J Photochem Photobiol B, 1997 Nov, 41(1-2), 60 - 6 Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA; Padula M et al.; The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac181 . Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg . The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites . Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions . To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisiae . In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain . However, the transforming efficiency was not affected in Fpg-deficient strains . In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14::URA3 strain . The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities . The results show that the yeast strain defective in the nucleotide excision repair pathway (rad14::URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain . It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14::URA3 mutant only exhibits a minor oxygen dependence . To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro- and eukaryotic cells by the nucleotide excision repair pathway. Biophys Chem, 1997 Nov, 69(1), 85 - 96 A high-angle neutron fibre diffraction study of the hydration of deuterated A-DNA; Shotton MW et al.; A high-angle neutron fibre diffraction study of the hydration of A-DNA has been performed using the single-crystal diffractometer D19 at the Institut Laue-Langevin (Grenoble, France) . The sample was prepared using deuterated DNA extracted from E . Coli cells cultured on deuterated nutrients . In common with our previous neutron fibre diffraction studies of DNA, this work exploits the ability to isotopically replace H2O around the DNA by D2O . However this study benefitted additionally from the fact that the hydrogen atoms which are covalently bonded to carbon atoms in the DNA sugars and bases were replaced by deuterium so that incoherent scattering and absorption effects were minimised . Successive cycles of Fourier synthesis and Fourier difference synthesis allowed water peaks to be identified and their positional and occupancy parameters to be refined against the observed diffraction data . The results confirm the main hydration features noted in our earlier studies with a clear network of water running along the inside edge of the major groove linking successive OI phosphate oxygen atoms . The central core of water running along the axis of the double helix is very much clearer in this work . Additionally this study shows chains of ordered water lying in the centre of the major groove. Trans Am Ophthalmol Soc, 1997, 95, 111 - 25; discussion 126-9 Cloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C; Gottsch JD et al.; PURPOSE: The primary structure of a cornea-associated antigen (CO-Ag) has been identified and has been implicated in the pathogenesis of Mooren's ulcer . The study designs were to isolate full-length clones encoding CO-Ag from a bovine and a human corneal cDNA library so that complete sequence analyses might further define the possible role of this protein in Mooren's ulcer . METHODS: DNA fragments of bovine and human CO-Ag were generated using unique oligonucleotide primers and reverse transcription polymerase chain reaction . These fragments were used as probes to obtain cDNA clones from a bovine and a human corneal cDNA libraries . The clones with the longest cDNA inserts were selected for sequence analyses . Human cDNA fragment was digested with Stu I and Hind III and cloned into a expression vector, pPROEXHT, at the same restriction enzyme sites . The plasmid was transformed into E . coli cells . Correct cloning and the full-length sequence of human CO-Ag were determined by sequencing the insert cDNA . RESULTS: The bovine cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail . The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal . This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons . Plasmid expression vector, pPROEXHT-CO-Ag, was constructed that direct the synthesis of human CO-Ag in E . coli as fusion protein . Human CO-Ag fusion protein was purified to 90% pure with a yield of 17.2 mg per liter of the bacterial cell lysate . The nucleotide sequence of the CO-Ag cDNA insert was completely identical to human neutrophil calgranulin C . The deduced amino acid sequence was completely identical to a Ca(2+)-binding protein isolated on the surface of filarial nematodes . CONCLUSIONS: The isolation and analysis of cDNA clones containing the complete coding sequence of bovine and human CO-Ag proteins is reported . The proteins identified by deduced amino acid sequences demonstrate 100% sequence homology with human and bovine calgranulin C . Immune recognition of calgranulin C to a filarial nematode may lead to a hyperactive autoimmune response to CO-Ag in the cornea leading to a Mooren's ulcer. J Physiol Pharmacol, 1997 Sep, 48 Suppl 4, 59 - 65 Perspectives of anti-H . pylori vaccination; Saldinger PF et al.; Mucosal vaccination using different antigens in conjunction with adjuvants has been used for the prevention and even cure of Helicobacter infection in animal models . A phase I-II trial was recently performed on infected volunteers with urease and the heat labile enterotoxin from E . coli (LT) . A significant decrease in bacterial density but no cure of infection was observed in some patients . The immune effectors which prevent or cure infection with Helicobacter are not well understood and will need to be more clearly defined in order to improve vaccination strategies . Future developments will likely include the following: generation of new mucosal adjuvants without gastrointestinal toxicity; combination of two or three different antigens in order to ensure broader efficacy; use of different routes of administration such as nasal or rectal; coadministration of anti-Helicobacter treatment and vaccine; development of alternate vaccine methods which do not require a mucosal adjuvant, i.e . antigen expression by live carriers or by DNA vaccination; combination of different vaccination methods, for instance DNA vaccination followed by a mucosal boost. Hum Genet, 1997 Dec, 101(3), 333 - 8 Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W; Vreken P et al.; Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype . Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity . In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug . Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the delta C1897 frame-shift mutation, revealed the presence of a novel missense mutation, R235W . Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity . The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype . We show that the E . coli expression system is a valuable tool for examining DPD enzymatic variants . In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified . Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency. Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 187 - 90 Avidin-FITC topological studies with three cysteine mutants of equinatoxin II, a sea anemone pore-forming protein; Anderluh G et al.; Equinatoxin II (EqtII) is a cysteinless pore-forming protein from sea anemone Actinia equina . Three cysteine mutants were produced in an E . coli expression system in order to study the topology of lysine 77, arginine 126, and alanine 179 . Accessibility of an introduced thiol group in the water soluble mutants was studied by using the thiol specific reagent fluorescein maleimide . In aqueous solution all three mutants were readily modified with the probe, indicating their accessibility to the solvent . Mutants were also biotinylated with biotin maleimide, enabling coupling with avidin-fluorescein isothiocyanate (avidin-FITC) . After binding and insertion of biotinylated toxins into liposomes, avidin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intra- or extravesicularly located thiols . All the mutated residues are found to be located on the outside of the lipid vesicles . The results proved the biotin-avidin system as suitable for topological studies of proteins creating pores in membranes. Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 79 - 83 Oligomerization of N-terminal domain of carcinoembryonic antigen (CEA) expressed in Escherichia coli; Krop-Watorek A et al.; The N-terminal domain of CEA, which is essential for cell adhesion activity and lacks cysteine residue, was expressed in Escherichia coli and purified from the solubilized inclusion bodies by DEAE-Sepharose and gel filtration chromatographies . The purified N-domain migrated in SDS-PAGE as a single 13-kDa band, whereas it migrated in non-SDS-PAGE as five distinct bands . The N-domain, analyzed by two-dimensional PAGE after cross-linking with DSS, migrated in multiple forms ranging from monomer to pentamer, showing unequivocally the presence of multimers in each band . The amount of monomer was distinctively the least among the oligomers in the non-SDS-PAGE . These results suggest that the N-domain of CEA molecule has a strong tendency to self-assemble that may convey the homophilic cell adhesion of CEA. Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 71 - 8 Modulation of the Escherichia coli tryptophan repressor protein by engineered peptides; Fenton C et al.; We have used the E . coli tryptophan repressor (TrpR) as a model protein for modulation by engineered peptides both in vivo and in vitro . The tryptophan operator promoter-lacZ reporter system was used to investigate the in vivo ability of several synthetic peptides to modulate TrpR function . GMSA (gel mobility shift analysis) was used to study the in vitro ability of the peptides to modulate binding of the TrpR protein to the operator DNA . Peptides WRW, DRW, DW, RW enhanced TrpR binding to the operator in vivo at 100 microM concentrations . The same peptides enhanced TrpR binding to the operator in vitro at 1 mM concentrations . The peptide RRW reduced TrpR binding to the tryptophan operator both in vivo and in vitro . Thus the peptide RRW acted more as an inducer than corepressor . The peptide WR could neither enhance nor impede binding between TrpR and the operator in vivo or in vitro, suggesting that the presence of a carboxyl tryptophan residue may be necessary for binding to the TrpR protein . Thin layer chromatography was used to ensure that the peptides had not been subject to proteolysis during the in vitro gel mobility shift assays. Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 67 - 70 Effects of intracellular glutathione on sensitivity of Escherichia coli to mercury and arsenite; Latinwo LM et al.; The effect of intracellular glutathione on sensitivity to mercuric cations and arsenite anions was studied in Escherichia coli mutants that lack glutathione (gshA) with or without an additional mutation affecting the osmotregulant trehalose . The absence of glutathione increased cellular sensitivity to both Hg2+ and AsO2- . The double mutant was more sensitive to Hg2+ than the single mutant strain . The addition of plasmid resistance determinants of Hg2+ and AsO2- showed additivity between chromosomal genes and plasmid genes . Mercury resistance was increased in the plasmid-containing cells but not up to the level of wild-type cells . Plasmid arsenite resistance was not expressed in the gshA mutant of E . coli. Arch Biochem Biophys, 1998 Jan 1, 349(1), 153 - 60 Characterization of S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase involved in floral scent production in Clarkia breweri; Wang J et al.; Eugenol, isoeugenol, methyleugenol, and isomethyleugenol are volatiles found in the floral scent of Clarkia breweri . With their distinct aromas, they are used in many perfumes and food seasonings . Here we report the purification and characterization of (iso)eugenol O-methyltransferase (IEMT), the enzyme that methylates eugenol or isoeugenol to make methyleugenol or isomethyleugenol, respectively, using S-adenosyl-L-methionine as the methyl donor . C . breweri IEMT was copurified with caffeic acid O-methyltransferase (COMT) from petals and purified to homogeneity from a bacterial expression system . IEMT is active as a homodimer with a subunit molecular mass of 40 kDa . It is stable at temperatures up to 35 degrees C . It shows optimum activity at pH 7.5, and it does not require any cofactors for enzymatic activity . Plant-purified IEMT has K(m) values of 7 and 58 microM for eugenol and isoeugenol, respectively, and 27 microM for SAM (30, 74, and 19 microM, respectively, for the plant IEMT expressed in Escherichia coli) . By substituting coding regions from COMT into IEMT, it was determined that the regions in IEMT involved in substrate specificity are located in the first half of the protein sequence and that a small segment of 82 amino acids (amino acids 92-173) accounts for the main differences between IEMT and COMT in both substrate specificity and methylation regiospecificity. Arch Biochem Biophys, 1998 Jan 1, 349(1), 47 - 52 The active site of pyrophosphate-dependent phosphofructo-1-kinase based on site-directed mutagenesis and molecular modeling; Hinds RM et al.; Despite a low level of overall sequence identity between PPi-dependent and ATP-dependent phosphofructo-1-kinases (PFKs), similarities in active-site residues permit a convincing amino acid alignment of these two groups of kinases . Employing recent protein sequence and site-directed mutagenesis data along with the known three-dimensional coordinates of Escherichia coli ATP-dependent PFK, a model of the active site of PPi-dependent PFK was proposed . In addition to providing compatible placement of residues shown to be important by earlier mutagenesis studies, the model predicted an important role for two arginyl residues that are conserved in all known PPi-PFK sequences . Replacement by site-directed mutagenesis of these two residues with neutral amino acids in the PPi-PFK of Naegleria fowleri resulted in a substantial reduction in kcat while not altering the global structure of the enzyme . While the data indicate many similarities in the active-site structures and mechanisms of ATP-dependent and PPi-dependent PFKs, subtle differences, such as the relative roles of Arg residues in the active sites, have evolved in the development of these two subgroups of the PFK family. Biosci Biotechnol Biochem, 1997 Dec, 61(12), 2125 - 6 Periplasmic secretion of functional ovotransferrin N-lobe in Escherichia coli; Miyauchi T et al.; The cytoplasmic and periplasmic production systems of ovotransferrin N-lobe in Escherichia coli were constructed . The periplasmic, but not cytoplasmic product, was found to have the iron-binding function. Biosci Biotechnol Biochem, 1997 Dec, 61(12), 2119 - 21 Regulatory region of expression of Thiobacillus ferrooxidans leuB gene in Escherichia coli; Kawaguchi H et al.; The regulatory region of Thiobacillus ferrooxidans leuB gene was analyzed . The deletion analysis indicated that the promoter sequences CATCCG and TATTAT, which were similar to the consensus -35 and -10 sequences of Escherichia coli, respectively, existed . The transcriptional initiation site was located at the position of cytosine -26 . The deletion analysis of the upstream region suggested the existence of a regulatory region by leucine and the region related to transcription of the gene. Biosci Biotechnol Biochem, 1997 Dec, 61(12), 2076 - 9 Molecular cloning of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli; Saito K et al.; The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotinovorans GS-9, and expressed in Escherichia coli . It was found that a single open reading frame consisted of 1554 base pairs that encoded a polypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M(r) 53,152), and it was also found that a putative ribosome-binding site was present in the upstream from the ORF . The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic regions of other fructosylhydrolases . The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with alpha-peptide of the lacZ gene of pUC18 . An E . coli transformant carrying pLFT-BB1 expressed six times as much activity of levan fructotransferase as that of the original strain, A . nicotinovorans GS-9. Biosci Biotechnol Biochem, 1997 Dec, 61(12), 2046 - 50 Purification and characterization of N-acetylneuraminate