu, Trp-29 into Phe, Asp-31 into His, and Arg-33 into Glu, with no concomitant change in secondary structure . These residues appear to be important elements of the curaremimetic function of Ea . Thus, a picture of the contribution of conserved residues to the function of a curaremimetic toxin is proposed on the basis of experimental evidence.

J Biol Chem, 1993 Jan 15, 268(2), 880 - 6
Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V . The position of the alpha NH2 moiety affects catalytic activity; Schrock RD 3rd et al.; Reductive methylation of the alpha NH2 moiety of the DNA repair enzyme T4 endonuclease V has been shown previously to eradicate both the N-glycosylase and apyrimidinic/apurinic lyase activities of the enzyme (Schrock, R . D., III, and Lloyd, R . S . (1991) J . Biol . Chem . 266, 17631-17639) . The present study uses the technique of site-directed mutagenesis to investigate the important parameters involved in the cleavage mechanism . The prediction was that the addition of an amino acid in the immediate NH2-terminal region of the protein would alter the proximity of the alpha NH2 moiety of Thr2 to its target, thereby severely compromising the enzyme's catalytic activity . However, substitutions in this region generally should be tolerated . To test this hypothesis, three substitutions of the NH2-terminal amino acid were produced: Ser2 (T2S), Val2 (T2V), and Pro2 (T2P) . An addition mutant was also produced by adding a glycine between the first and second amino acids of the protein (Thr2-Gly-Arg3) (+Gly) . The T2P and +Gly mutants had negligible pyrimidine dimer-specific N-glycosylase activity as well as negligible pyrimidine dimer-specific nicking activity in vitro . Conversely, the T2S enzyme exhibited wild type levels of activity and the T2V exhibited intermediate levels of activity in vitro . Results from ultraviolet (UV) survival studies of the mutant enzymes indicated that the in vivo activities of these enzymes were directly correlated to the enzymes' ability to cleave at pyrimidine dimers in vitro . These results indicate that a critical parameter for the functionality of endonuclease V is the relative distance between the primary alpha NH2 group in the active site of the enzyme and those elements responsible for DNA binding and pyrimidine dimer recognition.

J Biol Chem, 1993 Jan 15, 268(2), 867 - 72
The gamma subunit of the Escherichia coli ATP synthase . Mutations in the carboxyl-terminal region restore energy coupling to the amino-terminal mutant gamma Met-23-->Lys; Nakamoto RK et al.; The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M . (1992) J . Biol . Chem . 267, 20835-20839) . In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys . By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation . H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored . Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased . Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable . Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.

J Biol Chem, 1993 Jan 15, 268(2), 815 - 22
Escherichia coli fumarate reductase frdC and frdD mutants . Identification of amino acid residues involved in catalytic activity with quinones; Westenberg DJ et al.; Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate . The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones . Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis . Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E . coli DW35 (delta frdABCD sdhC::kan) under selective conditions requiring functional enzyme . Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities . Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone . Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol . Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His-80 . The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex . In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.

J Biol Chem, 1993 Jan 15, 268(2), 775 - 8
GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein; Chuang TH et al.; The majority of the GTP-binding proteins of the Ras superfamily hydrolyze GTP to GDP very slowly . A notable exception to this are the Rac proteins, which have intrinsic GTPase rates at least 50-fold those of Ras or Rho . A protein (or proteins) capable of inhibiting this GTPase activity exists in human neutrophil cytosol . Since Rac appears to exist normally in neutrophils as a cytosolic protein complexed to (Rho)GDI, we examined the ability of (Rho)GDI to inhibit GTP hydrolysis by Rac . (Rho)GDI produced a concentration-dependent inhibition of GTP hydrolysis by Rac1 that paralleled its ability to inhibit GDP dissociation from the Rac protein . Maximal inhibition occurred at or near equimolar concentrations of the GDI and the Rac substrate . The ability of two molecules exhibiting GTPase activating protein (GAP) activity toward Rac to stimulate GTP hydrolysis was also inhibited by the presence of (Rho)GDI . The inhibitory effect of the GDI could be overcome by increasing the GAP concentration to levels equal to that of the GDI . (Rho)GDI weakly, but consistently, inhibited GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) dissociation from Rac1, confirming an interaction of (Rho)GDI with the GTP-bound form of the protein . These data describe an additional activity of (Rho)GDI and suggest a mechanism by which Rac might be maintained in an active form in vivo in the presence of regulatory GAPs.

J Biol Chem, 1993 Jan 15, 268(2), 771 - 4
Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor; Schroder I et al.; Integration host factor protein (IHF) was shown to be required for Fnr- and NarL-dependent activation of the nitrate reductase (narGHJI) operon of Escherichia coli in response to nitrate availability and anaerobiosis . Using a narG-lacZ reporter fusion to evaluate narGHJI expression in vivo both the nitrate and anaerobic dependent controls were severely impaired in a himA mutant compared with the wild type strain . IHF was also required for Fnr-independent anaerobic control of narGHJI expression . In vitro, purified IHF protein was shown to bind to a narG promoter fragment with an apparent dissociation value of 5 nM by use of a gel shift assay . DNase I footprinting studies revealed that IHF protects a 37-base pair region centered 125 base pairs 5' of the narG transcription site . These studies suggest that the IHF protein performs a DNA bending function at the narG promoter to allow nitrate-dependent activation by the NarL regulatory protein, and second, it enhances the Fnr-dependent expression from the narG promoter under anaerobic cell growth conditions . A model whereby three transcriptional activators, NarL, IHF, and Fnr, induce expression of a sigma 70-dependent promoter for the narGHJI operon is discussed.

J Biol Chem, 1993 Jan 15, 268(2), 1488 - 93
Lipopolysaccharide interaction with S2 subunit of pertussis toxin; Lei MG et al.; Using radioiodinated, photoactivable, reducible cross-linker conjugated bacterial endotoxic lipopolysaccharide (125I-ASD-LPS), we have demonstrated that LPS selectively binds to the S2 subunit of pertussis toxin (PT) . Since LPS also interacts with the S2 subunit of the B-oligomer of the toxin, the binding of LPS to PT is not A-protomer (S1 subunit) dependent . The binding can be inhibited with native underivatized LPS and with purified lipid A, suggesting that the binding is mediated through the lipid A moiety of the LPS molecule . The binding of PT to LPS can be inhibited by bovine fetuin glycoprotein . Since PT has been demonstrated to interact specifically with N-linked oligosaccharide side chains of fetuin, the interaction of LPS with the S2 subunit of PT may involve carbohydrate-dependent interactions of the disaccharide backbone of lipid A with S2 . Additional studies have documented that LPS binding to PT may be competitively inhibited by lysozyme but not by polymyxin B . Sequence analysis has allowed identification of a high degree of amino acid sequence similarity between the S2 subunit of PT and hen egg white lysozyme at the N-terminal 80-residue regions . Shared N-terminal sequence similarity between lysozyme, PT-S2, and a third LPS-binding protein alpha-lactalbumin allows tentative identification of a second family of LPS binding proteins.

J Biol Chem, 1993 Jan 15, 268(2), 1462 - 9
Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli; Jonsson CB et al.; The forward and reverse reactions for integration were characterized for the Moloney murine leukemia virus integrase (M-MuLV IN) protein . The M-MuLV IN was recombinantly produced in Escherichia coli, and was purified to greater than 90% homogeneity by a one-step affinity purification scheme . M-MuLV IN was highly active for integration as measured by in vitro cleavage and strand transfer assays . Furthermore, the integration of a model viral substrate into lambda concatamers by IN correctly produced the flanking 4-base pair duplications characteristic of M-MuLV IN . The reverse reaction of integration, disintegration, was also catalyzed by the recombinant M-MuLV IN . Two products were generated, a 3'-recessed long terminal repeat and a ligated target DNA, from a model integration-intermediate substrate in the presence of M-MuLV IN . The requirements and optimal conditions for maximal integration and disintegration activity for M-MuLV IN were determined . The forward and reverse reactions required different concentrations of manganese ion and reductant . Salt was also titrated for the forward and reverse reactions . Sodium chloride inhibited integration, but had little affect on disintegration . Low concentrations of potassium chloride enhanced integration, but had no affect on disintegration . The dinucleotide cleavage, strand transfer, and the disintegration reactions each had a unique pH profile of activity.

J Biol Chem, 1993 Jan 15, 268(2), 1414 - 23
Mutational analysis of G protein alpha subunit G(o) alpha expressed in Escherichia coli; Slepak VZ et al.; G protein-mediated signal transduction is dependent on alpha subunit interactions with beta gamma subunits, receptors, effectors, magnesium ions, and guanine nucleotides . The interdependence of these interactions can be probed by mutational analysis . We developed large scale screening procedures in recombinant Escherichia coli to identify and characterize novel mutations in G(o) alpha . Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of G(o) alpha . Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E . coli . beta gamma interactions were assayed by pertussis toxin mediated ADP-ribosylation . Efficacy of the screening procedures was evaluated by studying properties of wild-type G(o) alpha and site-directed mutations that were characterized previously in other G proteins . Several novel mutants with altered GTP binding characteristics and reduced ability to interact with beta gamma had been isolated from the randomly generated mutant library . ADP-ribosylation of mutants R10G, K21N, and K35E was significantly reduced, whereas two of the mutants bearing multiple amino acid substitutions were refractory to modification . Mutant K35E also exhibited reduced affinity to guanosine 5'-(3-O-thio)triphosphate at submicromolar concentrations of magnesium . These experiments demonstrate the feasibility of using large scale random mutagenesis in the studies of G protein function.

J Biol Chem, 1993 Jan 15, 268(2), 1332 - 7
Structure of hepatitis B virus core and e-antigen . A single precore amino acid prevents nucleocapsid assembly; Schodel F et al.; The hepatitis B virus core gene codes for two polypeptides: the core protein, which assembles to form particles (HBcAg), and the secreted precore protein (HBeAg) . Expression vectors directing the synthesis in Escherichia coli of a recombinant HBeAg corresponding in sequence to serum-derived HBeAg encompassing the 10 precore amino acids remaining after cleavage of the precursor and residues 1-149 of HBcAg (PC-HBeAg) were constructed . Recombinant PC-HBeAg, HBcAg, and C-terminally truncated HBcAg were isolated from E . coli and analyzed by sucrose velocity sedimentation, electron microscopy, anti-HBc/e specific monoclonal antibody analysis, and for immunogenicity . HBcAg and truncated HBcAg formed 27-nm particles and displayed HBc antigenicity . In contrast, PC-HBeAg was nonparticulate and did not band in sucrose gradients . PC-HBeAg was recognized efficiently by HBeAg-specific antibodies and displayed little HBc antigenicity . Immunogenicity studies including T and B cell recognition confirmed that PC-HBeAg demonstrates HBe antigenicity . The presence of the 10 precore amino acids therefore prevented particle formation . To analyze which precore amino acids might be responsible for the prevention of particle formation a cysteine to glutamine substitution at amino acid position -7 was introduced into PC-HBeAg (-7C-->Q)PC-HBeAg . This single amino acid change at position -7 restored particle formation and HBc antigenicity . The evolutionarily conserved cysteine at position -7 thus appears responsible for the prevention of particle assembly in the HBeAg biosynthesis pathway.

J Biol Chem, 1993 Jan 15, 268(2), 1326 - 31
The tRNA-(m5U54)-methyltransferase of Escherichia coli is present in two forms in vivo, one of which is present as bound to tRNA and to a 3'-end fragment of 16 S rRNA; Gustafsson C et al.; The enzyme tRNA-(m5U54)-methyltransferase (EC 2.1.1.35) of Escherichia coli catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to uridine in position 54 of the T psi-loop of all E . coli tRNA species, forming 5-methyluridine (m5U) . In vivo, this enzyme is present both as a native polypeptide of 42 kDa and as a TrmA.RNA complex . The TrmA.RNA complex is not dissociated during strong denaturing conditions such as boiling in 8 M urea or 6 M guanidine HCl, consisting with that the RNA is covalently bound to the protein . After sequencing and Southern blot analyses, the RNA was identified to be a subset of undermodified tRNA species as well as the 3' terminus of 16 S rRNA . However, the complex is not associated with the ribosome and the covalently bound RNA does not affect the tRNA methylating activity of the enzyme.

J Biol Chem, 1993 Jan 15, 268(2), 1292 - 7
Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation; Rehrauer WM et al.; The hydrolysis of the nucleoside triphosphates, such as ATP or GTP, plays a central role in a variety of biochemical processes; but, in most cases, the specific mechanism of energy transduction is unclear . DNA strand exchange promoted by the Escherichia coli recA protein is normally associated with ATP hydrolysis . However, we advanced the idea that the observed ATP hydrolysis is not obligatorily linked to the exchange of DNA strands (Menetski, J . P., Bear, D . G., and Kowalczykowski, S . C . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 21-25); instead, ATP binding resulting in an allosteric transition to an active form of the recA protein is sufficient . In this paper, we extend this conclusion by introducing a mutation within a highly conserved region of the recA protein that, on the basis of sequence similarity, is proposed to interact with the pyrophosphate moiety of a bound NTP molecule . The conservative substitution of an arginine for the invariant lysine at position 72 reduces NTP hydrolysis by approximately 600-850-fold . This mutation does not significantly alter the capacity of the mutant recA (K72R) protein either to bind nucleotide cofactors and single-stranded DNA or to respond allosterically to nucleotide cofactor binding . Despite the dramatic attenuation in NTP hydrolysis, the recA (K72R) protein retains the ability to promote homologous pairing and extensive exchange of DNA strands (up to 1.5 kilobase pairs) . These results both identify a component of the catalytic domain for NTP hydrolysis and demonstrate that the recA protein-promoted pairing and exchange of DNA strands mechanistically require the allosteric transition induced by NTP cofactor binding, but not the energy educed from NTP hydrolysis.

J Biol Chem, 1993 Jan 15, 268(2), 1166 - 73
Phosphorylation of recombinant tau by cAMP-dependent protein kinase . Identification of phosphorylation sites and effect on microtubule assembly; Scott CW et al.; Tau protein is an integral component of paired helical filaments, a pathological feature of Alzheimer's disease . tau extracted from these filaments displays decreased electrophoretic mobility due to aberrant phosphorylation . Here we show that recombinant human tau can be phosphorylated by cAMP-dependent protein kinase resulting in decreased electrophoretic mobility . Phosphorylation of tau by cAMP-dependent protein kinase caused a 92% decrease in the maximum rate of tau-induced microtubule assembly . The sites of phosphorylation were identified by digesting phosphorylated tau with proteases, separating the peptides by reversed-phase HPLC, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase N-terminal sequencing . Five phosphorylation sites were identified, two of which were located within microtubule binding domains . One site was previously shown to be the sole phosphorylation site for CaM kinase II; phosphorylation at this site by CaM kinase II was sufficient to cause decreased electrophoretic mobility (Steiner, B., Mandelkow, E . M., Biernat, J., Gustke, N., Meyer, H . E., Schmidt, B., Mieskes, G., Soling, H . D., Drechsel, D., Kirschner, M . W., Goedert, M., and Mandelkow, E . (1990) EMBO J . 9, 3539-3544) . Thus two different second messenger-dependent protein kinases can phosphorylate tau at the same site and induce a shift in tau mobility like that seen in Alzheimer's disease.

Gene, 1993 Jan 15, 123(1), 63 - 8
A new lambda RES vector with a built-in Tn1721-encoded excision system; Altenbuchner J; A new lambda replacement vector for construction of genomic libraries was developed which allows the excision of cloned fragments by site-specific recombination from the lambda DNA and conversion into autonomously replicating plasmids . The vector system, derived from lambda EMBL4, is called lambda RES . It contains two recognition sites for site-specific recombination from Tn1721 on both sides of the replacement fragment of lambda EMBL4 . Additionally, on one side, there is a plasmid replication origin from Rtsl with a kanamycin-resistance (KmR) marker . DNA fragments in the range of 8-14 kb may be inserted between BamHI or Sall sites in the lambda vector . Efficient excision and conversion of plaque-forming units into KmR colonies are obtained by infection of Escherichia coli strains harbouring Tn1739tnpR on a F' plasmid . Tn1739tnpR is a derivative of Tn1721 with a chloramphenicol-resistance-encoding gene (CmR), the lambda cI repressor gene, and a further copy of the resolvase-encoding tnpR gene under control of the tac promoter.

