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 b