synthase from Escherichia coli K1-M12; Komaki E et al.; N-Acetylneuraminate (NeuAc) synthase, which catalyzes NeuAc synthesis by condensation of N-acetyl-D-mannosamine (ManNAc) and phosphoenolpyruvate (PEP), was purified from a cell extract of Escherichia coli K1-M12 to electrophoretically homogeneity by serial column chromatographies . The molecular weight of native enzyme was estimated to be 106,000 by gel filtration . After denaturation in sodium dodecyl sulfate, the molecular weight was reduced to 52,000, indicating the existence of 2 identical subunits . The optimum pH was 7.5 and the stable pH range was 7.0 to 10.0 . The enzyme was thermostable up to 30 degrees C . No metal ion was required for the enzyme activity . SH-inhibitors such as p-chloromercuribenzoic acid and mercury chloride were potent inhibitors . The K(m) for ManNAc and PEP were 5.6 mM and 0.04 mM, respectively. J Biol Chem, 1997 Dec 19, 272(51), 32337 - 44 Purification, cDNA cloning, and gene mapping of the small subunit of human DNA polymerase epsilon; Li Y et al.; HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication and DNA repair, consists of a catalytic subunit of 261 kDa and a tightly bound peptide with a relative molecular mass of 55 kDa . The cDNA of the 261-kDa polypeptide has been independently cloned, sequenced, and then overexpressed in insect cells to give a soluble, but catalytically unstable protein, suggesting that the small subunit of HeLa pol epsilon might be important for stability . HeLa pol epsilon has been isolated by immunoaffinity purification to obtain sequence information which enabled the cloning of a full-length human cDNA encoding the small subunit . The clone encoded nine proteolytic peptides obtained from the subunit . The 59,434-Da predicated polypeptide has 26% identity and 44% homology to the yeast pol epsilon 80-kDa subunit, DPB2 . Using fluorescence in situ hybridization, the human pol epsilon p59 locus (DPE2) was assigned to chromosome 14q13-q21. J Biol Chem, 1997 Dec 19, 272(51), 32230 - 9 Characterization of Escherichia coli endonuclease VIII; Jiang D et al.; Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R . J., Hatahet, Z., Kow, Y . W., Ide., H., and Wallace, S . S . (1994) Biochemistry 33, 1255-1264) . Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil . Endo VIII cleaved the damage-containing DNA strand by beta,delta-elimination as does formamidopyrimidine DNA glycosylase (Fpg) . Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site . Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R . J., and Wallace, S . S . (1997) J . Bacteriol . 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg . In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM . Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3' to the reduced AP site. J Biol Chem, 1997 Dec 19, 272(51), 32158 - 62 Calorimetric observation of a GroEL-protein binding reaction with little contribution of hydrophobic interaction; Aoki K et al.; Binding of Escherichia coli chaperonin, GroEL, to substrate proteins with non-native structure, reduced alpha-lactalbumin (rLA) and denatured pepsin, were analyzed by isothermal titration calorimetry at various temperatures in the presence of salt (0.2 M KCl) . Both proteins bound to GroEL with 1:1 stoichiometry and micromolar affinity at all temperatures tested . However, thermodynamic properties of their binding to GroEL are remarkably different from each other . While heat capacity changes (DeltaCp) of rLA-GroEL binding showed large negative values, -4.19 kJ mol-1 K-1, that of denatured pepsin-GroEL binding was only -0.2 kJ mol-1 K-1 . These values strongly indicate that the hydrophobic interaction is a major force of rLA-GroEL binding but not so for denatured pepsin-GroEL binding . When salt was omitted from the solution, the affinity and DeltaCp of the rLA-GroEL binding reaction were not significantly changed whereas denatured pepsin lost affinity to GroEL . Thus, in the non-native protein-GroEL binding reaction, thermodynamic properties, as well as the effect of salt, differ from protein to protein and hydrophobic interaction may not always be a major driving force. J Biol Chem, 1997 Dec 19, 272(51), 32071 - 7 Molecular determinants of selectivity in 5-hydroxytryptamine1B receptor-G protein interactions; Bae H et al.; The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli . Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine1B (5-HT1B) receptor to discriminate between G protein heterotrimers containing Galphai1 or Galphat . Gi1 can interact with the 5-HT1B receptor and stabilize a high affinity agonist binding state of this receptor, but Gt cannot . A series of Galphat/Galphai1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N . P., Bae, H., and Hamm, H . E . (1996) J . Biol . Chem . 271, 413-424) . We have tested the functional coupling abilities of the Galphat/Galphai1 chimeras to 5-HT1B receptors using high affinity agonist binding and receptor-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding . In the presence of betagamma subunits, amino acid residues 299-318 of Galphai1 increase agonist binding to the 5-HT1B receptor and receptor stimulation of GTPgammaS binding . Moreover, Galphai1 containing only Galphat amino acid sequences from this region does not show any coupling ability to 5-HT1B receptors . Our studies suggest that the alpha4 helix and alpha4-beta6 loop region of Galphas are an important region for specific recognition between receptors and Gi family members. J Biol Chem, 1997 Dec 19, 272(51), 31974 - 8 Cloning and expression of a cDNA encoding bovine lipoyltransferase; Fujiwara K et al.; Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the specific lysine residue of the lipoate-dependent enzymes . We have isolated lipoyltransferase I (LipTI) and II (LipTII) from bovine liver (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y . (1994) J . Biol . Chem . 269, 16605-16609) . N-terminal amino acid sequences of LipTI and LipTII were identical except that LipTI had an additional Asn residue on the N terminus . We cloned LipTII cDNA from a bovine liver cDNA library . The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids including a mitochondrial targeting signal of 26 amino acids . The calculated molecular mass of the mature protein is 39,137 Da . The predicted amino acid sequence showed 35% identity with that of Escherichia coli lipoate-protein ligase A . Northern and Southern blot analyses showed a single band, and a single species of mRNA for lipoyltransferase was found by reverse transcription-polymerase chain reaction . Recombinant LipTII was expressed in E . coli and purified to apparent homogeneity . The Kmapp values of the recombinant enzyme for lipoyl-AMP and apoH-protein were comparable with those of native LipTII . An antibody raised against recombinant enzyme cross-reacted with LipTI and LipTII in a similar manner . The results suggest that LipTI and LipTII are derived from the same translated product but processed differently. Biochemistry, 1997 Dec 23, 36(51), 16338 - 44 Characterization of the interthiol acyltransferase reaction catalyzed by the beta-ketoacyl synthase domain of the animal fatty acid synthase; Witkowski A et al.; The enzyme activity responsible for translocation of saturated acyl chains from the 4'-phosphopantetheine of the acyl carrier protein to the active site cysteine of the beta-ketoacyl synthase in the animal fatty acid synthase has been identified . An enzyme assay was devised that allows uncoupling of the interthiol transfer step from the condensation reaction . Experiments with various fatty acid synthase mutants indicate clearly that catalysis of the transfer of saturated acyl moieties from the 4'-phosphopantetheine thiol to the active site cysteine thiol, Cys-161, is an inherent property of the beta-ketoacyl synthase domain . Catalytic efficiency of the interthiol transferase increases from C2 to C12 and decreases with increasing chain-lengths beyond C12 . Malonyl, beta-hydroxybutyryl, and crotonyl thioesters are not substrates for the transferase, whereas the beta-ketobutyryl moiety is a poor