J Biol Chem, 1993 Jan 15, 268(2), 1081 - 6
Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli; Iglesias AA et al.; cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADP-glucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrophosphorylase activity . Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity . Maximal enzyme activity required both subunits . The expressed ADP-glucose pyrophosphorylase was purified and characterized . The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber . The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum . 3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol . In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A0.5 value of 57 microM . The recombinant ADP-glucose pyrophosphorylase was less sensitive to P(i) inhibition and more sensitive to heat denaturation than the potato tuber enzyme . Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.

FEMS Microbiol Lett, 1993 Jan 15, 106(2), 217 - 22
Clonal relationships among bovine pathogenic Escherichia coli producing surface antigen CS31A; Contrepois M et al.; Forty-two Escherichia coli strains producing surface antigen CS31A isolated from bovine infections were characterized with respect to OKH serotypes, outer membrane protein (OMP) electrophoretic patterns, allozymes for esterases A, B, C, I and biotypes . A large majority of the strains could be clustered in a limited number of groups of clonally related strains with diverse O serogroups . CS31A producing Escherichia coli strains thus appear to have a common genetic background and are representative of an important part of bovine pathogenic Escherichia coli.

Eur J Biochem, 1993 Jan 15, 211(1-2), 27 - 35
High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli; Pompejus M et al.; A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis . The gene sequence was designed for convenient manipulation in future protein engineering experiments . In-frame fusion of bos to the lacZ454 gene allowed high-yield production in Escherichia coli of a beta-Gal454/BO fusion protein, deposited as intracellular inclusion bodies . These were enriched by virtue of their insolubility in 0.5% Triton X-100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the beta-Gal454/BO boundary . Pure BO could be obtained from the mixture of water-insoluble cleavage products by selective extraction into organic solvent . The yield was in the range 30-50 mg pure protein/l culture medium, depending on individual preparation . This material could be used for reconstitution of fully functional bacteriorhodopsin . Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.

Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 242 - 9
Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells; Chang HH et al.; Rhodostomin (Rho) from snake venom, a potent inhibitor of platelet aggregation, contains 68 amino acids having an RGD sequence and 12 cysteine residues . A chemically synthesized Rho gene was cloned and expressed in Escherichia coli . The expression of Rho gene fused with the glutathione S-transferase (GST) gene was about 10-30% of total cell proteins . The Rho-fusion protein could be recognized by antibodies raised against either a native Rho peptide or a synthetic peptide . The purified GST-Rho coated on culture plates facilitated the attachment of human hepatoma cells, which was inhibitable by co-incubation with a synthetic hexapeptide GRGDSP but not with a related peptide of GRGESP, suggesting that the E . coli-expressed Rho-fusion protein was properly folded and biologically functional.

Carbohydr Res, 1993 Jan 15, 238, 261 - 70
Structure of the O56 antigen of Escherichia coli, a polysaccharide containing 7-substituted alpha-N-acetylneuraminic acid; Kogan G et al.; The O56 polysaccharide moiety of the O56 antigen (LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid in the molar ratios 1:1:1:1 . Methylation analysis, periodate oxidation, mild acid hydrolysis, as well as 1H and 13C NMR spectroscopy showed that the O56 polysaccharide has the primary structure {formula: see text}

Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 457 - 61
Potent anti-CD5 ricin A chain immunoconjugates from bacterially produced Fab' and F(ab')2; Better M et al.; We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30) . This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines . An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography . An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab' . Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG . An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30 . The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane . These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.

J Biol Chem, 1993 Jan 15, 268(2), 1349 - 54
The gene PPG encodes a novel yeast protein phosphatase involved in glycogen accumulation; Posas F et al.; Degenerate oligonucleotides were used to selectively amplify yeast genomic sequences related to Ser/Thr protein phosphatases . Among the sequences obtained, clone ST4-2 was found to code for a novel sequence related to previously known phosphatases . A size-selected yeast genomic library was constructed and screened using clone ST4-2 as probe, and one positive clone, named PPG, was isolated . DNA sequencing of a 1.8-kilobase pair fragment of this clone revealed an open reading frame of 1104 base pairs which codes for a 368-amino acid protein . On the basis of its amino acid sequence, the product of gene PPG would be an acidic protein, structurally more related to type 2A than to type 1 or 2B phosphatases, and is characterized by an extension of about 50 amino acids at the carboxyl terminus . The gene, which is located in chromosome XIV, is expressed as a 1.3-kilobase mRNA and is not essential for growth . Haploid mutants carrying a disrupted copy of the gene were able to grow in glucose as well as in other carbon sources, but they accumulated less glycogen than the wild type strain . However, the state of activation of glycogen synthase was essentially identical in wild type and mutant cells . The finding that, in early exponential phase, mutant cells contain higher levels of glycogen phosphorylase a, in addition to a lower amount of total glycogen synthase activity observed in medium-late exponential phase, could account for the difference found in glycogen accumulation.

J Biol Chem, 1993 Jan 15, 268(2), 1132 - 40
Evidence for autoinhibitory regulation of the c-src gene product . A possible interaction between the src homology 2 domain and autophosphorylation site; Fukami Y et al.; In the previous study (Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y . (1990) Biochem . Biophys . Res . Commun . 171, 1152-1159), we found a synthetic peptide, termed peptide A, that inhibited the kinase activity of p60v-src . The peptide A sequence corresponds to residues 137 to 157 of p60v-src which are included in the amino-terminal portion of the src homology 2 domain . In this study, we attempted to specify the inhibitory sequence in this domain and to identify its target site . The most potent peptide A derivative was one that corresponds to residues 140 through 157 . The target site of peptide A was assumed to reside in the autophosphorylation site of p60v-src, since synthetic peptides containing the sequence Phe424-Pro-Ile-Lys-Trp428 which is present downstream of the autophosphorylated Tyr416 partially counteracted the inhibitory effect of peptide A . An antibody was prepared against one of such target peptides, termed pepY . Cross-linking experiments showed that 125I-labeled peptide A could bind to p60v-src blotted on a membrane, and the binding was blocked by the anti-pepY antibody but not by other anti-p60v-src antibodies . Conversely, immunoblotting of p60v-src with anti-pepY antibody was blocked by the cross-linking of peptide A to p60v-src . To our surprise, anti-pepY antibody did not affect the p60v-src activity . Furthermore, p60c-src was activated 2- to 6-fold by this antibody . These results suggest that the pepY region in the catalytic domain of p60v-src or of p60c-src is not essential for the catalytic activity but rather is involved in the negative regulation of the kinase activity of p60c-src.

Biochemistry, 1993 Jan 12, 32(1), 83 - 90
Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region; Ariansen S et al.; The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied . Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored . Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity . To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences . The amino acids C-terminal to Cys186 were found not to be required for translocation . Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation . The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.

Biochemistry, 1993 Jan 12, 32(1), 260 - 7
L-29, an endogenous lectin, binds to glycoconjugate ligands with positive cooperativity; Massa SM et al.; The soluble mammalian lactose-binding lectins L-14-I and L-29 are both secreted and bind to oligosaccharides on laminin, a large extracellular matrix glycoprotein containing polylactosamine chains . Because of the potential functional significance of these lectin-laminin interactions, we compared quantitative aspects of L-14-I and L-29 binding to immobilized laminin using recombinant lectins labeled with 125I . We report that the concentration-dependent binding of L-29 exhibits positive cooperativity whereas binding of L-14-I does not . Cooperative binding of L-29 can also occur on glycoconjugate substrates other than laminin and is not dependent on cystine bond formation or aggregation in solution . L-29 contains repetitive sequences within the N-terminal domain not present in L-14-I . This domain is not required for binding activity, but is required for positive cooperativity . Though the precise mechanism of interaction of L-29 with laminin remains to be determined, it apparently results in assembly of a lectin aggregate on the substrate surface, which could have important functional consequences.

Biochemistry, 1993 Jan 12, 32(1), 232 - 40
Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase; Miles BW et al.; Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons . These three histidine residues were individually mutated to asparagine residues . The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500) . By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia . The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate . This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction . The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme . The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jan 12, 32(1), 2 - 6
Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA; Toney JH et al.; A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or "transformation" occurs upon ligand binding as a first step toward regulation of gene transcription . In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli . Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT . These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA . These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway.

Biochemistry, 1993 Jan 12, 32(1), 191 - 8
Comparison of the B-pentamers of heat-labile enterotoxin and verotoxin-1: two structures with remarkable similarity and dissimilarity; Sixma TK et al.; We have compared the B-subunit pentamers of Escherichia coli heat-labile enterotoxin (LT) and verotoxin-1 (VT-1) . The B-subunits of these bacterial toxins of the AB5 class have virtually no sequence identity and differ considerably in size (69 amino acids in VT-1 versus 103 in LT) . They share a number of functional properties: pentamer formation, association with an A-subunit, binding to carbohydrate-containing lipids, and interaction with membranes . The structures of these proteins are very similar in some respects and very different in others . They can be superimposed with an rms deviation of only 1.29 A on the main chain atoms of 52 amino acids (0.98 A on 47 C alpha) . Seven out of eight secondary structure elements are retained in the two toxins; only the N-terminal helix of LT is absent in VT-1 . A disulfide bridge, which is essential for pentamer formation, is found in both structures, but in slightly different locations . However, the VT-1 B-subunit is much shorter on one side of the toxin, where the proposed membrane binding site of both VT-1 and LT is located . The monomer-monomer interface in the pentamer is much larger in LT than in VT-1, making the LT pentamer more stable . The central pores have a different character, and the sugar binding sites are not conserved between the toxins . The evolutionary relationship of the toxins is discussed.

Nucleic Acids Res, 1993 Jan 11, 21(1), 21 - 6
Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme; Gaur RK et al.; A new approach for modification interference studies is presented . It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification . This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E . coli RNase P RNA (M1 RNA) . The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1) . This site was already known to interfere with RNase P cleavage, if modified . We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs . Two unpaired regions were involved in both pre-tRNAs . Phosphorothioates interfered at the transition between acceptor- and D-arms . The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage . These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P . We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.

FEBS Lett, 1993 Jan 11, 315(3), 343 - 6
Cloning and sequence analysis of cDNA for the Ca(2+)-activated photoprotein, clytin; Inouye S et al.; Clytin is a member of the aequorin family of photoproteins . It is made up of 189 amino acid residues, contains 3 Ca(2+)-binding sites, and shows 62% homology in amino acid residues to those in aequorin . The cysteine, tryptophan, and histidine residues, and the C-terminal proline, that are conserved in aequorin and clytin may be involved in the Ca(2+)-activated bioluminescence of the two proteins . Clytin may also prove useful in the determination of Ca2+.

Nucleic Acids Res, 1993 Jan 11, 21(1), 51 - 7
Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P; Tallsjo A et al.; The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E . coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions . The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release . Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release . Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5 . In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme . In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules . A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro . We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.

J Mol Biol, 1993 Jan 5, 229(1), 26 - 36
Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase; Meinnel T et al.; To be aminoacylated by Escherichia coli methionyl-tRNA synthetase, a tRNA requires the presence of the methionine anticodon (CAU sequence) . However, the importance in this reaction of the other nucleotides of tRNAs(Met) has still to be described . In this work, through the study of more than 35 variants of tRNAs(Met), it is shown, firstly, that the parameters of the aminoacylation reaction remain independent of the mutations affecting either the sequences or the sizes of the D-loop, D-stem and variable loop . This conclusion is illustrated by the construction and study of a tRNAf(MetCAU) with the D-stem, D-loop and very long variable loop of a class II tRNA . The resulting chimaeric tRNA is methionylated as efficiently as tRNAf(MetCAU) or tRNAm(MetCAU) . Secondly, mutations affecting base 73 and base pairs 2.71 and 3.70 in the acceptor stem of tRNAf(MetCAU), as well as bases 32, 33 and 37, adjacent to the anticodon, cause a strong reduction of the rate of the aminoacylation reaction . Thirdly, it is shown that, provided it is given the acceptor stem of tRNAm(MetCAU) or tRNAf(MetCAU), a tRNA having the anticodon loop of tRNA(Met) can be converted into a substrate for methionyl-tRNA synthetase as efficient as tRNAf(MetCAU) or tRNAm(MetCAU) . Finally, it is proposed that, beyond the binding of the anticodon loop to the synthetase, the sequence of the acceptor stem may strongly influence the rate of the catalytic step of the aminoacylation reaction by properly orientating the 3'-end of the tRNA towards the catalytic centre.

J Mol Biol, 1993 Jan 5, 229(1), 1 - 7
Spacing requirements between LexA operator half-sites can be relaxed by fusing the LexA DNA binding domain with some alternative dimerization domains; Oertel-Buchheit P et al.; The dimerization domain of the LexA repressor has been replaced by two heterologous dimerization motifs: the "leucine zipper" from the jun oncogene product and the carboxy-terminal oligomerization domain of Escherichia coli lac repressor . The corresponding hybrid proteins LexA1-87-Jun zipper and LexA1-87-lac repressor have been purified and their DNA binding properties have been studied using gel mobility shift assays . Both fusion proteins form stable specific complexes with a short DNA duplex harboring the CTGT(at)4ACAG consensus sequence of the LexA repressor . This conserved DNA binding capacity distinguishes these two fusion proteins from many others containing a LexA DNA binding domain fused to different heterologous transactivation and/or dimerization domains . However the fusion proteins LexA1-87-Jun zipper and LexA1-87-lac repressor behave differently from native LexA repressor in that these fusion proteins tolerate the insertion of additional base-pairs between the two invertedly repeated CTGT motifs . LexA1-87-Jun zipper requires two CTGT motifs and tolerates the insertion of at least two additional base-pairs between these motifs, whereas LexA1-87-lac repressor requires in fact only a single CTGT motif for the formation of a specific complex detectable in gel mobility shift assays . The inability of the normal LexA repressor to form well-defined complexes with operators containing additional base-pairs in the center suggests that the LexA "hinge region" between the amino-terminal DNA binding and the carboxy-terminal dimerization domain might not be entirely flexible . In an attempt to remove a hypothetical interaction between the LexA cleavage site (which is situated within the hinge region) and the catalytic cleavage center (situated within the carboxy-terminal domain) a LexA mutant repressor containing five simultaneous mutations in the hinge region has been constructed and purified . Surprisingly this mutant repressor failed to form stable complexes detectable by the gel mobility shift assay even with the normal consensus sequence, suggesting that the LexA hinge region is more than a simple connector between the two structural domains and that its chemical nature is important not only for LexA cleavage, but also for the formation of stable LexA-DNA complexes.

J Biol Chem, 1993 Jan 5, 268(1), 699 - 705
Reticulocalbin, a novel endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence; Ozawa M et al.; A novel Ca(2+)-binding protein, tentatively designated reticulocalbin, has been identified and characterized . Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence staining . The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids . The protein has six repeats of a domain containing the high affinity Ca(2+)-binding motif, the EF-hand . Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them . Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+ . The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus . This is similar to the Lys-Asp-Glu-Leu sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells . A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into medium in transient expression assays.

J Biol Chem, 1993 Jan 5, 268(1), 584 - 90
Expression of mammalian 5-aminolevulinate synthase in Escherichia coli . Overproduction, purification, and characterization; Ferreira GC et al.; 5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes . A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D . S., and Curtis, P.J . (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein . Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate . A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture . Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml . Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources . These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor . Removal of the pyridoxal 5'-phosphate led to complete loss of activity . However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate . The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme . This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E . coli and its purification from an overproducing strain . The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.

J Biol Chem, 1993 Jan 5, 268(1), 52 - 8
Metalloregulated expression of the ars operon; Wu J et al.; The plasmid-borne arsenical resistance (ars) operon encodes an arsenical-translocating ATPase and confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the toxic compounds from the cytosol . The trans-acting regulatory ArsR protein was shown to bind to a fragment of DNA containing the ars promoter . Hybrid formation of the ArsR protein with a ArsR-beta-lactamase chimeric protein suggested that the active form of the ArsR repressor is a dimer . From footprinting analysis the binding site was defined as a region of imperfect dyad symmetry just upstream of the -35 site . In vivo the operon was derepressed by oxyions of +III oxidation state of arsenic, antimony, and bismuth, as well as arsenate (As(V)), whereas in vitro ArsR protein-operator interaction was reduced by each of those compounds except arsenate, as determined by gel retardation and DNase I and hydroxyl radical footprinting experiments . This indicates that arsenate is not a true inducer and must be reduced to arsenite in vivo to induce . An operator mutant obtained by deletion of the in vitro ArsR-protected DNA sequence exhibited constitutive ars promoter activity, demonstrating that the binding site is the functional target for the ArsR repressor in vivo.