substrate . These features of the substrate specificity are exactly as predicted for a transferase that fulfills the proposed role in the fatty acid synthase reaction sequence and indicate that this activity plays an important role in determining the overall specificity of the beta-ketoacyl synthase reaction. Biochemistry, 1997 Dec 23, 36(51), 16300 - 8 Use of Trp mutations to evaluate the conformational behavior and membrane insertion of A and B chains in whole diphtheria toxin; Wang Y et al.; The structure of diphtheria toxin was examined using its Trp fluorescence . To examine the interactions of the A and B chains of the toxin independently, mutants were constructed in which Trp residues were restricted to either the A or the B chain . The conformation and stability of the mutants were very similar to those of the wild-type protein . In addition, they underwent the low-pH conformational transition and membrane insertion at about the same pH as wild-type toxin . This shows Trp do not play a critical role in these processes which are necessary for the translocation of toxin across endosomal membranes in vivo . There was a shift in fluorescence of the Trp mutants which showed the low-pH-induced transition increases exposure of both the A and B Trp to a more polar environment . This supports a model in which the interdomain interactions present at neutral pH break down at low pH . To evaluate the location of the A and B chains in the membrane, the fluorescence quenching of model membrane inserted toxin was measured . Comparison of the amount of quenching by lipid labeled with nitroxides localized at shallow, medium, or deep depths within the bilayer demonstrated that both the A and B chains insert deeply, but the A chain Trp are somewhat less deeply inserted . Trp on the A chain are also less exposed to lipid than on the B chain, as judged by their weaker quenching by the nitroxide-labeled lipid . This conclusion was supported by the observation that the Trp of membrane-inserted isolated A chain is more lipid-exposed than when the A chain is part of the whole toxin . Both the A and B chain Trp become less exposed to lipid after neutralizing pH . However, both chains remain inserted, with at least part of the B chain remaining deeply inserted . These results support the "partial wrapper" model in which both the A and B chains are inserted but contacts between the two chains significantly reduce the exposure of the A chain to lipid. Biochemistry, 1997 Dec 23, 36(51), 16259 - 66 Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB; Butler CS et al.; The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy . Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M . Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost . A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent . The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration . These results are consistent with the binding of up to two NO molecules at CuBII . This has been confirmed by studies with the Cl- adduct of fast cytochrome bo . MCD evidence shows that heme o remains ligated by histidine and water . Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal . Hence chloride ion binds to CuB, blocking the binding of a second NO molecule . These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group. Biochemistry, 1997 Dec 23, 36(51), 16221 - 30 Redox potentials and quinone reductase activity of L-aspartate oxidase from Escherichia coli; Tedeschi G et al.; l-Aspartate oxidase (EC 1.4.3.16) is a flavoprotein that catalyzes the first step in the de novobiosynthetic pathway to pyridine nucleotides both under aerobic and under anaerobic conditions . Despite the physiological importance of this biosynthesis particularly in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen . In this report, evidence is presented which suggests that in vitro quinones can play such a role . L-Aspartate oxidase binds menadione and 2, 3-dimethoxy-5-methyl-p-benzoquinone with Kd values of 11.5 and 2.4 microM, respectively . A new L-aspartate:quinone oxidoreductase activity is described in the presence and in the absence of phospholipids, and its possible physiological relevance is discussed . Moreover, considering the striking sequence similarity between L-aspartate oxidase and the highly conserved family of succinate-fumarate oxidoreductases, the redox properties of L-aspartate oxidase were investigated in detail . A value of -216 mV was calculated for the midpoint potential of the couple FAD/FADH2 bound to the enzyme . This result perfectly explains why L-aspartate oxidase may be considered as a very particular fumarate reductase unable to use succinate as the electron donor. Biochemistry, 1997 Dec 23, 36(51), 16155 - 65 X-ray structure of motor and neck domains from rat brain kinesin; Sack S et al.; We have determined the X-ray structure of rat kinesin head and neck domains . The folding of the core motor domain resembles that of human kinesin reported recently {Kull, F . J., et al . (1996) Nature 380, 550-554} . Novel features of the structure include the N-terminal region, folded as a beta-strand, and the C-terminal transition from the motor to the rod domain, folded as two beta-strands plus an alpha-helix . This helix is the beginning of kinesin's neck responsible for dimerization of the motor complex and for force transduction . Although the folding of the motor domain core is similar to that of a domain of myosin (an actin-dependent motor), the position and angle of kinesin's neck are very different from those of myosin's stalk, suggesting that the two motors have different mechanisms of force transduction . The N- and C-terminal ends of the core motor, thought to be responsible for the directionality of the motors {Case, R . B., et al . (1997) Cell 90, 959-966}, take the form of beta-strands attached to the central beta-sheet of the structure. Biochemistry, 1997 Dec 23, 36(51), 16141 - 6 Energetics of heme binding to native and denatured states of cytochrome b562; Robinson CR et al.; Cytochrome b562 is a four-helix bundle protein containing a noncovalently bound b-type heme prosthetic group . For the first time, energetics of heme binding to an apocytochrome were measured by isothermal titration calorimetry . The heme is tightly bound to native apocytochrome b562, with a dissociation constant (Kd) of approximately 9 nM (DeltaG degrees = 11 kcal mol-1) at 25 degrees C . Unexpectedly, the thermally denatured apoprotein is also capable of specifically binding heme with modest affinity (Kd = 3 microM, DeltaG degrees = 7.6 kcal mol-1) . This interaction results in the dependence of holocytochrome b562 stability on protein concentration in the submicromolar range. Biochemistry, 1997 Dec 23, 36(51), 16134 - 40 The alrestatin double-decker: binding of two inhibitor molecules to human aldose reductase reveals a new specificity determinant; Harrison DH et al.; It is generally expected that only one inhibitor molecule will bind to an enzyme active site . In fact, specific drug design theories depend upon this assumption . Here, we report the binding of two molecules of an inhibitor to the same active site which we observed in the 1.8 A resolution structure of the drug Alrestatin bound to a mutant of human aldose reductase . The two molecules of Alrestatin bind to the active site in a stacked arrangement (a double-decker) . This stack positions the carboxylic acid of one drug molecule near the NADP+ cofactor at a previously determined anion binding site and the carboxylic acid of the second drug molecule near the carboxy-terminal tail of the enzyme . We propose that interactions of inhibitors with the carboxy-terminal loop of aldose reductase are critical for the development of inhibitors that are able to discriminate between aldose reductase and other members of the aldo-keto reductase superfamily . This finding suggests a new direction for the introduction of specificity to aldose reductase-targeted drugs. Biochemistry, 1997 Dec 23, 36(51), 16087 - 96 Structural studies of the Escherichia coli signal transducing protein IIAGlc: implications for target recognition; Feese MD et al.; In Escherichia coli, the glucose-specific phosphocarrier protein of the phosphotransferase system (PTS), IIAGlc (IIIGlc in older literature), is also the central regulatory protein of the PTS . Depending upon its state of phosphorylation, IIAGlc binds to a number of different proteins that display no apparent sequence homology . Previous structural studies suggested that nonspecific hydrophobic interactions, specific salt bridges, and an intermolecular Zn(II) binding site contribute to the wide latitude in IIAGlc binding sites . Two new crystal forms of IIAGlc have been solved at high resolution, and the models were compared to those previously studied . The major intermolecular contacts in the crystals differ in detail, but all involve the hydrophobic active site of IIAGlc interacting with a hydrophobic patch on a neighbor and all are shown to be surprisingly similar to the physiologically relevant regulatory interaction of IIAGlc with glycerol kinase . In two crystal forms, a helix on one molecule interacts with the face of another, while in the other crystal form, the primary crystal contact consists of a strand of beta-sheet that contributes to an intermolecular Zn(II) binding site with tetrahedral ligation identical to that of the zinc peptidase thermolysin . Thus, relatively nonspecific hydrophobic interactions combined with specific salt bridges and an intermolecular cation binding site (cation-promoted association) permit a regulatory protein to bind to target proteins that have little or no sequence or structural homology with one another . It is suggested that signal transduction by IIAGlc is a binary switch in which phosphorylation at the active site directly controls binding to target molecules. Biochemistry, 1997 Dec 23, 36(51), 16040 - 8 Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments; Chaffotte AF et al.; The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association {Tasayco, M . L., & Chao, K . (1995) Proteins: Struct., Funct., Genet . 22, 41-44} . Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association {kon = (1330 +/- 54) M-1 s-1}, and two apparent unimolecular rate constants {k1 = (0 . 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1} . The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants . An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used . Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total . Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively . Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment. Biochem J, 1997 Dec 1, 328 ( Pt 2), 677 - 87 Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum; Mohrle JJ et al.; PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing . The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified . The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases . There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases . The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids . Both inserts are larger than their homologues in eIF-2alpha kinases . The sequence of PfPK4 has one putative haemin-binding site . The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases . Autophosphorylation and substrate phosphorylation are inhibited by haemin . Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase . Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa . Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites . One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters . Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters . PfPK4 co-localizes with P . falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1. Biochem J, 1997 Dec 1, 328 ( Pt 2), 643 - 7 Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles; Gazaryan IG et al.; Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane . In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis . Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms . The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima . The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms . This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site . In the model structure obtained, three of eight glycosylation sites were screened . This might explain the absence of dimeric structures in native enzyme peroxidase . The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme . Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures. Biochem J, 1997 Dec 1, 328 ( Pt 2), 483 - 7 Bovine cytosolic IMP/GMP-specific 5'-nucleotidase: cloning and expression of active enzyme in Escherichia coli; Allegrini S et al.; A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE) . Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx . 2951 bp . The similarity in the coding region to that of the human 5'-nucleotidase cDNA sequence {Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem . Biophys . Res . Commun . 205, 917-922}, indirectly identified as a 5'-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical . The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5'-nucleotidase/phosphotransferase purified from calf thymus . Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues . The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE . It exhibited both 5'-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus . This is the first time that a fully active recombinant 5'nucleotidase has been described. Biochem J, 1997 Dec 1, 328 ( Pt 2), 377 - 82 Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2: role of the hydratase reaction in bile acid synthesis; Qin YM et al.; Rat liver peroxisomes contain two multifunctional enzymes: (1) perMFE-1 {2-enoyl-CoA hydratase 1/Delta3,Delta2-enoyl-CoA isomerase/(S)-3-hydroxyacyl-CoA dehydrogenase} and (2) perMFE-2 {2-enoyl-CoA hydratase 2/(R)-3-hydroxyacyl-CoA dehydrogenase} . To investigate the role of the hydratase activity of perMFE-2 in beta-oxidation, a truncated version of perMFE-2 was expressed in Escherichia coli as a recombinant protein . The protein catalyses the hydration of straight-chain (2E)-enoyl-CoAs to (3R)-hydroxyacyl-CoAs, but it is devoid of hydratase 1 {(2E)-enoyl-CoA to (3S)-hydroxyacyl-CoA} and (3R)-hydroxyacyl-CoA dehydrogenase activities . The purified enzyme (46 kDa hydratase 2) can be stored as an active enzyme for at least half a year . The recombinant enzyme hydrates (24E)-3alpha,7alpha,12alpha-trihydroxy- 5beta-cholest-24-enoyl-CoA to (24R,25R)-3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholestanoyl-CoA, which has previously been characterized as a physiological intermediate in bile acid synthesis . The stereochemistry of the products indicates that the hydration reaction catalysed by the enzyme proceeds via a syn mechanism . A monofunctional 2-enoyl-CoA hydratase 2 has not been observed as a wild-type protein . The recombinant 46 kDa hydratase 2 described here survives in a purified form under storage, thus being the first protein of this type amenable to application as a tool in metabolic studies. Nucleic Acids Res, 1997 Dec 1, 25(23), 4786 - 91 Dynamics in the isomerization of intramolecular DNA triplexes in supercoiled plasmids; Shindo H et al.; We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids . Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form) . The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time . The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+ . The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates . Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5 . Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids . The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form. Nucleic Acids Res, 1997 Dec 1, 25(23), 4703 - 9 Non-canonical sequence elements in the promoter structure . Cluster analysis of promoters recognized by Escherichia coli RNA polymerase; Ozoline ON et al.; Nucleotide sequences of 441 promoters recognized by Escherichia coli RNA polymerase were subjected to a site-specific cluster analysis based on the hierarchical method of classification . Five regions permitting promoter subgrouping were identified . They are located at -54 +/- 4, -44 +/- 3, -35 +/- 3 (-35 element), -29 +/- 2 and -11 +/-4 (-10 element) . Promoters were independently subgrouped on the basis of their sequence homology in each of these regions and typical sequence elements were determined . The putative functional significance of the revealed elements is discussed on the basis of available biochemical data . Those promoters that have a high degree of homology with the revealed sequence elements were selected as representatives of corresponding promoter groups and the presence of other sequence motifs in their structure was examined . Both positive and negative correlations in the presence of particular sequence motifs were observed; however, the degree of these interdependencies was not high in all cases, probably indicating that different combinations of the signal elements may create a promoter . The list of promoter sequences with the presence of different sequence elements is available on request by Email: ozoline@venus.iteb . serpukhov.su. Naunyn Schmiedebergs Arch Pharmacol, 1997 Oct, 356(4), 500 - 4 Involvement of nitric oxide in the in vivo effects of lipopolysaccharide on the contractile and electrical properties of mouse diaphragm; Liu SH et al.; The contractile and electrical properties of the mouse diaphragm during endotoxemia were studied, and the possible role of nitric oxide (NO) on these changes was investigated . The mice were injected intraperitoneally with E . coli . lipopolysaccharide (endotoxin, LPS) at various times before isolation of the diaphragm to induce endotoxemia . It was observed that direct twitch tension was slightly increased, and that there was a significant increase in tetanic tension when compared with controls . The potentiation of direct twitch tension induced by a Cl--channel blocker (9-anthracene carboxylic acid), but not the potentiation by a Na+-channel activator (veratridine) or by K+-channel blockers (uranyl ion, 4-aminopyridine and tetraethylammonium ion), was attenuated in the diaphragm of LPS-treated mice . Moreover, the resting membrane potential was significantly reduced and the membrane input resistance was significantly increased, largely due to a decrease in Cl--conductance . However, the membrane K+-conductance remained unaltered . These results imply that the sarcolemmal Cl--channel is markedly affected in the mouse diaphragm during endotoxemia . These changes of contractile and electrical characteristics of the mouse diaphragm during endotoxemia could be reversed by treatment with dexamethasone and N(G)-nitro-L-arginine (NO synthase inhibitors) . On the other hand, in in vitro studies, LPS (20 microg/ml), by itself, applied directly to the diaphragm, did not alter the muscle contractions or the membrane potentials . A NO donor, added to the diaphragm bath, increased the tetanus/twitch ratio and induced a transient depolarization . All of these findings suggest that LPS may, at least in part, affect the sarcolemmal electrical properties and muscle contractions during endotoxemia through the L-arginine:NO pathway. Pediatr Nephrol, 1997 Dec, 11(6), 737 - 40 Gentamicin-induced Bartter-like syndrome; Landau D et al.; Gentamicin is well known to be associated with nephrotoxicity, including acute renal failure and renal tubular dysfunction . A Bartter-like syndrome has also been described as a toxic manifestation of gentamicin therapy in adults, but this nephrotoxic syndrome has not been well characterized in children . In this report we describe the clinical course of four patients with gentamicin-associated Bartter-like syndrome . These patients ranged in age from 4 months to 17 years; they all demonstrated evidence of renal tubulopathy, primarily affecting the distal nephron . Hypocalcemia, hypomagnesemia, alkalosis, and hypokalemia were the main manifestations in these patients . After discontinuation of gentamicin, recovery of the renal tubular functions and resolution of the electrolyte abnormalities were complete in all patients. Poult Sci, 1997 Dec, 76(12), 1682 - 7 Adverse effects of Escherichia coli infection of turkeys were not alleviated by supplemental dietary vitamin E; Sell JL et al.; Two experiments were conducted to determine the influence of dietary vitamin E on the response of young male turkeys to Escherichia coli infection . A complete factorial arrangement of two concentrations of supplemental dietary vitamin E (12 or 300 IU/kg as dl-alpha-tocopheryl acetate) and infection or no infection of turkeys with E . coli was used in both experiments . In Experiment 1, each dietary treatment was fed to four pens of turkeys from 1 to 28 d of age . At 28 d, turkeys in two pens per dietary treatment received an injection of 3.0 x 10(7) E . coli cells into the left and right thoracic air sacs . All turkeys were necropsied 7 d after E . coli injection and the incidence and severity of lesions in air sacs, lungs, pericardium, and liver were determined . The same dietary vitamin E treatments were used in Experiment 2 . Each diet was fed to eight pens of turkeys from 1 to 47 d of age . At 47 d, turkeys in four pens per dietary treatment received an injection of 3.0 x 10(7) cells of the same E . coli used in Experiment 1 into the left and right thoracic air sacs . All turkeys were necropsied as in Experiment 1 at 54 d of age . Weight gain and efficiency of feed utilization were impaired markedly by E . coli infection during the 7 d after injection . Livability also was decreased by E . coli infection in Experiment 1 but not in Experiment 2 . Adverse effects of E . coli on performance and livability were not affected by dietary vitamin E concentration . Lesions observed in turkeys that received E . coli injection ranged from mild to severe, with the most severe lesions observed in air sacs . Lung lesions were observed frequently but were less severe than in air sacs . Dietary concentration of vitamin E had no effect on incidence or severity of lesions in air sacs or lungs . Overall, the results of these experiments show that adding 300 IU of vitamin E/kg of diet did not alleviate the adverse effects of E . coli infection in young turkeys. Plant Cell, 1997 Dec, 9(12), 2281 - 9 The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter; Santa-Maria GE et al.; The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies . However, major components of this system remain to be identified . We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters . HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots . HvHAK1 also mediated low-affinity Na+ uptake . Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated . Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family . Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters. Vet Immunol Immunopathol, 1997 Oct 6, 59(1-2), 65 - 78 Bovine stem cell factor: production of a biologically active protein and mRNA analysis in cattle infected with Trypanosoma congolense; Mertens B et al.; The cDNA coding for the soluble form of bovine stem cell factor (boSCFAla165) was cloned and recombinant protein was produced in bacteria as a histidine tagged-protein . The protein was purified from the inclusion bodies in one step by metal chelation chromatography under denaturing conditions . Recombinant bovine SCF was shown to act synergistically with interleukin 3 (IL-3) and erythropoietin (EPO) in stimulating the growth of bone marrow progenitor cells such as colony forming units-granulocyte macrophage (CFU-GM) and burst forming units-erythroid (BFU-E) . Analysis of SCF mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the transcripts were detectable in bone marrow, lymph nodes and spleen of cattle, and that the level of transcription was upregulated in lymph nodes of cattle infected with Trypanosoma congolense . Two isoforms of SCF mRNA were amplified by RT-PCR . The availability of recombinant bovine SCF provides a valuable tool for studying the role of SCF in the development, growth and differentiation of bovine hematopoietic cells. Chem Res Toxicol, 1997 Dec, 10(12), 1351 - 8 Synthesis and miscoding specificity of oligodeoxynucleotide containing 8-phenyl-2'-deoxyguanosine; Kohda K et al.; Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts . 8-Phenyl-2'-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques . A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo+) or 3'-->5' exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha (pol alpha) . Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products . In contrast, primer extensions catalyzed by pol alpha were strongly blocked opposite the lesion . The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG . The exo- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP . Two-base deletions were also observed . In contrast, the exo+ Klenow fragment incorporated dCMP opposite the lesion . When pol alpha was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions . The duplex containing 8-PhdG.dG was thermally and thermodynamically more stable than dG.dG . The duplex containing 8-PhdG.dA was thermodynamically more stable than dG.dA . We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G-->T and G-->C transversions and deletions in cells. Nat Struct Biol, 1998 Jan, 5(1), 15 - 9 Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase; Nakatsu T et al.; The crystal structure of E . coli asparagine synthetase has been determined by X-ray diffraction analysis at 2.5 A resolution . The overall structure of the enzyme is remarkably similar to that of the catalytic domain of yeast aspartyl-tRNA synthetase despite low sequence similarity . These enzymes have a common reaction mechanism that implies the formation of an aminoacyl-adenylate intermediate . The active site architecture and most of the catalytic residues are also conserved in both enzymes . These proteins have probably evolved from a common ancestor even though their sequence similarities are small . The functional and structural similarities of both enzymes suggest that new enzymatic activities would generally follow the recruitment of a protein catalyzing a similar chemical reaction. Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 638 - 45 A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase; Wrobel JA et al.; By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome . After expression in Escherichia coli, two phenotypic assays were performed . The first assay tested for RNA-dependent DNA polymerase activity . The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit . The resulting phenotypic data provided a "genetic" means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface . Several HIV-1 RT crystal structures were used to evaluate the mutational analysis . Our genetic map correlates well with the crystal structures . Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues . The important functional residues are found near the enzyme active site . Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior . In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general . This strategy should be useful for studying proteins for which no crystallographic data are available. RNA, 1998 Jan, 4(1), 47 - 54 Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides; Pel HJ et al.; Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2 . Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro . Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors . These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis . Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2 . This cooperativity is absent, however, in the presence of GDPCP . The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction. Can J Microbiol, 1997 Nov, 43(11), 1036 - 43 Survival of Escherichia coli exposed to visible light in seawater: analysis of rpoS-dependent effects; Gourmelon M et al.; We investigated the effect of visible light on Escherichia coli in seawater microcosms . Escherichia coli lost its ability to form colonies in marine environments when exposed to artificial continuous visible light . Survival of illuminated bacteria during the stationary phase was drastically reduced in the absence of the sigma factor (RpoS or KatF) that regulates numerous genes induced in this phase . In the stationary phase, double catalase mutants katE katG and mutants defective in the protein Dps (both catalase and Dps are involved in resistance to hydrogen peroxide (H2O2)), were more sensitive to light . In the exponenital phase, a mutation in oxyR, the regulatory gene of the adaptive response to H2O2, increased sensitivity to light, further suggesting that deleterious effects might be associated with H2O2 production . However, in the stationary phase, the katE katG dps mutant was considerably more resistant to visible light than the rpoS mutant, suggesting rpoS-dependent protection against deleterious effects other than those related to H2O2 . The deleterious action of visible light was less important when the salinity decreased . In freshwater, rpoS and katE katG dps mutants did not show a drastic difference in sensitivity to light suggesting that osmolarity sensitizes E . coli to those deleterious effects of visible light that are unrelated to H2O2. Vet Immunol Immunopathol, 1997 Sep 19, 58(3-4), 321 - 33 Expression and functional characterization of recombinant chicken interferon-gamma; Song KD et al.; A cDNA encoding chicken interferon-gamma (chIFN-gamma) was cloned from a CD4+ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli, COS- and CEC-32 fibroblast cell lines . In general, recombinant chicken IFN-gamma (rchIFN-gamma) expressed in the COS- and CEC-32 cell lines showed high bioactivity in vitro . The kinetics of IFN-gamma gene expression were examined in concanavalin A (Con A)-activated spleen lymphocytes by Northern blot and RT-PCR . IFN-gamma mRNA was detected as early as 30 min after Con A activation, reached peak expression at 2 h and then decreased starting at 4 h post Con A activation . A rabbit serum made to a synthetic peptide of IFN-gamma immunoprecipitated a 60 kDa E . coli maltose-binding fusion protein of recombinant IFN-gamma (MBP-IFN) and a 26-27 kDa secreted protein from COS cells and Con A-activated spleen cells . IFN-gamma inhibited vesicular stomatitis virus (VSV) mediated cytotoxicity of chicken embryonic fibroblast (CEF) cells and upregulated the expression of many macrophage cell surface antigens, including class I and class II major histocompatibility complex (MHC) proteins . These results show that chicken IFN-gamma possesses anti-viral activity and immunoregulates macrophage activities. Lung, 1998, 176(1), 25 - 34 Effects of concanavalin A on Na(+)-dependent and Na(+)-independent mechanisms for H+ extrusion in alveolar macrophages; Bidani A et al.; Alveolar macrophages (m phi) possess two parallel mechanisms for plasmalemmal H+ extrusion: a V-type H+ pump (V-ATPase) and a Na+/H+ exchanger (NHE) . To investigate the coordinated functioning of the H+ extruders for m phi intracellular pH (pHi) regulation, we investigated the effects of the plant lectin concanavalin A (ConA) on resident alveolar m phi from rabbits . ConA (1 microM, 30-min pretreatment) activated the m phi for phagocytosis of opsonized Escherichia coli . ConA activation did not affect the baseline pHi of m phi or the initial rate of pHi recovery (dpHi/dt) from an intracellular acid load (acid-loaded pHi nadir approximately 6.9) . However, the contributions of Na(+)-independent H+ transport (i.e . V-ATPase activity) and Na(+)-dependent H+ transport (i.e . NHE activity) to dpHi/dt were altered significantly . The lectin stimulated Na+/H+ exchange and inhibited V-ATPase activity . In control m phi, V-ATPase-mediated H+ extrusion was responsible for > 80% of dpHi/dt . Conversely, in ConA-treated m phi, Na+/H+ exchange was responsible for approximately 65% of dpHi/dt, and V-ATPase activity was responsible for only 35% of dpHi/dt . These results underscore the complex mechanisms and signaling pathways that coordinate the activities of cellular acid-base transporters in m phi pHi regulation. Lung, 1998, 176(1), 1 - 13 The importance of polymorphonuclear leukocytes in lipopolysaccharide-induced superoxide anion production and lung injury: ex vivo observation in rat lungs; Tsuji C et al.; The purpose of this study is to determine if the polymorphonuclear leukocyte (PMN) is a major causative agent for