J Biol Chem, 1993 Jan 5, 268(1), 455 - 63
DNA splicing by an active site mutant of Flp recombinase . Possible catalytic cooperativity between the inactive protein and its DNA substrate; Serre MC et al.; Each strand transfer catalyzed by the Flp recombinase is the composite of two transesterification reactions . The active nucleophilic species in the two reactions are the catalytic site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl from the Flp-nicked DNA substrate, respectively . A "half recombination site" is capable of undergoing this pair of transesterifications in the presence of Flp . When the substrate is a half-site containing a chiral phosphorothioate at the exchange point, the Flp reaction yields a product in which the phosphate chirality is retained . A mutant of Flp that lacks the active site tyrosine, Flp(Y343F), is incapable of mediating strand transfer in a full-recombination site but can execute strand transfer in a half-site . The efficiency of this reaction is about 2% of that of the wild type reaction . The activity of Flp(Y343F) is critically dependent on the length of the half-site spacer . Furthermore, in this reaction, the strand cleavage and strand exchange steps cannot be uncoupled . These results strongly suggest a direct attack by the 5'-hydroxyl of the half-site spacer on the phosphodiester at the normal strand transfer point.

J Biol Chem, 1993 Jan 5, 268(1), 269 - 75
Active sites of the cytochrome p450cam (CYP101) F87W and F87A mutants . Evidence for significant structural reorganization without alteration of catalytic regiospecificity; Tuck SF et al.; Ferricyanide oxidation of the aryl-iron complexes formed by the reaction of cytochrome P450 enzymes with arylhydrazines causes in situ migration of the aryl group from the iron to the porphyrin nitrogen atoms . The regiochemistry of this migration, defined by the ratio of the four possible N-arylprotoporphyrin IX isomers, provides a method for mapping the topologies of cytochrome P450 active sites . The method has been validated by using it to examine the active site of cytochrome P450cam (CYP101), for which a crystal structure is available . In agreement with the crystal structure, reaction with phenylhydrazine gives a 5:25:70 ratio of the NA:NC:ND (subscript indicates pyrrole ring) N-phenylprotoporphyrin IX isomers . Naphthylhydrazine, however, yields exclusively the NC regioisomer and 4-(phenyl)phenylhydrazine the NA:NC:ND isomers in a 14:40:46 ratio . These isomer ratio differences are readily explained by topological differences between the upper and lower reaches of the active site . Having validated the aryl-iron shift as a topological probe, we used it to investigate the structural changes caused by mutation of Phe-87, a residue that provides the ceiling over pyrrole ring D in the crystal structure of cytochrome P450cam . Mutation of Phe-87 to a tryptophan causes no detectable change in the regiochemistry of camphor hydroxylation and only minor changes in the N-aryl isomer ratios . However, mutation of Phe-87 to an alanine, which was expected to open up the region above pyrrole ring D, severely decreased the proportion of the ND in favor of the NA isomer . Less rather than more space is therefore available over pyrrole ring D in the F87A mutant despite the fact that the regiochemistry of camphor hydroxylation remains unchanged . These results provide evidence for significant structural reorganization in the upper regions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant.

J Biol Chem, 1993 Jan 5, 268(1), 161 - 7
ATP synthase of yeast mitochondria . Isolation and disruption of the ATP epsilon gene; Guelin E et al.; The nuclear gene encoding the subunit epsilon of the catalytic sector F1 of the yeast Saccharomyces cerevisiae ATP synthase was cloned and sequenced . Degenerated oligonucleotide primers were constructed from primary structure data . A part of the ATP epsilon gene was amplified by polymerase chain reaction from yeast genomic DNA . From the amplified DNA sequence a nondegenerated oligonucleotide probe was constructed and used for isolating a 2040-base pair EcoRI fragment bearing the whole gene . A 186-base pair open reading frame encoding a 62-amino acid polypeptide is described . The deduced amino acid sequence was one amino acid longer than the mature protein . A null mutant was constructed . The mutant strain was unable to grow on glycerol medium . The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity . The catalytic sector appeared unstable during purification but F0-subunits were still bound to F1 . The mutation promoted a highly oligomycin-sensitive uncoupling of the mitochondrial respiration rate.

J Biol Chem, 1993 Jan 5, 268(1), 146 - 52
Characteristics of the operon for a putrescine transport system that maps at 19 minutes on the Escherichia coli chromosome; Pistocchi R et al.; The nucleotide sequence of the operon for the putrescine transport system that maps at 19 min on the Escherichia coli chromosome was determined . It contained four open reading frames encoding potF, -G, -H, and -I proteins . The potF protein (M(r) = 38,000) was inferred to be a putrescine-specific binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein . The potG protein (M(r) = 45,000) had consensus amino acid sequences for the nucleotide-binding site . The potH (M(r) = 35,000) and potI (M(r) = 31,000) proteins consisted of six putative transmembrane-spanning segments linked by hydrophilic segments of variable length as shown by hydropathy profiles . The spermidine-putrescine transport system, which is mainly involved in spermidine transport, consisted of potA, -B, -C, and -D proteins (Furuchi, T., Kashiwagi, K., Kobayashi, H., and Igarashi, K . (1991) J . Biol . Chem . 266, 20928-20933) . The homologies of the corresponding two proteins between those two systems, F and D, G and A, H and B, and I and C, were 35, 42, 37, and 36%, respectively . The initiation point of the transcription of the operon for the putrescine transport system was determined by primer extension and S1 nuclease mapping . Transcription started from the T residue located either 149 or 150 nucleotides upstream from the initiator AUG codon of potF protein mRNA . By making several subclones and a mutant lacking the potF gene, we showed that the expression of all four proteins was necessary for maximal putrescine transport activity . These results indicate that the putrescine transport system can also be defined as a bacterial periplasmic transport system.

J Biol Chem, 1993 Jan 5, 268(1), 100 - 6
Regulation of elongation factor G GTPase activity by the ribosomal state . The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA; Voigt J et al.; The elongation factor G (EF-G) is responsible for the translocation of the ribosome along the mRNA chain . Under in vitro conditions, EF-G exhibits a very active uncoupled GTPase activity which is dependent on the presence of ribosomes and is modulated by mRNA-dependent binding of tRNA . In the absence of tRNA, uncoupled EF-G GTPase is inhibited by initiation factors IF1 and IF3, but not by initiation factor IF2 . In the presence of N-fMet-tRNAfMet and poly(A,U,G) or in the presence of N-acetyl-Phe-tRNAPhe and poly(U), initiation factor IF2 causes an additional decrease of the uncoupled EF-G GTPase activity . This effect, however, is dependent on the presence of IF1 and IF3 and is obviously due to the mRNA- and initiation factor-dependent binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, respectively, to the ribosomal P-site . Non-enzymatic binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, however, causes a stimulation of uncoupled EF-G GTPase activity . The same effects are observed for Met-tRNA, Phe-tRNAPhe and uncharged tRNA . These findings are discussed in the light of the three-site model of the ribosome and the mechanism of translocation.

J Mol Biol, 1993 Jan 5, 229(1), 67 - 78
Strand-specific binding to duplex DNA ends by the subunits of the Escherichia coli RecBCD enzyme; Ganesan S et al.; RecBCD enzyme of Escherichia coli unwinds DNA from duplex DNA ends to produce single-stranded DNA, a central intermediate in the major (RecBCD) pathway of homologous recombination . To help elucidate the mechanism of unwinding we studied the complex of RecBCD enzyme bound to duplex DNA ends in the absence of ATP, a cofactor required for unwinding . In this complex the terminal 16 or 17 nucleotides of the 3' terminated strand and the terminal 20 or 21 nucleotides of the 5' terminated strand were protected from DNase I cleavage . u.v.-irradiation of the complex cross-linked the RecB subunit to the 3' terminated strand and the RecC and RecD subunits of the 5' terminated strand . Studies using pyridoxal 5-phosphate, an inhibitor of the RecBCD enzyme, corroborated the cross-linking studies and revealed a conformational change in the enzyme upon binding to DNA . Based on these results and proposals by others, we present a model for the mechanism of DNA unwinding by RecBCD enzyme.

J Biol Chem, 1993 Jan 5, 268(1), 88 - 92
Role of histidine 124 in the catalytic function of ribonuclease HI from Escherichia coli; Oda Y et al.; The role of the conserved histidine residue (His124) in the catalytic function of Escherichia coli ribonuclease HI was probed by the use of the pH titration experiments with 1H NMR and site-directed mutagenesis . His124 is located close to the catalytic triad formed by the three carboxylates . The C2H proton resonance of His124, as well as those of four other His residues, in the enzyme were assigned by comparing the 1H NMR spectrum of the wild-type enzyme to the spectra of the five mutant enzymes, each of which lacked a different His residue . From the analysis of the pH titration shifts of these resonances, the pK alpha values of all His residues were determined . The pK alpha value of His124 was 7.1, which is slightly higher than, but close to, the normal value of the imidazole group . This result suggests that His124 is located in an acidic environment but does not interact directly with a carboxyl group in the solution structure . Three mutant enzymes, in which His124 was replaced by Lys, Gln, or Glu were constructed . The kinetic parameters of these mutant enzymes were determined by using the M13 DNA/RNA hybrid as a substrate . Substitution of Lys, Gln, or Glu for His124 dramatically lowered the Vmax value by 30-100-fold, without significantly affecting the Km value, as compared to those of the wild-type enzyme . In addition, any mutation impaired the in vivo function of the enzyme . These results indicate that His124 is involved in the catalytic function of the enzyme . We propose that His124 changes its conformation in the catalytic reaction and enhances the catalytic efficiency by removing a proton from a catalytically essential carboxylate.

J Biol Chem, 1993 Jan 5, 268(1), 633 - 9
An exopolyphosphatase of Escherichia coli . The enzyme and its ppx gene in a polyphosphate operon; Akiyama M et al.; A gene, ppx, that encodes a novel exopolyphosphatase of 513 amino acids (58,133 Da) was found downstream of the gene for polyphosphate kinase, ppk . Transcription of the ppx gene depends on the ppk promoters, indicating a polyphosphate (polyP) operon of ppk and ppx . Exopolyphosphatase, purified to homogeneity from overproducing cells, is judged to be a dimer of 58-kDa subunits . Orthophosphate is released processively from the ends of polyP approximately 500 residues long, but chains of approximately 15 residues compete poorly with polyP as substrate; ATP is not a substrate . Mg2+ (1 mM) and a high concentration of K+ (175 mM) support optimal activity.

J Biol Chem, 1993 Jan 5, 268(1), 541 - 6
Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA) . Evidence for a novel MGSA receptor; Horuk R et al.; Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells . Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4 . These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology . In this study, we report the high level expression of recombinant human MGSA in Escherichia coli . The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing . Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells . Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell . These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8 . The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner . In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8 . Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA . Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux . The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.

J Biol Chem, 1993 Jan 5, 268(1), 133 - 7
Dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II by protein phosphatase 2C; Fukunaga K et al.; It has been demonstrated that okadaic acid-insensitive protein phosphatases are involved in dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in rat cerebellar granule cells (Fukunaga, K., Rich, D . P., and Soderling, T . R . (1989) J . Biol . Chem . 264, 21830-21836) . In the present study, recombinant rat protein phosphatase 2C (PrP-2C) expressed in Escherichia coli could dephosphorylate both Thr286/287 and Thr305/306 phosphorylation sites of CaM kinase II, which are responsible for the generation of Ca(2+)-independent activity and the inhibition of the total activity, respectively . The dephosphorylation of Thr286/287 and Thr305/306 was accomplished within 15 min at 0 degrees C and totally dependent on Mg2+ . Phosphopeptide mapping of the CNBr-cleaved 32P-labeled CaM kinase II revealed that PrP-2C was relatively specific for dephosphorylation of Thr286/287 and Thr305/306 in the autophosphorylated CaM kinase II . These results suggest that PrP-2C has a role in the regulation of the Ca(2+)-independent activity of CaM kinase II in the neural cells.

J Biol Chem, 1993 Jan 5, 268(1), 613 - 9
Relative roles of CCAAT/enhancer-binding protein beta and cAMP regulatory element-binding protein in controlling transcription of the gene for phosphoenolpyruvate carboxykinase (GTP); Park EA et al.; The gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is expressed in a tissue-specific manner in the liver, kidney, and adipose tissue and is regulated by hormones including cAMP and insulin . Previous studies have shown that the CCAAT/enhancer-binding protein alpha (C/EBP alpha) binds to several sites on the PEPCK promoter and activates transcription from the promoter in hepatoma cells . Here, we report that a second member of the C/EBP family, C/EBP beta, bound to the same sites on the PEPCK promoter . However, C/EBP beta stimulated transcription primarily through the cAMP-responsive element (CRE), which maps between positions -77 to -94, but not at the more 5'-binding sites . In addition, the nuclear factor-1 site, which is immediately adjacent to the CRE in the PEPCK promoter, was also required for the full response of the promoter to cotransfected C/EBP beta . In gel mobility assays, antibodies to both C/EBP beta and the cAMP regulatory element-binding protein (CREB), but not to C/EBP alpha, "supershifted" DNA-protein complexes formed between a synthetic CRE oligomer and proteins prepared from rat liver nuclei . C/EBP beta mRNA was expressed at low levels in both the periportal and pericentral regions of the liver lobule, whereas expression of the gene for C/EBP alpha was confined to the pericentral region of the liver lobule . PEPCK gene transcription is greatest in the periportal region of the liver . CREB also bound to the CRE and stimulated transcription of a PEPCK-CAT vector in the presence of an expression vector for the catalytic subunit of protein kinase A . C/EBP beta and CREB bound to the CRE with similar affinities, both of which were greater than the affinity of C/EBP alpha . Within 90 min after the administration of dibutyryl cAMP to rats, there was a marked increase in the hepatic concentration of C/EBP beta mRNA and a decrease in the level of mRNA for C/EBP alpha . These studies indicate that C/EBP beta can regulate PEPCK gene transcription by acting through the CRE and that C/EBP beta, together with CREB, may contribute to the cAMP responsiveness of the PEPCK promoter.

J Biol Chem, 1993 Jan 5, 268(1), 320 - 4
A new member of the ras superfamily, the rac1 homologue from Caenorhabditis elegans . Cloning and sequence analysis of cDNA, pattern of developmental expression, and biochemical characterization of the protein; Chen W et al.; A new member of the ras superfamily, designated CErac1 has been identified . The CErac1 cDNA clone was isolated from a Caenorhabditis elegans mixed stage library and encodes a protein of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively . The CErac1 cDNA maps to a position on C . elegans chromosome IV in close proximity to cha-1, a choline acetyltransferase gene . The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kilobases) . Their expression is developmentally regulated, that of the more abundant 1.7 kilobases being highest at the embryonic stage and decreasing dramatically during development with 10% of the embryonic level in adult nematodes . The glutathione-S-transferase/CErac1 fusion protein expressed in Escherichia coli binds GTP and exhibits intrinsic GTPase activity . The GTPase activity of the CErac1 protein is stimulated by human n-chimaerin, a GTPase-activating protein for p21 rac1 . These data suggest a role of CErac1 in C . elegans early development . The conserved biochemical properties indicate that further characterization of CErac1 by genetic analysis will be helpful in elucidating not only its role in the signal transduction, but also the biological function of its mammalian homologues.

J Biol Chem, 1993 Jan 5, 268(1), 276 - 81
Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives . Potency, binding characteristics, and effect of template sequence; Carroll SS et al.; The inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by pyridinone compounds has been investigated using as templates synthetic RNA with sequences based on the HIV-1 genome sequence . In reactions catalyzed by the enzyme that incorporated more than one nucleotide per primer, inhibition by a representative pyridinone inhibitor, 3-{2-(1,3-benzoxazol-2-yl)ethyl}-5-ethyl-6-methyl-pyridin-2(1H)one (L-696,229), was noncompetitive against deoxynucleotide triphosphate . For reactions that incorporated one deoxynucleotide per primer, IC50 values ranged from 20 to 200 nM, depending on the position of incorporation of the incoming deoxynucleotide base on the template . Inhibition of synthesis on a set of four templates differing only at the template base complementary to the incoming nucleotide had similar IC50 values . These results demonstrate that inhibitory potency is dependent on the primary structure of the template and that inhibitory potency is largely independent of the identity of the incoming nucleotide base . The inhibition of HIV-1 reverse transcription by L-696,229 also displayed slow-binding characteristics . The slow-binding aspect was exploited to gauge the interaction between inhibitor and enzyme . By titrating the reduction in the extent of the burst of synthesis observed in a reaction incorporating dideoxythymidine monophosphate into poly(rA)-oligo(dT)18, the apparent equilibrium constant for dissociation of the reverse transcriptase-L-696,229 complex was estimated to be 400 nM.

FEBS Lett, 1993 Jan 4, 315(2), 173 - 7
Effect of metal ions on the activity of casein kinase II from Xenopus laevis; Gatica M et al.; Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X . laevis CKII, produced in E . coli from the cloned cDNA genes, were tested with different divalent metal ions . The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+ . Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+ . In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+ . The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+ . Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit . Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

FEBS Lett, 1993 Jan 2, 315(1), 11 - 5
The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system; van Nuland NA et al.; The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (II(Amt1)) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC) . Fourteen of the eighty-five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of II(Amt1) while a further seventeen residues experience lesser shifts . Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr . Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of II(Amt1) resulted in chemical shift changes in a sub-set of the above residues; these were located more in the vicinity of the active site phospho-histidine . Phosphorylation of the HPr/II(Amt1) complex resulted in a HSQC spectrum which was indistinguishable from the P-HPr spectrum in the absence of II(Amt1) indicating that, as expected, the complex P-HPr/P-II(Amt1) does not exist even at the high concentrations necessary for NMR.

Res Exp Med (Berl), 1993, 193(2), 81 - 8
Altered energy metabolism and oxidative injury following endotoxemia in rats with normal or cirrhotic livers; Ouchi K et al.; The release of oxygen-derived free radicals has been implicated in endotoxin-mediated hepatic injury . The effect of hepatic lipid peroxidation on tissue energy reserves in the livers of normal and cirrhotic rats was studied following administration of E . coli endotoxin . Before endotoxin injection, the basal hepatic energy charge was lower and levels of hepatic malondialdehyde (MDA) and total glutathione (GSH) higher in cirrhotic rats than in normal rats . Virtually identical levels of blood endotoxin were obtained in the two groups 24 h after injection of LD50 doses of endotoxin (20 mg/kg and 1 mg/kg in normal and cirrhotic rats, respectively) . Hepatic energy charge, tissue blood flow, GSH and glutathione peroxidase (GPX) were consistently or transiently decreased up to 24 h after the injection of endotoxin in both normal and cirrhotic rats . MDA, significantly increased in normal rats 1 h after injection of endotoxin, returned to normal levels 3-12 h after endotoxin administration, but was again elevated at 24 h . Cirrhotic rats did not show any significant change in MDA following endotoxin injection . In normal rats, endotoxin appears to trigger the liberation of free radicals accelerating depletion of hepatic energy reserves, over and above the effect of decreased hepatic blood flow . In contrast, increased lipid peroxidation was not detected in cirrhotic rats despite GSH and GPX consumption during endotoxemia (indicating oxygen radical generation) . Cirrhotic livers were apparently protected against oxygen radical injury by higher levels of endogenous GSH and GPX . Reduced hepatic blood flow may be mainly responsible for the alteration in energy metabolism of the cirrhotic liver.

Transgenic Res, 1993 Jan, 2(1), 1 - 13
Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives; Shaw-White JR et al.; Transgenes in mice often exhibit different expression patterns in different transgenic lines . While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression . Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites . In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of a lacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome . Expression of lacZ was assayed by histochemical staining for Escherichia coli beta-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation . Several of the randomly integrated cell lines expressed lacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells . Targeted cell lines with lacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines . Targeted cell lines transcribing lacZ in the same orientation as HPRT transcription did not express high levels of lacZ in any differentiated cell type . Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter.

Mol Gen Mikrobiol Virusol, 1993 Jan-Feb, (1), 24 - 7
{Synthesis, cloning, and determination of the primary structure of a full-length DNA copy of the gene for influenza A/Alma-Ata/1417/84 virus (H1N1-serovariant HSW1N1) hemagglutinin}; Beklemishev AB et al.; The full-length copy of the hemagglutinin gene RNA of the influenza virus A/Alma Ata/1417/84 (Hsw1 N1-serovariant) has been synthesized and cloned on Escherichia coli plasmid pBR327 . The complete nucleotide sequence of the cloned DNA copy was determined by the Maxam-Gilbert procedure . The predicted amino acid, sequence of HA1 hemagglutinin subunit was compared with the sequences of HA1 subunits from other H1N1-subtype influenza virus strains . It has been found that the structure of the HA1-subunit of the studied strain is most similar to the structure of the identical region of the A/New Jersey/18/76 hemagglutinin.

Mol Gen Mikrobiol Virusol, 1993 Jan-Feb, (1), 17 - 20
{Immunobiological characteristics of Brucella outer membrane proteins, genetic determinants of which are expressed in Escherichia coli cells}; Seliutina DF et al.; The influence of the brucella OMP preparations obtained by the genetic engineering technique (18, 38 and 18 + 38 kD) on the formation of the specific protection and progress of infectious process in brucellosis in the in vivo experiments has been studied . The OMPs synthesized in Escherichia coli cells GSE579 and having mol mass 18 and 38 kD are common antigens for a number of brucella species . The 18 kD OMP was found to protect 66.7% of experimental animals against brucellosis, while the protection by the commercial live vaccine was 78.0% . The 38 kD OMP did not possess the protective activity with the index of infectivity in the experimental group of animals being higher than the one in the control group (77.9% and 53.4% respectively) . The indexes of colony forming units in the mice spleen were also higher in the experimental group of animals . The obtained results suggest that the 38 kD OMP may be a factor of brucella virulence.

Membr Biochem, 1993 Jan-Mar, 10(1), 61 - 70
Isolation of lactose permease mutants which recognize arabinose; Goswitz VC et al.; In the present study lactose permease mutants were isolated which recognize the monosaccharide, L-arabinose . Although the wild-type permease exhibits a poor recognition for L-arabinose, seven independent mutants were identified by their ability to grow on L-arabinose minimal plates . When subjected to DNA sequencing, it was found that all seven of these mutants were single-site mutations in which alanine 177 was changed to valine . The wild type and valine 177 mutant were then analyzed with regard to their abilities to recognize and transport monosaccharides and disaccharides . Free L-arabinose was shown to competitively inhibit {14C}-lactose transport yielding a Ki value of 121 mM for the Val177 mutant and a much higher value of 320 mM for the wild-type . Among several monosaccharides, D-glucose as well as L-arabinose inhibited lactose transport in the Val177 mutant to a significantly greater extent, while D-arabinose and D-xylose only caused a slight inhibition . On the other hand, kinetic studies with sugars which are normally recognized by the wild-type permease such as {14C}-galactose and {14C}-lactose revealed that the Val177 mutant and wild-type strains had similar transport characteristics for these two sugars . Overall, these results are consistent with the notion that the Val177 substitution causes an enhanced recognition for particular sugars (i.e . L-arabinose) but does not universally affect the recognition and unidirectional transport for all sugars . This idea is further supported by the observation that site-directed mutants containing isoleucine, leucine, phenylalanine, or proline at position 177 also were found to possess an enhanced recognition for L-arabinose.

Methods Enzymol, 1993, 218, 587 - 609
In situ detection of DNA-metabolizing enzymes following polyacrylamide gel electrophoresis; Longley MJ et al.; We have presented several protocols for producing an in situ activity gel that allows detection of various DNA-metabolizing enzymes . Both nondenaturing polyacrylamide and SDS-polyacrylamide activity gel electrophoresis procedures were detailed . Combining the use of defined {32P}DNA substrates with product analysis, these procedures detected a wide spectrum of enzymatic activities . The ability to detect 7 different catalytic activities of 15 different enzymes provides encouragement for expanded applications . It is hoped that others will find this technique applicable for detecting these enzymes and other activities in different biological systems . The modification of DNA in situ and the creation of intermediate substrates within activity gels should prove extremely useful for dissecting the enzymatic steps of DNA replication, repair, recombination, and restriction, as well as the metabolic pathways of other nucleic acids.

J Hepatol, 1993, 17 Suppl 3, S20 - 3
Regulation of hepatitis B virus gene expression; Huan B et al.; Human hepatitis B virus (HBV) mainly infects hepatocytes . HB viral gene expression has been demonstrated to be liver-specific using DNA transfection methods . This liver-specific gene expression is regulated by promoter/enhancer elements . HBV contains two enhancer elements . Enhancer element I has been studied in detail at the DNA-protein level . This is further substantiated by DNA transfections of liver and non-liver cell lines with expression plasmids containing enhancer elements controlling the transcription of reporter genes . Genetic analysis of the enhancer elements defined the minimal sequences which play a key role in the regulation of enhancer function . One of the factors binding in this region is RXR alpha . Using only the DNA binding domain of the liver-specific RXR alpha expressed in E . coli, we demonstrated binding of RXR alpha to the putative retinoic acid receptor response element (RARE) in the HBV enhancer . Our studies implicate a potentially important role of retinoic acid and its receptor in the liver-specific regulation of HBV gene expression and the disease pathogenesis associated with infection.

Mikrobiologiia, 1993 Jan-Feb, 62(1), 37 - 45
{Role of the energy status and putrescine transport in the maintenance of the intracellular pH homeostasis in the course of alkaline and acidic shifts in Escherichia coli}; Tkachenko AG et al.; Alkaline and acidic shifts in exponentially grown Escherichia coli cultures were accompanied by an abatement of the cell energetic status (decline of ATP and energy charge and increase of the ADP and AMP pools) . This event led to a suspension of the growth at (the means of) pH 9.0 and 6.0 accordingly . An increment of ornithine decarboxylase activity and an efflux of putrescine from the cells were observed in the course of alkaline shift . Environmental acidification was accompanied by intensification of the cell respiration rate and H+ATPase activity in the absence of the putrescine efflux from the cells . The involvement of the polyamine synthesizing system in the regulation of cell K+ content and the role of the primary proton pumps in the maintenance of pH homeostasis are discussed.

Adv Pharmacol, 1993, 24, 1 - 20
Antibody engineering using Escherichia coli as host; Ward ES; The expression of immunoglobulin fragments with antigen binding activities in E . coli is now routinely possible . Using such expression systems, Fv, Fab, and scFv fragments and single VH domains can be produced as secreted proteins in yields of the order of milligrams per liter . Moreover, expression systems are being rapidly developed for the production of antibody scFv or Fab fragments by repertoire cloning followed by selection . Diverse repertoires of genes encoding VH and VL domains can be isolated by the PCR and cloned for expression using these systems, which allow the selection of recombinants that produce fragments with the desired antigen binding specificities . This technology is rapidly evolving and, coupled with the development of systems for the random mutagenesis and selection of higher-affinity antibody fragments, could, in the longer term, provide an alternative rapid route to hybridoma technology.

Sci China B, 1993 Jan, 36(1), 60 - 7
Cloning and structural-functional studies for 7.5k promoter of Tiantan strain of vaccinia virus; Wang SP et al.; The 7.5k promoter of vaccinia virus Tiantan strain has been cloned by DNA polymerase chain reaction (PCR) method . Results of DNA sequencing analysis showed that in comparison with P7.5k of WR strain, three site mutations and 7 bp of natural deletion existed in the 155 bp fragment of P7.5k of Tiantan strain; four of 7 base pairs deleted were in the late transcriptional initiation region . Although the total mutation rate was high to 6.45%, these two 7.5k promoters of Tiantan strain & WR strain were all early-late promoters and not obviously different in their activities and functional phases . The results above confirmed further that it is a mechanism to keep their genetic stability that some genes of vaccinia virus have multiple transcriptional initiation sites and produce many mRNA with heterologous 5' ends.

Ann Urol (Paris), 1993, 27(2), 93 - 6
{Pseudotumoral lipomatosis of the renal sinus}; Chaabouni MN et al.; Two cases of pseudotumoral lipomatosis in the renal sinus and responsible for clinical symptoms and radiological renal alterations are reported . Emphasis is put on the misleading features of this disease which may suggest a malignancy . Outcome is consistently favorable . Computed tomography ensures the preoperative diagnosis, avoiding an unnecessary nephrectomy.

Photochem Photobiol, 1993 Jan, 57(1), 139 - 42
Pigment complexes of light-harvesting chlorophyll a/b binding protein are stabilized by a segment in the carboxyterminal hydrophilic domain of the protein; Paulsen H et al.; In order to identify segments of light-harvesting chlorophyll a/b-binding protein (LHCP) that are important for pigment binding, we have tested various LHCP mutants regarding their ability to form stable pigment-protein complexes in an in vitro reconstitution assay . Deletion of 10 C-terminal amino acids in the LHCP precursor, pLHCP, did not significantly affect pigment binding, whereas deletion of one additional amino acid, a tryptophan, completely abolished the formation of stable pigment-protein complexes . This tryptophan, however, can be exchanged with other amino acids in full-length pLHCP without noticeably altering the stability or spectroscopic properties of pigment complexes made with these mutants . Thus, the tryptophan residue is not likely to be involved in a highly specific interaction stabilizing the complex . A double mutant of LHCP lacking 66 N-terminal and 6 C-terminal amino acids still forms pigmented complexes that are virtually identical to those formed with the full-length protein concerning their pigment composition and spectroscopic properties . We conclude that about 30% of the polypeptide chain in LHCP is not involved in pigment binding.

Environ Mol Mutagen, 1993, 21(4), 365 - 71
Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 Chinese hamster cells; Lee YW et al.; Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful . In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E . coli gpt gene . This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants . When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background . Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR) . The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred . When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background . This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

Biochem Mol Biol Int, 1993 Jan, 29(1), 113 - 21
Peculiarities of the binding of restriction endonuclease EcoRII to synthetic DNA duplexes; Karpova EA et al.; The binding of restriction endonuclease EcoRII to synthetic oligodeoxyribonucleotide substrates 11 to 30 bp long was investigated by gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the absence of Mg2+ ions . Irrespective of the substrate's length, two types of specific DNA-protein complexes were shown to be formed . Their mobility in gel was close to that of the monomer and the dimer of the marker ovalbumin . The number of such complexes in solution depended on the ratio of the molar concentrations of restriction endonuclease EcoRII and the DNA duplex . The possible structure of the complexes is discussed.

HPB Surg, 1993, 6(3), 151 - 62
Reticuloendothelial system function following acute liver failure induced by 90% hepatectomy in the rat; Wang X et al.; Sepsis and bacterial infections are frequent complications of acute liver failure and following major liver resection . The mechanisms underlying this phenomenon are unclear . In this study, RES function and blood clearance of radiolabelled E . coli was immediately impaired following 90% hepatectomy, although the reduction in liver volume resulted in an increase in splenic (temporary) and pulmonary (persisting) uptake . A significant correlation between liver function and host RES function was observed . The uptake capacity of the RES in the liver remnant and spleen did not correlate with subserosal blood flow . The uptake in the brain gradually increased with time, paralleling an increased leakage across the blood-brain barrier . Thus, a significantly impaired RES function resulted from experimental 90% hepatectomy-induced acute liver failure, which might explain the high incidence of septic events in the clinical situation.

Cytokine, 1993 Jan, 5(1), 16 - 23
Generation of monoclonal antibodies against HILDA/LIF and their use in the quantitative assay of the cytokine; Godard A et al.; An adoptive transfer immunization/fusion protocol in mice has been successfully used to raise a series of monoclonal antibodies directed against the Human Interleukin for DA-1a (HILDA)/Leukemia Inhibitory Factor (LIF) cytokine . These antibodies which were raised using recombinant HILDA/LIF purified from conditioned medium of transfected Chinese Hamster Ovary (CHO) cells also react with natural HILDA/LIF from the HSB2 T lymphoma cell line and unglycosylated HILDA/LIF produced in E . coli . They define four separate epitopes, one of which is involved in receptor binding and induction of biological activity . A sensitive sandwich immunoradiometric assay which is linear up to 5 ng/ml HILDA/LIF and can detect as low as 25 pg/ml of the cytokine has been developed.

Xenobiotica, 1993 Jan, 23(1), 5 - 10
Inhibition of beta-glucuronidase by natural glucuronides of kampo medicines using glucuronide of SN-38 (7-ethyl-10-hydroxycamptothecin) as a substrate; Narita M et al.; 1 . 7-Ethyl-10-{4-(piperidino)-1-piperidino} carbonyloxycamptothecin (CPT-11), a potent anticancer agent currently under development for clinical use, is metabolized in vivo to 7-ethyl-10-hydroxycamptothecin (SN-38), which is subsequently conjugated to 7-ethyl-10-hydroxycamptothecin glucuronide (SN-38-glucuronide) . The SN-38-glucuronide was hydrolysed by beta-glucuronidase from E . coli to aglycones and glucuronic acid . 2 . Four purified natural glucuronides including baicalin, wogonoside, luteolin-3'-glucuronide, and glycyrrhizin, inhibited beta-glucuronidase using SN-38-glucuronide as substrate . The inhibition potencies of these natural glucuronides toward beta-glucuronidase were similar to that of saccharic acid 1,4-lactone . 3 . These results indicate that plant materials of Kampo (Japanese herbal) medicines containing these glucuronides could be used in vivo to decrease the enterohepatic circulation of SN-38 and possibly that of other drugs.

Mol Biol (Mosk), 1993 Jan-Feb, 27(1), 143 - 52
{Acyclic analogs of 2',3'-dideoxy-2',3'-didehydronucleoside-5'-triphosphates--terminators of DNA synthesis, catalyzed by a broad set of DNA polymerases}; Viktorova LS et al.; O-4'-nor-2', 3'-dideoxy-2', 3'-didehydronucleoside 5'-triphosphates are shown to be effective termination substrates of DNA biosynthesis catalyzed by human placental DNA polymerases alpha and epsilon, rat liver DNA polymerase beta, reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus, and calf thymus terminal deoxynucleotidyl transferase . These compounds do not interact only with the Escherichia coli DNA polymerase I (Klenow fragment) . The probable reasons of interaction of acyclo-d4NTP with the DNA synthesizing complexes are discussed.

Mol Biol (Mosk), 1993 Jan-Feb, 27(1), 120 - 31
{Differential expression of recombinant hybrid proteins containing amino- and carboxy-terminal regions of the exterior large glycoprotein (gp46) of the human T-cell leukemia virus (HTLV-I) in Escherichia coli cells}; Pavlish OA et al.; Plasmid clones capable of expressing a recombinant fusion proteins containing the anthranilate synthase of E . coli (TrpE) and different regions of gp46 HTLV-I were constructed on the basis of pATH-vectors . A high extent of TrpE-gp46 proteolytic degradation took place independently of the bacterial La-protease . Fusion proteins containing an N-terminal part of gp46 were more stable and could be purified in preparative quantities but were less antigenic . On the contrary, a TrpE-gp46 protein encoded by the TaqI-TaqI DNA fragment and containing only 35 C-terminal amino acids was still susceptible to degradation but possessed good serologic reactivity . Some of the recombinant proteins obtained can be useful for diagnostics and for preparing monoclonal or polyclonal antibodies.

Mol Biol (Mosk), 1993 Jan-Feb, 27(1), 103 - 8
{Expression of the v-src oncogene in Escherichia coli affects the rate of cell growth and leads to phosphorylation of cellular proteins}; Ambartsumian NS et al.; The possible influence of expression of v-src oncogene harboring tyrosine-specific protein kinase activity on the physiological state of E . coli cells, carrying plasmid which expresses src oncoprotein was studied . The realization of this activity in the cells brings to the phosphorylation of tyrosine in some additional cellular proteins and, as a consequence, to an increased proliferative activity of cells expressing src oncoprotein.

Dev Genet, 1993, 14(2), 92 - 102
Activity of a microinjected inducible murine hsp68 gene promoter depends on plasmid configuration and the presence of heat shock elements in mouse dictyate oocytes but not in two-cell embryos; Bevilacqua A et al.; After fertilization in the mouse, the zygotic genome is activated in two-cell embryos by the spontaneous expression, among other genes, of the major inducible heat shock gene, hsp68, in the absence of heat-inducibility of heat shock genes . To obtain information on this phenomenon, we have probed one- and two-cell embryo's ability to express microinjected reporter DNA constructs, containing the Escherichia coli lacZ gene driven by promoters from early SV40 genes, the human beta-actin gene, and the normal or HSE-deleted mouse hsp68 gene . Activity of these promoters was also tested in mouse granulosa cells and dictyate oocytes, as a function of circular/linear construct configuration and occurrence of heat shock . The hsp68 promoter was heat-inducible in both granulosa cells and oocytes . Its heat activation required the presence of HSEs and, in the oocytes, of construct linear configuration . In the embryos however, this promoter was expressed independently of the presence of HSEs and of construct configuration, and its activity was not affected by heat shock . When constructs with early SV40 and beta-actin promoters were injected into one-cell embryos, they appeared to be inactivated with the first embryonic cleavage, in agreement with previous observations {Wiekowski et al., 1992} . By contrast, both normal and HSE-deleted hsp68 promoters maintained their activity through the first cleavage, providing the first evidence of a gene escaping such transcriptional repression . Present results confirm previous findings on hsp68 expression during early mouse development, and suggest that this activation is mediated by a factor(s) other than HSF.

Circ Shock, 1993 Jan, 39(1), 74 - 9
Adrenergic blockade attenuates endotoxin-induced hepatic glucose uptake; Ottlakan A et al.; The purpose of the present study was to elucidate the role of catecholamines in mediating the endotoxin-induced increase in glucose uptake of individual tissues . In vivo glucose utilization by selected tissues, assessed by the 2-deoxyglucose (2dGlc) tracer technique, was determined 3 hr following the i.v . injection of Escherichia coli lipopolysaccharide (LPS; 100 micrograms/100 g bw) or saline . Catecholamine action was inhibited by the combined administration of alpha and beta receptor antagonists, phentolamine and propranolol . Adrenergic antagonists alone did not change plasma glucose levels or the glucose metabolic rate (Rg) of the investigated tissues; however, adrenergic blockage resulted in mild hypoglycemia in endotoxemic animals . LPS administration increased in vivo Rg by the liver (571%), lung (229%), spleen (210%), intestine (76%), skin (82%), fat (181%), gastrocnemius muscle (70%), and kidney (61%) . There was a significant elevation in the glucose metabolic clearance rate (MCR) by these tissues as well . LPS did not increase Rg by brain and testis . Adrenergic blockade completely prevented the LPS-induced Rg increase in the liver and partially inhibited the elevation in other tissues . The LPS-induced increase in the MCR in spleen, lung, intestine, skin, fat, muscle, and kidney was not altered by adrenergic blockade, indicating that the attenuated Rg in these tissues was the consequence of the decreased plasma glucose concentration observed under this condition . However, in the liver, adrenergic antagonists markedly inhibited the LPS-induced increase in both Rg and MCR . Thus our data indicate that the glucose metabolic response to LPS is partially mediated by catecholamines through the accompanying changes in plasma glucose concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Am Surg, 1993 Jan, 59(1), 9 - 12
Role of the leukocyte in endotoxin-induced alterations of the red cell membrane . Second place winner of the Conrad Jobst Award in the Gold Medal paper competition; Todd JC 3rd et al.; Sepsis and endotoxemia are known to be associated with alterations in the red cell membrane that result in diminished flexibility . This decreased flexibility may be responsible, in part, for the microcirculatory abnormalities accompanying sepsis . The etiology of these sepsis-associated changes remains unclear . This study evaluates the role of the white blood cell in these abnormalities . Specimens were obtained from 44 volunteers and divided into two treatment groups . Group I specimens were incubated with Escherichia coli endotoxin (2 micrograms/ml) followed by removal of the white blood cells . The white blood cells were removed from group II specimens before endotoxin incubation . Paired, saline-incubated samples served as controls . After incubation, washed erythrocytes were evaluated for deformability and membrane viscosity . Deformability was assessed by filtration through 4.7-microns membranes . Red cell deformability was expressed as filtration rate (volume of cells per second per square centimeter) . Membrane viscosity was assessed by fluorescent spectroscopy of cells into which the membrane probe 1(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene had been incorporated . Results were expressed as anisotropy . Endotoxin resulted in a significant increase in erythrocyte membrane viscosity (experimental, 0.296 +/- 0.002 vs . control, 0.284 +/- 0.002, P < 0.001) . This was reflected by a significant decrease in cellular deformability (experimental, 142.55 +/- 6.55 vs . control, 157.86 +/- 8.63, P < 0.01) . However, these alterations are not a direct effect of endotoxin, but require the presence and participation of the white blood cell and/or its mediators (experimental, 0.301 +/- 0.002 vs . control, 0.300 +/- 0.001, P = NS).

J Basic Microbiol, 1993, 33(1), 47 - 51
A genetic study of apramycin-resistant mutants of Escherichia coli; Vasiljevic B et al.; The apramycin-resistant mutants of E . coli were isolated spontaneously . Mutations, aprA and aprB, conferring resistance to apramycin were located at 72 min on the E . coli genetic map where most of ribosomal genes were mapped . One of the mutants, carrying the aprB mutation, has an altered translational fidelity expressed as severe restriction of amber suppressor activity in vivo . The chromosome location and altered translational fidelity indicate that the apramycin-resistant phenotype could be a consequence of an alteration of the ribosomal structure.

No To Shinkei, 1993 Jan, 45(1), 49 - 56
{Brain injury induced by continuous infusion of endotoxin in rats--protective effects of methylprednisolone on intracerebral blood vessels}; Tamada F; Hemorrhagic intracerebral lesions, analogous to multiple punctate hemorrhagic necrosis seen in the brain of human disseminated intravascular coagulation (DIC), can be induced in rats by continuous intravenous infusion of E . coli endotoxin lipopolysaccharide (ET) at 88.5 micrograms/hour . The occurrence of cerebral lesions increases with time of ET infusion initially, but levels off after 120 hours of the continuous administration . To investigate protective effects of methylprednisolone (MP) against intracerebral vascular injury, 122 male rats were divided into the basic endotoxemic rats without MP medication (Group 1), and MP medication of 0.2mg/kg body weight, 1.0mg/kg, 2.0mg/kg and 20.0mg/kg immediately before induction of endotoxemia (Groups 2-5), one dose of 20.0mg/kg MP at 48 hours and 2 doses at 48 and 72 hours after induction of endotoxemia (Groups 6-7), and 3 doses each of 2.0mg/kg, 20.0mg/kg and 100.0mg/kg MP for 3 days immediately before and at 24 and 48 hours after induction of endotoxemia (Groups 8-10) . All surviving rats were autopsied after 120 hours of ET infusion and histologic sections were made . Multiple hemorrhagic intracerebral lesions developed in 83% (10/12) of Group 1 rats, whereas the frequency of brain lesions in Groups 4, 5, 8, 9 and 10 was significantly lower than that in Group 1 (p < 0.05) . Electron microscopically, the frontal lobe cortex of Group 1 after 120 hours of ET infusion showed subendothelial dilatation containing macrophages, and perivascular accumulation of erythrocytes (diapedesis) . In contrast, the frontal lobe cortex of Group 5 revealed no appreciable electron microscopic changes of intracerebral blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticancer Res, 1993 Jan-Feb, 13(1), 263 - 6
Antiplasmid and carcinogenic molecular orbitals of benz{c}acridine and related compounds; Molnar J et al.; Effect of K- and L- molecular orbital regions on expression of antiplasmid and carcinogenic activity was studied with various benz{c}acridine derivatives, tricyclic compounds (acridine orange, phenothiazines) and dibenzoazepine (imipramine) . Antiplasmid compounds showed the out-of-phase of molecular orbital in the L-region . On the other hand, mutagenic and carcinogenic compounds showed the out-of-phase in the L-region, and their energy was accumulated in the K-region of the molecular orbitals . The present study suggests that the molecular orbitals responsible for the expression of antiplasmid and carcinogenic effects might be basically different.

Prikl Biokhim Mikrobiol, 1993 Jan-Feb, 29(1), 150 - 4
{Biosynthesis of bovine growth hormone in Escherichia coli DH1 cells and its purification on a pilot scale}; Nikolaeva VM et al.; The biosynthesis of the bovine growth hormone (BGH) in E . coli DH1 cells containing the plasmid pbGH(1-119)ptrp was studied . It was found that BGH accumulated in the cytoplasm of bacterial cells as optically dense granules that can be isolated by low-speed centrifugation . A fraction of purified protein granules was used for further purification of BGH . A purified BGH preparation contained two polypeptides with molecular weights of 18,000 and 22,000 D . The state of plasmid was investigated during cultivation and storage of the culture.

Microbiol Immunol, 1993, 37(1), 85 - 9
Genetic transformation in Helicobacter pylori; Tsuda M et al.; Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs . Six out of the eight strains exhibited the natural competence for incorporation of H . pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells with a glycerol solution . The much higher frequency of transformation was obtained in each strain by means of electroporation . Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H . pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H . pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H . pylori.

Methods Enzymol, 1993, 217, 483 - 509
Optimizing the biolistic process for different biological applications; Sanford JC et al.; The biolistic process is still rapidly evolving . We do not anticipate further major improvements in biolistic apparatus . There will probably still be further major improvements in particles, DNA coating, and vectors, as well as significant further advances in understanding of biological determinants of cell penetration and survival . The technology has currently reached the point at which it can be readily and reliably used for a wide range of applications . Given the information presented in this chapter, new applications can be optimized fairly readily.

Methods Enzymol, 1993, 217, 414 - 31
Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner; Rye HS et al.; The stable complexes between highly fluorescent, polyfunctional intercalators and dsDNA can be used to detect dsDNA in agarose gels at picogram levels and for multicolor detection of multiplexed dsDNA fragments . Development of additional DNA-binding fluorophores with appropriate spectroscopic properties will expand the range of applications . In principle, the DNA-dye intercalation complexes represent a more sensitive alternative to an established approach to fluorescent labeling and detection of restriction fragments by ligation to single-stranded short oligonucleotides labeled with different fluorochromes, followed by separation on denaturing polyacrylamide gels . The latter technique gives near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases, and has been successfully applied to cosmid mapping . Detection of DNA fragments as intercalation complexes requires that the separations be performed on agarose gels under nondenaturing conditions . Such conditions have been used for extensive mapping of yeast cosmids with postelectrophoresis staining with ethidium bromide . For the patterns on agarose gels, the magnitude of the "error window," which specifies how similar two fragments must be before the corresponding fragments in different digests are paired, was reported to be strongly size dependent . The error window was expanded by a factor of 1.3 for fragments from 400 to 600 bp, 1.2 for fragments from 600 to 800 bp, and 1.1 for fragments from 800 to 1000 bp . Moreover, it was necessary to introduce corrections for systematic differences between size estimates taken from two different gels . For the multiplexing procedure described here, the size estimates for fragments from 600 bp to over 23 kbp were in close agreement with actual sizes as determined from DNA sequence (Table I), and certainly within the error windows given above . The multiplexing procedure should also minimize errors introduced by gel-to-gel variations in mobility, because the standard and unknowns are always run in the same lanes . Kohara et al . established a physical map of almost the entire Escherichia coli chromosome by analysis of a large genomic library . In this case, partial restriction digests were used to generate patterns of fragments and the mapping was performed by agarose gel electrophoresis . The disadvantage of this approach is that fewer fragments are generated . However, this is compensated for by the fact that partial digests reveal the order of the fragments produced and thus greatly increase the amount of information relevant to the question of overlap between different DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Biometals, 1993 Spring, 6(1), 37 - 44
A novel purification of ferric citrate receptor (FecA) from Escherichia coli UT5600 and further characterization of its binding activity; Zhou XH et al.; In our earlier paper, it was demonstrated that the FecA receptor protein from Escherichia coli UT5600/pBB2 (leu-, proC-, trpE-, entA-, rpsl-, delta (ompT-fepA)-/Ampr, fepA) binds with ferric enterobactin . In order to explore this further the outer membrane receptor protein, FecA, has been isolated from UT5600 (fepA-) and purified to homogeneity by DE-52-cellulose anion exchange chromatography followed by MonoPFPLC chromatofocusing . Partially purified FecA and homogeneous FecA show binding activity to {55Fe}ferric enterobactin and the binding is specific . Binding activity of FecA can be enhanced by ferric citrate . Lipopolysaccharide-free FecA as ascertained by silver staining and the endotoxin test still retains the same activity . In vivo uptake studies using different strains of E . coli suggest that FecA in E . coli plays an important role in ferrienterobactin transport.

Biometals, 1993 Spring, 6(1), 25 - 35
Purification of outer membrane iron transport receptors from Escherichia coli by fast protein liquid chromatography: FepA and FecA; Zhou XH et al.; Fast protein liquid chromatography (FPLC) with DEAE-Sepharose Fast Flow, PBE-94 and Q-Sepharose Fast Flow columns are applied to the purification of the ferric enterobactin protein receptor (FepA) . The apparent single band of FepA on SDS-PAGE is isolated and purified into two proteins with very similar molecular weights . The two proteins are identified to be FepA and ferric citrate protein receptor (FecA) by N-terminus amino acid determination and a computer search with the Gene Bank file . The assay of binding activities of these proteins shows that both FepA and FecA bind ferric enterobactin, with the former having about double the activity of the latter . Competition studies shows that Fe-MECAM is competitively bound to both proteins and that ferric parabactin only slightly competes with {55Fe}ferric enterobactin . It is found that ferrichrome A has no effect on the binding of the receptor proteins with ferric enterobactin.

Biokhimiia, 1993 Jan, 58(1), 98 - 107
{mRNA with an extended Shine-Dalgarno sequence is translated independently of ribosomal protein S1}; Balakin AG et al.; The role of the ribosomal protein S1 in translation of a mRNA containing an extended Shine-Dalgarno sequence has been investigated . Using the toe printing technique, the formation of a ternary initiation complex with both S1-depleted 30S subunits and subunits treated with anti-S1 antibodies and mini-mRNA containing an lambda-cro-mRNA translational initiation region with a very long (9 nucleotides) Shine-Dalgarno sequence, has been demonstrated . It is concluded that initiation of translation on mRNA with extended SD-sequence is S1-independent . By means of an E . coli cell-free system of translation (S-100 extract), the translation of mini-cro-mRNA by S1-depleted ribosomes has been shown . In contrast with mini-cro-mRNA, the 30S subunits without protein S1 are inactive both in the ternary initiation complex formation and in cell-free translation with MS2 or fr phage RNAs and RNA protein III of phage fd.

Arch Virol, 1993, 129(1-4), 349 - 56
Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses; Li Y et al.; The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined . It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene . The cp gene was cloned into the expression vector pET8c and expressed in E . coli . The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses . The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions . Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.

Acta Anat (Basel), 1993, 146(2-3), 86 - 9
Kinematic gait analysis in equine carpal lameness; Back W et al.; Gait analysis plays a major role in the clinical evaluation of equine lameness . It is generally accepted that the clinician expresses the grade of lameness as a subjective score . In this study lameness was objectively assessed using a standardized transient lameness model, in which lameness was induced by intra-articular injection of bacterial endotoxin into the radiocarpal joint of ponies . Lameness was scored by an experienced clinician, and locomotion was recorded simultaneously using a CODA-3 apparatus . The obtained kinematic gait parameters correlated well with the clinical lameness score and also provided possibilities to objectively study the locomotor disturbances of the lame limb in more detail at the walk and trot.

Surg Today, 1993, 23(3), 234 - 40
K252a, a potent protein kinase inhibitor, improves endotoxic lethality and glucose dyshomeostasis; Inaba H et al.; To investigate whether the inhibition of protein kinases including protein kinase C can antagonize endotoxicosis, the in vivo effects of K252a, a potent inhibitor of protein kinases, on endotoxin-induced lethality and glucose dyshomeostasis were determined in conscious rats . Sprague-Dawley rats (260-340 g) were divided into the following four groups: Group DS, 2.5% dimethyl sulfoxide (DMSO), 6 ml/kg iv + 0.9% saline, 2 ml/kg iv; group KS, K252a in 2.5% DMSO, 4 mg/kg iv + 0.9% saline; group DE, 2.5% DMSO + endotoxin (E . coli), 15 mg/kg iv; and group KE, K252a in 2.5% DMSO + endotoxin . A quarter of DMSO or K252a solution was continuously infused over a 15 min period before a bolus injection of either saline or endotoxin . The remaining dose was administered over a 180 min period after saline or endotoxin . All animals in the DS and KS groups survived for 24 hrs . K252a significantly improved endotoxic lethality . It attenuated the initial hyperglycemia, and late hypoglycemia, hyperlactacidemia, and base deficit after endotoxin . However, K252a had no influence on the endotoxic alterations of blood pressure, PaCO2 or PaO2 . These results suggest that the activations of protein kinases, particularly protein kinase C, are involved in the pathogenesis of lethal endotoxicosis and sepsis.

Bioessays, 1993 Jan, 15(1), 51 - 9
Mechanism of action of the Escherichia coli UvrABC nuclease: clues to the damage recognition problem; Van Houten B et al.; During the process of E . coli nucleotide excision repair, DNA damage recognition and processing are achieved by the action of the uvrA, uvrB, and uvrC gene products . The availability of highly purified proteins has lead to a detailed molecular description of E . coli nucleotide excision repair that serves as a model for similar processes in eukaryotes . An interesting aspect of this repair system is the protein complex's ability to work on a vast array of DNA lesions that differ widely in their chemical composition and molecular architecture . Here we propose a model for damage recognition in which the UvrB protein serves as the component that confers enhanced specificity to a preincision complex . We hypothesize that one major determinant for the formation of a stable preincision complex appears to be the disruption of base stacking interactions by DNA lesions.

Adv Biochem Eng Biotechnol, 1993, 48, 29 - 52
Host-vector interactions in Escherichia coli; Bailey JE; Introduction of a DNA vector into E . coli for the purposes of cloned gene expression can perturb native cell functions at many levels . The presence of foreign DNA can alter regulation of chromosomal DNA replication, regulation of transcription of chromosomal genes, ribosome functions and RNA turnover, activities of regulatory proteins, chaperone and protease levels, membrane energetics and protein post-translational processing, as well as energy and intermediary metabolism of the cell . The combined effects of these interactions on the metabolic characteristics of the host-vector system have major implications for yields of cloned biotechnological products and interactions of genetically engineered organisms with their environment.

Int Rev Exp Pathol, 1993, 34 Pt B, 209 - 16
Clinical experience with Escherichia coli rHuGM-CSF; Stern AC et al.; The use of rHuGM-CSF has resulted in patient benefit as shown by reduced infections (MDS and AA), reduced days in intensive care (ABM transplant), better adherence to cancer chemotherapy protocols, and the ability to use full doses of antiviral drugs in AIDS and cytomegalovirus retinitis . The adverse reactions are significant when high doses are used, therefore high doses should be avoided (there is a plateau in the dose-effective biological responses) . At recommended doses, GM-CSF is well tolerated and is a valuable adjunctive therapy in the management of patients with conditions of dysmyelopoiesis and myeloid hypoplasia associated with myelotoxic therapy, or after bone marrow transplantation.

Clin Ther, 1993 Jan-Feb, 15(1), 19 - 29; discussion 18
Clinical properties of yeast-derived versus Escherichia coli-derived granulocyte-macrophage colony-stimulating factor; Dorr RT; Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be expressed in yeast, bacteria, or mammalian cells . Expression in each system results in a protein that differs, to a varying extent, from native GM-CSF . Like the native protein, yeast-expressed GM-CSF is glycosylated and has 127 amino acids, but differs from native GM-CSF in molecular mass and in the substitution of leucine for proline at position 23 . GM-CSF expressed in Escherichia coli bacteria is not glycosylated, has six fewer amino acids than the native protein, and an extra methionine at position 1 . A review of laboratory studies shows that these differences in physiochemical properties result in variations in the pharmacokinetics, biologic activity, and immunogenicity of GM-CSF expressed in different host cells . These variations may lead to an increased clinical toxicity with GM-CSF expressed in E coli versus that produced in yeast . A total of 32 clinical trials were reviewed to determine the relative frequency of adverse events in patients treated with GM-CSF expressed in E coli versus that expressed in yeast . In general, the median reported frequency of adverse events was higher in patients treated with E coli-derived GM-CSF . The median frequencies of fluid retention, dyspnea, fever, myalgias/bone pain/joint pain, and rash were 8.3%, 13.4%, 21.7%, 16%, and 14.3%, respectively, in patients receiving GM-CSF expressed in yeast, versus 18.4%, 55.2%, 40.7%, 28.5%, and 12.5%, respectively, in patients treated with GM-CSF expressed in E coli . Thus data in the literature support the view that the GM-CSF expression system influences the pharmacokinetic properties, biologic activity, and clinical toxicity of GM-CSF.

Mol Carcinog, 1993, 7(2), 126 - 34
Quantitation of 2-amino-3-methylimidazo{4,5-f}quinoline and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease; Nouso K et al.; 2-Amino-3-methylimidazo{4,5-f}quinoline (IQ) and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation . Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-IQ or N-acetoxy-MelQx, and the adduct levels in the 5' dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization . Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37 degrees C, 60 min) to incise DNA at IQ and MelQx adduct sites . The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes . Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MelQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA . IQ and MelQx adduct levels were the same in the 5' DHFR gene and in the downstream region . Southern hybridization analysis of pBR322 containing known levels of IQ or MelQx adducts showed that the efficiency of cutting IQ or MelQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively . 32P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MelQx . The results indicate that IQ and MelQx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.

J Biochem (Tokyo), 1993 Jan, 113(1), 81 - 7
Structural analysis of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor; Yamanishi K et al.; The recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor ({3-153}M-CSF) consists of 302 amino acid residues and has a molecular mass of about 32 kDa, as estimated by SDS-PAGE . Two covalently linked subunits constitute a bioactive homodimer . The structure of the purified protein, expressed in Escherichia coli and refolded from inclusion bodies, was studied . The amino acid sequence was determined by automated Edman degradation of fragments obtained from degradation with CNBr and iodosobenzoic acid as well as by digestion with Glu-C endopeptidase of reduced and alkylated M-CSF . The absence of free thiol groups in the molecule was confirmed with Ellman reagent, which indicated the presence of seven disulfide linkages per homodimer . Sequence analysis of cystine-containing peptides, identified by comparing the peptide maps from unmodified and performic acid-oxidized pepsin digests, gave the following results . (1) Six out of seven disulfide linkages were formed between Cys 7 and Cys 90, Cys 48 and Cys 139, and Cys 102 and Cys 146 at each pair of positions as either intra- or inter-chain disulfides . (2) The remaining disulfide linkage linked Cys 31 of one subunit to Cys 31 of the second subunit of M-CSF . Based on our findings, a two-dimensional model is proposed in which the possible covalent linkage is suggested between the two subunits of the bioactive {3-153}M-CSF molecule.

J Biochem (Tokyo), 1993 Jan, 113(1), 74 - 80
Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase; Gekko K et al.; To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis . Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type . Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation . These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation . The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively . The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.

J Biochem (Tokyo), 1993 Jan, 113(1), 48 - 54
Halobacterium halobium cytochrome b-558 and cytochrome b-562: purification and some properties; Fujiwara T et al.; Four different membrane-bound b-type cytochromes were found to occur in Halobacterium halobium strain L-33, and two of them, b-558 and b-562, were purified to homogeneity . Cytochrome b-558 showed absorption peaks at 414 and 526 nm in the oxidized form, and peaks at 425, 528, and 558 nm in the reduced form . Its alpha peak at 558 nm in the reduced form was asymmetric with a shoulder at around 554 nm . At liquid nitrogen temperature, the a the alpha peak was split into two peaks at 549 and 556 nm which appeared to be the alpha peaks of cytochromes c and b, respectively . The cytochrome contained 1 mol of protoheme in 28,500 g, and was composed of one molecule each of two subunits with molecular masses of 15.4 and 11.7 kDa, respectively . The heme seemed bound to the larger subunit . The cytochrome was very autoxidizable and its redox potential at pH 8.0 was -75 mV . Cytochrome b-562 showed absorption peaks at 417 and 530 nm in the oxidized form and peaks at 431, 531, and 562 nm in the reduced form . The cytochrome was composed of only one polypeptide (25 kDa) and seemed to contain one protoheme molecule per molecule.

Acta Otolaryngol Suppl, 1993, 500, 70 - 4
Animal model of otitis media with effusion; Hamada E et al.; A rat model of otitis media with effusion (OME) was developed by intratympanic injection of E . coli endotoxin and section of the third branch of the trigeminal nerve (V3) . The period of fluid retention induced by the endotoxin was prolonged for 5 days or longer, in cases when tubal function was impaired by cutting of V3 . Three Eustachian tube function tests (patency test of inflation-deflation tests, forced response test and negative pressure test) were carried out before and after the endotoxin inoculation and V3 sectioning . At 4 days after these procedures, passive opening pressure (Po), closing pressure (Pc) and tubal resistance (R2) were significantly lowered . The negative pressure test showed impaired capacity of active opening . This model of Eustachian tube dysfunction is considered to reveal functional obstruction, a condition similar to that of clinical cases of OME . The study shows that both inflammation in the middle ear and tubal dysfunction, such as functional obstruction, are factors in the development and prolongation of OME.

Proteins, 1993 Jan, 15(1), 42 - 9
Crystal structures of two mutants of adenylate kinase from Escherichia coli that modify the Gly-loop; Muller CW et al.; Two mutants of adenylate kinase from Escherichia coli have been crystallized and analyzed by X-ray diffraction at resolutions of 3.4 and 2.4 A, respectively . These mutants are Pro-9-->Leu and Gly-10-->Val . They were selected for their positions in the highly conserved Gly-loop forming a giant anion hole for the beta-phosphate of ATP (GTP) in adenylate kinases, H-ras-p21, and other nucleotide-binding proteins . Mutants at these positions of H-ras-p21 cause cancer . In adenylate kinase these mutations cause smallish changes at the active site . Relating the structural changes to the known changes in catalysis indicates that these mutants hinder the induced-fit movements . As a side result we find that mutant Pro-9-->Leu and wild-type form one very similar crystal packing contact that is crystallographic in one case and noncrystallographic in the other, while all other packing contacts and the space groups are quite at variance.

Life Sci, 1993, 52(14), 1209 - 15
Amino acid composition and N-terminal sequence of purified cystine binding protein of Escherichia coli; Butler JD et al.; Cystine Binding Protein (CBP), a commercially available crude protein extract obtained by osmotic shock of Escherichia coli (E . coli), was studied to characterize further its cystine binding properties and to elucidate its cystine transport activity . We report here the amino acid composition, the N-terminal amino acid sequence analysis and some binding characteristics of the purified cystine binding component of CBP . A search of the Swiss-Prot version 20 data base revealed that this sequence is unique.

J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 95 - 9
Immunogold localization of the DnaK heat shock protein in Escherichia coli cells; Bukau B et al.; Previously reported cell fractionation experiments have yielded conflicting information on the cellular localization of the DnaK heat shock protein of Escherichia coli . Here we used immunogold labelling of ultra-thin sections to determine the localization of DnaK in unstressed cells at 30 degrees C as well as in heat-shocked cells . In cells grown at 30 degrees C, gold particles were found predominantly in the cytoplasm, indicating that the majority of the DnaK molecules are cytoplasmic; however, a fraction of the gold particles was located in proximity to the membranes, raising the possibility that a subpopulation of DnaK proteins is membrane-associated . Heat shock of the cells did not induce detectable relocalization of DnaK.

J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 87 - 93
Cloning of genes for proline and leucine biosynthesis from Brucella abortus by functional complementation in Escherichia coli; Essenberg RC et al.; By selecting for growth of Escherichia coli mutant strains in the absence of the required amino acid, clones were found in a Brucella abortus library carrying genes for glutamyl phosphate reductase (proA) and beta-isopropylmalate dehydrogenase (leuB) . These clones hybridized to unique fragments in a genomic digest of B . abortus DNA . The proA-complementing DNA was found in a region of 1.3 kb, which directed the synthesis of a protein of 48,000 Da with a pI of 6.3 in maxicells . The leuB-complementing activity was in a region of 1.4 kb and directed synthesis of a protein of 46,000 Da with a pI of 5.9.

Comp Biochem Physiol B, 1993 Jan, 104(1), 55 - 61
Comparison of sequences of the 78 kDa gastrin-binding protein and some enzymes involved in fatty acid oxidation; Baldwin GS; 1 . The amino acid sequences of enzymes possessing enoyl-CoA hydratase activity have been aligned with each other and with the sequences of related proteins . 2 . The amino acid sequences of enzymes possessing 3-hydroxyacyl-CoA dehydrogenase activity have been similarly aligned . 3 . The N-terminal half of the 78 kDa gastrin-binding protein (Baldwin et al., J . biol . Chem . 261, 12,252-12,257, 1986) is related to the enoyl-CoA hydratase family, while the C-terminal half is related to the 3-hydroxyacyl-CoA dehydrogenase family . 4 . Evolutionary trees for the two families are presented.

Clin Infect Dis, 1993 Jan, 16(1), 82 - 5
Molecular analysis of multiply recurrent meningitis due to Escherichia coli K1 in an infant; Bingen E et al.; Bacterial DNA polymorphism was used to document the occurrence of three separate episodes of meningitis caused by Escherichia coli K1 in an infant . The methods employed included determination of the restriction fragment length polymorphism of total DNA and of ribosomal DNA regions as well as DNA fingerprinting by the arbitrarily primed polymerase chain reaction . By these three genotypic approaches, the three isolates obtained from the infant's cerebrospinal fluid on days 9, 34, and 70, respectively, were found to share the same patterns, which were different from the patterns of control strains . Thus, these three episodes of E . coli K1 meningitis were due to a single strain . DNA-based typing techniques seem extremely promising as tools to be used in unraveling the complex mechanisms of recurrent meningitis.

Vet Microbiol, 1993 Jan, 34(1), 7 - 18
Comparison of the in vitro adhesion of K88, K99, F41 and P987 positive Escherichia coli to intestinal villi of 4- to 5-week-old pigs; Cox E et al.; The adhesion of K88ab, K88ac, K88ad, P987, K99, F41 and K99/F41 positive Escherichia coli strains to duodenal, jejunal and ileal villi was studied using an in vitro adhesion assay . The villi were harvested from 4- to 5-week-old pigs . The K88+ strains adhered in large numbers (42 +/- 5 to 81 +/- 4 E . coli/250 microns villous length) to the villi from most pigs and in low to moderate numbers (5 +/- 2 to 24 +/- 7 E . coli/250 microns villous length) or not to villi of some pigs . The K99+ and F41+ strains either adhered in low numbers (1 +/- 1 to 11 +/- 2 E . coli/250 microns villous length) or did not adhere, whereas the P987+ and K99/F41+ strains always adhered in low to moderate numbers (2 +/- 1 to 26 +/- 2 E . coli/250 microns villous length) . The number of bacteria adhering to the villi was the highest for the K88ab+ and K88ac+ strains (55 +/- 5 to 81 +/- 4 E . coli/250 microns villous length) and decreasing in the following order: K88ad > P987 > K99/F41 > K99 > F41 (= 1 +/- 1 to 4 +/- 1 E . coli/250 microns villous length) . There was no difference in the adhesion of the villi of the different small intestinal segments for the P987+ and F41+ strains . The K99+ strains adhered significantly more to the villi of the caudal half of the small intestine, the K99/F41+ strain to jejunal and ileal and the K88+ strains to jejunal villi in comparison to duodenal ones.

Toxicon, 1993 Jan, 31(1), 53 - 60
Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix); Yeo MS et al.; The structural gene and cDNA encoding cardiotoxin in Naja naja sputatrix have been cloned and characterized with a view to study the gene protein relationships and also to produce pure protein in large amounts . Using the polymerase chain reaction on the total RNA isolated from the venom glands, the structural gene (180 bp) has been synthesized and expressed in Escherichia coli to produce a fusion protein with beta-galactosidase . Immunoblotting using polyclonal antibodies raised against the total venom in rabbits demonstrated the presence of cross-reacting proteins in plaques produced by recombinant lambda gt11 phages.

Mol Endocrinol, 1993 Jan, 7(1), 23 - 36
Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene; Rennie PS et al.; The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined . The DNA- and steroid-binding domains of the rat androgen receptor {glutathione-S-transferase (GST)-AR1} and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography . Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies . DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2) . Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3) . PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins . Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation . A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT . In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements . Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.

Mol Microbiol, 1993 Jan, 7(2), 311 - 22
Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR; DiRusso CC et al.; Fatty acid biosynthesis and fatty acid degradation in Escherichia coli are co-ordinately regulated at the level of transcription by the product of the fadR gene, FadR . In the present work we investigate FadR interaction with the fabA and fadL promoters . The FadR-responsive operator within fabA, OA, was localized to a region -47 to -31 base pairs relative to the start of transcription using DNase I protection studies . The promoter and untranslated leader within fadL had two binding sites for FadR, OL1 at -25 to -9 and OL2 at -1 to +16 relative to the start of transcription . The binding affinity of FadR for OA and OL1 or OL2 was lower than that for the single site within fadB (OB) as measured using protein-DNA gel retention assays . Overall, these experiments demonstrated that the affinity of FadR binding for DNA containing the fadB, fadL and fabA promoters was OB > OL1, OL2 > OA . We could not distinguish separate binding affinities for OL1 or OL2 . We demonstrated repression of fadL transcription and activation of fabA transcription in vitro using run-off transcription assays containing purified FadR and RNA polymerase.

Mol Microbiol, 1993 Jan, 7(2), 275 - 84
Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate; Tubulekas I et al.; We have found a simple relationship between bacterial growth rate and the translation elongation rate . Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate . When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed . The reductions and reversals are described by a unique linear relationship . We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction . We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome . The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1 . Interestingly, the reversal of the elongation and growth phenotypes is incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction . Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.

Protein Sci, 1993 Jan, 2(1), 103 - 12
Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase; Zhou BB et al.; Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites . The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme . Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state . Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu . As shown previously (Newell, J.O . & Schachman, H.K., 1990, Biophys . Chem . 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit . Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme . A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer . For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state . However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme . This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein . This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change . The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.

Int Immunol, 1993 Jan, 5(1), 63 - 70
Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells; Mages HW et al.; Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA) . We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression . Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA . The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133 . In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA . This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts . This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it.

Comp Immunol Microbiol Infect Dis, 1993 Jan, 16(1), 87 - 90
The isolation of cytotoxic necrotizing factor (CNF)-producing Escherichia coli from the intestinal contents of babies who died of sudden infant death syndrome (SIDS) and other causes as well as from the faeces of healthy babies; Bettelheim KA et al.; Strains of Escherichia coli producing cytotoxic necrotizing factor (CNF) have been isolated from intestinal contents of 16.8% of babies who died of Sudden Infant Death Syndrome (SIDS) and 16.5% of faeces from healthy babies . While no difference in CNF carriage was seen, it is noteworthy that these CNF-producing E . coli are present in such specimens . Some of the CNF-producing E . coli belonged to serotypes associated with this factor in other parts of the world.

Cell Motil Cytoskeleton, 1993, 24(2), 119 - 28
Incorporation of microinjected mutant and wildtype recombinant tropomyosins into stress fibers in fibroblasts; Ranucci D et al.; The structural requirements for assembly of tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle alpha-tropomyosins expressed in E . coli as fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of tropomyosin using indirect immunofluorescence . The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these tropomyosins . Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80-residue fusion protein {Hitchcock-DeGregori, S.E., and Heald, R.W . (1987): J . Biol . Chem . 262:9730-9735} . The pattern of incorporation was indistinguishable from that of tropomyosin isolated from chicken pectoral muscle . The striated alpha-tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells . Three recombinant mutant tropomyosins in which one-half, two-thirds, or one actin binding site was deleted were tested {Hitchcock-DeGregori, S.E., and Varnell, T.A . (1990): J . Mol . Biol . 214:885-896} . Only the fusion protein with a full actin binding site deleted incorporated into stress fibers . However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro . A fourth mutant, in which the conserved amino-terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant {Cho, Y.J., Liu, J., and Hitchcock-DeGregori, S.E . (1990): J . Biol . Chem . 265:538-545}.

Biol Chem Hoppe Seyler, 1993 Jan, 374(1), 69 - 74
Divergent binding sites in pyruvate kinases I and II from Escherichia coli; Valentini G et al.; Pyridoxal 5'-phosphate incorporation into pyruvate kinase II from E . coli was decreased by the substrate phosphoenolpyruvate and increased by the allosteric activator ribose 5-phosphate, the total incorporation being linearly related to inactivation . Four lysyl residues were substantially modified, whatever the incubation conditions were while two additional residues became reactive only in the presence of the allosteric activator . Six tryptic peptides containing modified lysines were purified and sequenced . They defined five regions of pyruvate kinase II, since one of them contained two labelled lysines and included a peptide which also appeared independently . Sequence comparison with E . coli type I, yeast and cat muscle pyruvate kinases shows that the binding regions of pyruvate kinase II are clearly divergent from those of pyruvate kinase I and of the eukaryotic enzymes.

Vaccine, 1993, 11(2), 227 - 33
Recombinant enterotoxins as vaccines against Escherichia coli-mediated diarrhoea; Aitken R et al.; A fusion protein, comprising the B subunit of the heat-labile enterotoxin and a portion of the precursor to the heat-stable enterotoxin of Escherichia coli, has been created by recombinant genetic techniques . It is exported successfully to the bacterial periplasm and assembles into pentamers which retain the ability to bind to GM1 ganglioside . Native toxin epitopes are displayed and the molecule can be easily purified from periplasmic extracts of cells expressing the gene fusion . Although the protein carries the natural sequence of the heat-stable enterotoxin, it is greatly attenuated in toxicity . Systemic immunization of mice or oral administration of the fusion elicits antibody responses against both classes of E . coli enterotoxin.

Mol Gen Genet, 1993 Jan, 236(2-3), 433 - 9
SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light; Tudek B et al.; In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mp18 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts . Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target . When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA . Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvr A fpg-1 double mutant than that seen in uvr A, fpg-1, or umuC single mutants or in the wild-type . Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions . The most frequent base change is the GC-->TA transversion . MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly G-->T transversions . Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli . The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.

Mol Gen Genet, 1993 Jan, 236(2-3), 409 - 14
Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonas reinhardtii; Durrenberger F et al.; The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron . Here we show that I-CreI generates a 4 bp staggered cleavage just downstream of the intron insertion site . The I-CreI recognition sequence is 19-24 bp in size and is located asymmetrically around the intron insertion site . Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.

Mol Gen Genet, 1993 Jan, 236(2-3), 245 - 50
SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus; Valentin K; Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli secA gene as well as two open reading frames (ORF 510, ORF 179) . In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization . None of these genes has been found in completely sequenced chlorophytic plastid genomes . SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secA Y may be ubiquitous in rhodophytes and chromophytes . The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.

Mol Gen Genet, 1993 Jan, 236(2-3), 179 - 86
Promoter sequences of a potato pathogenesis-related gene mediate transcriptional activation selectively upon fungal infection; Martini N et al.; Transcription of at least one member of the potato prp1 gene family, prp1-1, is activated at early stages of potato infection with the late blight fungus Phytophthora infestans . In this paper we present evidence that mRNA encoded by prp1-1 does not accumulate in response to abiotic environmental cues which stimulate transcription of other defence-related genes . Regulatory elements were identified in the 5' terminal region of prp1-1 by assaying the expression pattern of chimeric promoter/beta-glucuronidase gene constructs in transgenic potato . A 273 bp fragment comprising the promoter sequence between positions -402 and -130 was sufficient for rapid and strictly localized transcriptional activation at infection sites during the development of late blight disease . Like the native promoter, this truncated promoter did not mediate transcriptional activation in response to other abiotic stimuli . The use of the identified regulatory region to generate conditional mutations selectively at infection sites is discussed.

Mol Gen Genet, 1993 Jan, 236(2-3), 171 - 8
Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription; Ueguchi C et al.; The Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E . coli genes and operons . To gain an insight into the regulation of the hns gene itself, we constructed in this study a hns-lacZ transcriptional fusion gene and inserted a single copy at the att lambda locus on the E . coli chromosome . Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner . It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase . The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription . Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence . These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.

Mol Microbiol, 1993 Jan, 7(1), 39 - 47
Sequence-function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli; Dassa E; The malG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy-dependent transport of maltose and maltodextrins in Escherichia coli . The MalG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MalG subunit . Fifteen mutations have been isolated in malG by random linker insertion mutagenesis . Two regions essential for maltose transport have been identified . In particular, a hydrophilic region containing the peptidic motif EAA---G---------I-LP, highly conserved among inner membrane proteins from binding protein-dependent transport systems, is essential for maltose transport . The results also show that several regions of MalG are not essential for function . A region (residues 30-50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MalG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein . A region located at the middle of the protein (residues 153-157) is not essential for the function of the protein . A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C-terminus of the protein.

Mol Microbiol, 1993 Jan, 7(1), 29 - 38
Membrane topology of MalG, an inner membrane protein from the maltose transport system of Escherichia coli; Dassa E et al.; In Escherichia coli, the binding protein-dependent transport system for maltose and maltodextrins is composed of five proteins--LamB, MalE, MalF, MalG and MalK--located in the three layers of the bacterial envelope . Proteins MalF and MalG are hydrophobic inner membrane components mediating the energy-dependent translocation of substrates into the cytoplasm . In this paper, we analyse the topology of the MalG protein by using methods based on the properties of fusions between malG and 'phoA, a truncated gene encoding alkaline phosphatase lacking its translation initiation and exportation signals . Fusions were obtained by using either phage lambda TnphoA or by constructing in vitro fusions located randomly within the malG gene . The deduced topological model suggests that MalG spans the membrane six times and has its amino- and carboxy-termini in the cytoplasm . These results will be helpful for the interpretation of the phenotypes of mutants in malG.

Mol Microbiol, 1993 Jan, 7(1), 109 - 16
Signal transduction between the two regulatory components involved in the regulation of the kdpABC operon in Escherichia coli: phosphorylation-dependent functioning of the positive regulator, KdpE; Nakashima K et al.; The proteins KdpD and KdpE are crucial to the osmotic regulation of the kdpABC operon that is responsible for the high-affinity K+ ion transport system in Escherichia coli . We demonstrated previously that the response regulator, KdpE, is capable of undergoing phosphorylation mediated by the sensory protein kinase, KdpD . In this study, we obtained biochemical evidence supporting the view that when KdpE is phosphorylated, it takes on an active form that exhibits relatively high affinity for the kdpABC promoter, which in turn results in activation of the kdpABC operon . It was also suggested that the central hydrophobic domain of KdpD, which is conceivably responsible for membrane anchoring of this protein, plays a role in the signalling mechanism underlying KdpE phosphorylation in response to hyperosmotic stress.

Eur J Haematol, 1993 Jan, 50(1), 32 - 6
Comparative pharmacokinetics of single-dose administration of mammalian and bacterially-derived recombinant human granulocyte-macrophage colony-stimulating factor; Hovgaard D et al.; Pharmacokinetics of recombinant human non-glycosylated bacterially-synthesized (E . coli) granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied following single intravenous (i.v.) and subcutaneous (s.c.) bolus injection, and compared to equivalent doses of glycosylated mammalian-derived CHO-GM-CSF . Each route of administration gave a different GM-CSF concentration-time profile . The highest peak serum concentrations (Cmax) were observed following i.v . bolus injection . After i.v . administration, a two-phase decline in concentration was noted for both types of GM-CSF with a significantly shorter t1/2 alpha of 7.8 minutes for the E . coli GM-CSF versus 20.0 min for the CHO-GM-CSF, while no significant difference was observed for the terminal phase . Following s.c . administration of equivalent doses, a higher peak serum concentration was observed in the E . coli-treated patients and, again, a faster elimination where pretreatment serum levels were reached after 16-20 h, versus more than 48 h after administration of CHO-GM-CSF . Although the non-glycosylated E . coli GM-CSF thus seems to undergo a faster elimination that the glycosylated CHO-GM-CSF no significant difference could be demonstrated in the in vivo effect of corresponding doses of the two compounds with respect to stimulation of granulopoiesis--with reservation for small patient numbers and a large individual variations in response.

Arch Virol, 1993, 128(3-4), 195 - 210
Human T-cell leukemia virus type 1 protease protein expressed in Escherichia coli possesses aspartic proteinase activity; Saiga A et al.; We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector . DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease . To express a fusion protein of beta-galactosidase linked with the HTLV-1 protease in Escherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of a lacZ gene directed by a lambda phage Pr promoter and designated pUEX-pro . By Western blot analysis using anti-beta-galactosidase antibody, a bigger molecular size band than that of the control beta-galactosidase molecule was observed in E . coli cells transformed with pUEX-pro but not with control pUEX2, suggesting that the particular fusion protein was successfully expressed . This recombinant protease protein in the E . coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1 gag precursor polyprotein . The gag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme . Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously . Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.

Physiol Behav, 1993 Jan, 53(1), 127 - 31
Central and peripheral prostaglandins are involved in sickness behavior in birds; Johnson RW et al.; Many of the behavioral manifestations of mammals and birds following infection are now recognized as important mechanisms for maintaining homeostasis and promoting recovery . To investigate the role of prostaglandins (PGs) in the behavioral and physiological effects of lipopolysaccharide (LPS) in birds, chickens were injected with indomethacin (Ind) peripherally (IP, 5 mg) or centrally (ICV, 100 micrograms) and their behavior and body temperature following a challenge IP injection of LPS (2.5 mg) were assessed at 1 and 2 h, respectively . Pretreatment with Ind IP or ICV completely inhibited the hyperthermia caused by LPS . Ind injected IP but not ICV significantly attenuated the LPS-induced anorexia . The drowsiness caused by LPS was completely inhibited by Ind injected IP and partially inhibited by Ind administered ICV . These results are interpreted to indicate that LPS induces hyperthermia in the chicken by activating a PG system in the brain . Peripheral PGs appear to be involved in the anorectic response to LPS, whereas drowsiness caused by LPS may involve both peripheral and central PGs . These data are consistent with the hypothesis that multiple PG systems are activated during the acute-phase response, which may explain the dissociation between mechanisms controlling the behavioral and physiological responses to infection.

Protein Eng, 1993 Jan, 6(1), 81 - 4
Comparative stability of dihydrofolate reductase mutants in vitro and in vivo; Leontiev VV et al.; Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis . The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated . In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability . The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability . A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

Protein Eng, 1993 Jan, 6(1), 123 - 7
Overproduction and purification of the regulatory subunit of Escherichia coli aspartate transcarbamoylase; Dembowski NJ et al.; An Escherichia coli strain/plasmid system has been developed for the overexpression of the regulatory subunit of E . coli aspartate transcarbamoylase (ATCase) . Production of large quantities of regulatory subunit, by the method described here, should facilitate future experiments, such as X-ray crystallography, NMR and hybridization experiments, aimed at understanding the heterotropic mechanism that regulates the activity of ATCase . The plasmid used for the over-expression carries the gene for the regulatory subunit, pyrI, downstream from the strong pyrB promoter . The host strain, EK1104 {Nowlan, S.F . and Kantrowitz, E.R . (1985) J . Biol . Chem., 260, 14712-14716} carries a deletion in the pyrBI region of the chromosome, as well as a leaky pyrF allele . When this strain/plasmid system is grown under limiting pyrimidine levels, large quantities of the regulatory subunit of ATCase are produced without any trace of catalytic subunit or holoenzyme . A procedure for the purification of the regulatory subunit from cell extracts has also been developed yielding approximately 50 mg of purified regulatory subunit per liter of initial culture . The regulatory subunit produced in this fashion is fully competent in reassociation experiments with the native catalytic subunit . Furthermore, the reassociated holoenzyme exhibits kinetic properties identical to those of the wild type enzyme . In addition, we report the construction of a pUC119 based plasmid which carries a unique NdeI site at the fMet of the pyrB gene of ATCase . This plasmid, which was used in the construction of the system for the overexpression of the regulatory subunit of ATCase, has been shown to be of general use for the expression of foreign proteins in E . coli.

Protein Eng, 1993 Jan, 6(1), 109 - 22
The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment; Schmidt TG et al.; The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering . The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study . Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes . The streptavidin-affinity tag was engineered at the C-terminus of the VH domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli . Irrespective of whether it was displayed by the VH or the VL domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate . In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.

Protein Eng, 1993 Jan, 6(1), 101 - 8
Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli; Turner SL et al.; The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamide acetyltransferase subunits (E2p) of the Escherichia coli pyruvate dehydrogenase complex are connected by linker sequences which are characteristically rich in alanine and proline residues . By facilitating domain movement these linkers are thought to promote interactions between the three types of active site that participate in the catalytic cycle of the complex . To investigate functional constraints associated with linker composition and sequence, the natural linker of an E2p subunit containing one lipoyl domain was replaced by shorter sequences containing: mixtures of alanine plus proline residues; mainly alanine; mainly proline; and mainly charged residues . Each artificial linker possessed a central histidine residue for assessing linker flexibility by 1H-NMR spectroscopy . The resultant complexes exhibited 181% (proline), 74-79% (alanine plus proline), 63% (alanine) and 7% (charged residues) of parental activity compared with a value of 75% expected for a complex with a comparably shortened linker . The 1H-NMR spectra showed that the alanine plus proline linkers are flexible but the alanine linker and the proline linker are relatively inflexible . Substantial variations in linker sequence and composition were tolerated without loss of function, and the enhanced activity conferred by the proline linker was attributed to the combined effects of length and relative inflexibility.

J Photochem Photobiol B, 1993 Jan, 17(1), 57 - 61
UmuC product contributes to the inhibition of dimer excision produced by thymine-less-amino acid-less pretreatment in UV-irradiated Escherichia coli; Masek F et al.; In UV-irradiated Escherichia coli, predamaged by thymine-amino acid starvation or a UV predose, a large amount of dimers may remain unexcised and may be tolerated by an error-free mechanism, which requires the function of uvr, recA and lexA genes . A possible role of the umuC gene in both the inhibition of dimer excision and the toleration of unexcised dimers is investigated . Data suggest that the UmuC gene product is not absolutely necessary for the inhibition of dimer excision in UV-irradiated thymine-less-amino acid-less pretreated cells, but that it contributes to it . However, the UmuC product does not seem to be involved in the toleration of unexcised dimers which is dependent on uvr, recA and lexA.

Gut, 1993 Jan, 34(1), 63 - 7
Adhesive and hydrophobic properties of Escherichia coli from the rectal mucosa of patients with ulcerative colitis; Hartley MG et al.; The adherent properties and hydrophobicity of Escherichia coli isolates have been compared from the rectal mucosa of patients with active and inactive ulcerative colitis and from a control patient group . Patients with active colitis were colonised less frequently and with lower numbers of E coli than were control patients . Mannose resistant adhesion to HEp-2 cells was determined for 124 isolates of E coli and surface hydrophobicity was estimated by salt agglutination in 96 of these isolates . There was no significant difference in the distribution of adherent strains between the colitis patient groups or with disease activity . E coli from the control patients were marginally less adhesive than those from colitics . The hydrophobicity of isolates did not differ significantly between colitic and control groups nor were there significant differences correlated with disease activity . Furthermore, for these mucosal E coli isolates, hydrophobicity and mannose resistant adhesion were unrelated characteristics.

Can J Vet Res, 1993 Jan, 57(1), 45 - 8
Sensitive and specific polymerase chain reaction detection of Toxoplasma gondii for veterinary and medical diagnosis; MacPherson JM et al.; A polymerase chain reaction (PCR) method was developed for the detection of Toxoplasma gondii . A universal- and a T . gondii-specific primer was used to amplify a region of the small subunit ribosomal RNA gene . This approach allows for a theoretical detection limit of 0.01 zoite of T . gondii per sample assayed . Experiments showed that this PCR method could detect 0.1 pg of T . gondii DNA, which represents about one organism . Polymerase chain reaction tests using DNAs of cat, dog, swine, cattle, human, Sarcocystis cruzi, Eimeria ahsata, E . vermiformis, and Escherichia coli indicated no cross-reaction with nucleic acids of hosts, related coccidia, or bacteria . Data on the sensitivity and specificity suggest that this PCR assay could be extremely useful for the diagnosis of toxoplasmosis in human and veterinary medicine, as well as for food safety surveys.

Bioconjug Chem, 1993 Jan-Feb, 4(1), 63 - 8
Affinity purification and characterization of anti-Tac(Fv)-C3-PE38KDEL: A highly potent cytotoxic agent specific to cells bearing IL-2 receptors; Spence C et al.; A chimeric, single chain antibody fused immunotoxin, denoted anti-Tac(Fv)-C3-PE38KDEL, was engineered and expressed in Escherichia coli . The microbially expressed anti-Tac(Fv)-C3-PE38KDEL was solubilized from inclusion bodies using guanidine hydrochloride, and subsequently refolded in a redox buffer via thiol/disulfide exchange . The recombinant immunotoxin from the crude extract was purified employing receptor-affinity chromatography, which is based upon biological function and involved the immobilized p55 subunit of human IL-2 receptor . The cytotoxic activity of this immunotoxin was measured by the IL-2 dependent phytohemagglutinin (PHA) blast proliferation inhibition and HUT-102 protein synthesis inhibition assays, in which the IC50 values were 41.5 and 0.8 pM, respectively . The biochemical homogeneity and authenticity of the purified material were determined by gel permeation chromatography, amino acid composition and N-terminal sequence analyses, SDS-PAGE, isoelectric focusing, and Western blotting . The receptor-affinity-purified immunotoxin was shown to be highly effective in specifically killing cells bearing IL-2 receptors . Anti-Tac(Fv)-C3-PE38KDEL is a powerful immunosuppressant which may be a potentially useful therapeutic agent in the prevention of allograft rejection and in the treatment of autoimmune diseases . Another anticipated application of this fusion protein is as a chemotoxin in the treatment of some forms of cancer.

Vet Immunol Immunopathol, 1993 Jan, 35(3-4), 275 - 88
The influence of colostral leukocytes on the course of an experimental Escherichia coli infection and serum antibodies in neonatal calves; Riedel-Caspari G; Three hours post natum, 20 calves were orally infected with 10(9) colony forming units of an enteropathogenic E . coli . Immediately after infection and on 2 days subsequent, ten of the calves (COL-) received cell-deprived pooled colostrum and the other ten (COL+) pooled colostrum supplemented with colostral cells . The COL+ calves excreted significantly less bacteria of the infectious strain with their faeces in the first week after infection and reached the lower limit of detectability earlier than the COL- calves . Colostral leukocytes obtained from cows which developed clinical mastitis (n = 2), showed a marked reduction in the number of shed bacteria . The concentration of IgA and IgM specific antibodies against E . coli in the serum of the COL+ calves was significantly higher in the early postnatal period than in the serum of the COL- calves and remained at a slightly higher level throughout the whole investigative period . In addition to humoral substances of the colostrum, colostral leukocytes obviously contribute to the passive immunity and resistance of the newborn calf . The quality and quantity of the leukocytes seem to be of crucial importance to their efficiency.

Scand J Gastroenterol, 1993 Jan, 28(1), 53 - 62
Modification of reticuloendothelial function by muramyl dipeptide-encapsulated liposomes in jaundiced rats treated with biliary decompression; Ding JW et al.; Rats with 2 weeks of biliary obstruction, with and without 1 week of concomitant biliary decompression relieving the jaundice, were treated with physiologic saline, free muramyl dipeptide (MDP), placebo liposomes, or liposome-encapsulated MDP . Reticuloendothelial system (RES) function was evaluated by blood clearance of intravenously injected 125I-labelled Escherichia coli . The corrected phagocytic index (alpha) after 1 week of biliary decompression returned to normal levels in animals treated with MDP liposomes, whereas RES function was impaired (P < 0.05) in all other jaundiced and biliary-decompressed groups . In the biliary-decompressed, MDP-liposome-treated group, hepatic uptake of radiolabelled bacteria was significantly higher (P < 0.05) and renal entrapment of bacteria was significantly lower (P < 0.05) than in all other jaundiced and biliary-decompressed groups . We conclude that treatment with MDP liposomes improves the otherwise impaired RES function in rats with biliary obstruction and biliary decompression.

Scand J Gastroenterol, 1993 Jan, 28(1), 31 - 40
The influence of surgically induced acute liver failure on the intestine in the rat; Wang XD et al.; The influence of acute liver failure induced by 90% hepatectomy on the intestine was evaluated in the rat . Small-intestinal mucosal mass decreased 2 h after hepatectomy . Microvillous height decreased significantly from 1 h and on, and villous height and area in the distal small intestine from 2 h after operation . Ninety per cent hepatectomy resulted in a decrease in systemic arterial blood pressure and an increase in portal venous pressure . Subserosal microcirculation and small-arterial circulation in the proximal and distal small intestine and colon decreased significantly after 90% hepatectomy . Overgrowth and colonization of Escherichia coli occurred in the distal small intestine from 1 h and on after hepatectomy . Protein content in enterocytes and bile secretion from the liver remnant were markedly reduced in hepatectomized rats . Thus, the present study shows evidence of alterations in intestinal morphology and function that can contribute to explain the enteric bacterial translocation after surgically induced acute liver failure.

Gen Comp Endocrinol, 1993 Jan, 89(1), 51 - 61
Recombinant carp (Cyprinus carpio) growth hormone: expression, purification, and determination of biological activity in vitro and in vivo; Fine M et al.; Carp growth hormone (cGH) cDNA (Koren et al., 1989) was cloned under the control of lambda-phage PLOL promoter and lambda cll ribosomal binding site into pBR322 plasmid to enable its expression in Escherichia coli A1645 that produces constitutively the thermolabile lambda repressor c1857 . Temperature shift to 42 degrees abolished the repression, resulting in a high level of cGH expression . The bacterially expressed cGH protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on Q-Sepharose column by stepwise elution with NaCl . The bioactive fraction was eluted at 0.2 M NaCl at a yield of 10-15% . This fraction contained predominantly (95%) 21.5-kDa monomeric cGH . The activity of cGH in vitro was bioassayed using Nb2-11C lymphoma cells (containing lactogenic receptors) and 3T3-F442A preadipocyte cells (containing somatogenic receptors) . Bioactivity was found to be 0.01 and 6-10% that of human GH, respectively . In vivo cGH activity was measured by weekly ip injection in juvenile carp fed a low (23%) protein diet . Over a 6-week period, cGH increased the growth rate by 38% compared to fish injected with vehicle only . Identical injections with bovine GH yielded only a 21% increase.

EMBO J, 1993 Jan, 12(1), 9 - 16
The inactive form of recA protein: the 'compact' structure; Ruigrok RW et al.; When recA protein is enzymatically inactive in vitro, it adopts a more compact helical polymer form than that of the active protein polymerized onto DNA in the presence of ATP . Here we describe some aspects of this structure . By cryo-electron microscopy, a pitch of 76 A is found for both the self-polymer and the inactive complex with ssDNA . A smaller pitch of 64 A is observed in conventional electron micrographs . The contour length of complexes with ssDNA was used to estimate the binding stoichiometry in the compact complex, 6 +/- 1 nt/recA . In addition, the compact structure was observed in vivo in Escherichia coli: inclusion bodies produced upon induction of recA expression in an overproducing strain have a fibrous morphology with the structural parameters of the compact polymer.

EMBO J, 1993 Jan, 12(1), 45 - 52
Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element; Wang PZ et al.; The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein . The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication . The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity . We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required . Since the initiator is dimeric, we presume that it binds symmetrically to IR-III . A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III . In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III.

EMBO J, 1993 Jan, 12(1), 35 - 44
Variation of half-site organization and DNA looping by AraC protein; Carra JH et al.; The dimeric AraC protein of Escherichia coli binds specifically to DNA sequences upstream of promoters whose transcription is regulated by arabinose . Here we show with affinity measurements, DNase footprinting, dimethyl sulfate premethylation interference and dimethyl sulfate footprinting studies that AraC protein can recognize paired half-sites in direct repeat orientation or inverted repeat orientation . A similar high degree of flexibility was also seen in the ability of the protein in the absence of arabinose to bind tightly and specifically when the separation of its half-sites was increased by 10 or 21 bp . In the presence of arabinose the protein could specifically contact both half-sites of a +10 bp spacing construct but could not contact both in a +21 bp construct . Reduced extensibility of AraC protein in the presence of arabinose provides a simple mechanism for the protein's shift from a non-inducing, DNA looping state to an inducing, non-looping state that contacts two adjacent half-sites at the arapBAD promoter.

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