lipopolysaccharide (LPS)-induced lung injury and responsible for the excess production of superoxide anion in the lung . We measured superoxide anion production from the lung and pulmonary capillary permeability in rats with and without PMN depletion . The superoxide anion production from the lung was measured using a purpose-built ex vivo chemiluminescence apparatus . Pulmonary capillary permeability was evaluated by the Evans blue dye extravasation method . PMN sequestration was determined by counting PMNs in histologic tissue specimens using microscopy . All rats received 3 mg/kg LPS intravenously . Examinations were undertaken at 2, 6, and 12 h after the LPS injection . The PMN-depleted group received cyclophosphamide 4 days before the LPS injection, which resulted in a PMN count of less than 200 cells/microliter . In rats without PMN depletion, Evans blue dye extravasation increased significantly at 12 h after the LPS injection; PMN sequestration increased at 2, 6, and 12 h after the LPS injection; and superoxide anion production increased at 6 h and remained elevated at 12 h after the LPS injection . The increased permeability, PMN sequestration, and superoxide anion production were not seen in the PMN-depleted group . The contribution of the xanthine/xanthine oxidase system and alveolar macrophages to the observed superoxide anion production was negligible . We conclude that, in rats, the PMN is a major causative agent in LPS-induced lung injury and is responsible for the excess production of superoxide anion in the lung. J Med Chem, 1997 Dec 19, 40(26), 4208 - 21 Monoaryl- and bisaryldihydroxytropolones as potent inhibitors of inositol monophosphatase; Piettre SR et al.; The first successful preparation of mono- and disubstituted 3,7-dihydroxytropolone involves a four-step synthetic scheme . Thus, bromination of 3,7-dihydroxytropolone (8) followed by permethylation of the resultant products furnished gram quantities of intermediates 13-18 . Single or double Suzuki coupling reactions between these permethylated monobromo- and dibromodihydroxytropolone derivatives and a variety of boronic acids delivered the expected products whose deprotection yielded the desired compounds 1a-u and 26a-n, usually in fair to good yields . Tropolones 1 and 26 were found to be potent inhibitors of inositol monophosphatase with IC50 values in the low-micromolar range . The results are discussed in the context of the recently described novel mode of inhibition of the enzyme by 3,7-dihydroxytropolones. Cancer Immunol Immunother, 1997 Nov-Dec, 45(3-4), 193 - 7 Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer; Kufer P et al.; Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers . These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker . The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen . The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL17-1A-VH17-1A-VHCD3-VLCD3 . The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed . The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail . The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry . We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells . Most remarkable, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor alpha, interleukin-6 and interferon gamma . Maximal cytotoxicity (51Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies . CD8+ as well as CD4+ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics . CD8+ cells induced high 51Cr release within 4 h while CD4+ cells required a 20-h incubation . The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells . A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in vitro. Am J Physiol, 1997 Dec, 273(6 Pt 1), L1182 - 90 Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats; Lukaszewicz G et al.; During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine . This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia . It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones . To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats . Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS . Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above . A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA (P < or = 0.1) and a twofold increase in GS protein level 8 h postinjection . Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic (P < or = 0.1) and fourfold in nonneutropenic control rats (P < or = 0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction . In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold (P < or = 0.02) compared with sixfold in sham-operated control rats (P < or = 0.02) . However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold (P < or = 0.02) compared with fourfold in sham-operated control rats (P < or = 0.02) . Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone . In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively . The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30% . These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones. Am J Physiol, 1997 Dec, 273(6 Pt 1), G1304 - 11 Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells; Spolarics Z et al.; The present study investigated the effect of lipopolysaccharide (LPS; from Escherichia coli, 2 mg/kg body wt ip) on selected aspects of the antioxidant status in Kupffer and sinusoidal endothelial cells . Cells were isolated 18 h after the injection of saline or LPS . In fresh suspension cultures, cellular reduced glutathione (GSH) and H2O2 were determined by monochlorobimane, and 2',7'-dichlorofluorescein diacetate, respectively, using a fluorescence plate reader . LPS injection increased GSH content two- to threefold in Kupffer cells compared with cells from control rats . Cellular GSH content was higher in endothelial than Kupffer cells . However, LPS did not increase GSH content in endothelial cells . Addition of H2O2 (40-200 microM) to Kupffer or endothelial cells caused a transient decrease in GSH, which was more pronounced in cells from control rats (approximately 45% drop) than in LPS-exposed cells (approximately 25% drop) . Depleted GSH levels were accompanied by a proportional increase in cellular H2O2 . After inhibition of catalase by 3-amino-1,2,4-triazole, the presence of 0.2 mM H2O2 depleted GSH content by 75% and 40% in Kupffer cells from saline- or LPS-injected rats, respectively . The same treatments caused a similar 50% decrease in both activated and control endothelial cells . LPS decreased catalase activity by 45% in Kupffer cells, whereas it had no effect on catalase in endothelial cells . Glutathione reductase activity was not altered by LPS in either cell type . These data show that in activated Kupffer cells the elevated level of cellular glutathione plays an augmented role in the protection against reactive oxygen species, whereas the contribution of catalase to H2O2 detoxification is attenuated . In LPS-stimulated endothelial and Kupffer cells, the efficient maintenance of GSH is consistent with upregulated production of reducing power through the hexose phosphate shunt observed previously. Am J Physiol, 1997 Dec, 273(6 Pt 1), C1882 - 8 Urea inhibits inducible nitric oxide synthase in macrophage cell line; Prabhakar SS et al.; Macrophage dysfunction is considered an important contributory factor for increased propensity of infections in uremia . Because nitric oxide (NO) is believed to be an effector molecule of macrophage cytotoxicity, we propose that the dysfunction may be related to impaired NO synthesis . To verify this hypothesis, we evaluated macrophage NO synthesis in the presence of urea, a compound that accumulates in renal failure and is believed by some to be a uremic toxin . Macrophages (RAW 264.7 cells) were incubated with bacterial lipopolysaccharide to induce NO synthesis, whereas the test groups had various concentrations of urea in addition . NO synthesis was measured by assaying the supernatant for nitrites and nitrates by chemiluminescence . We observed that urea consistently produced a dose-dependent reversible inhibition of inducible NO production in macrophages, whereas parathormone, another toxin retained in uremia, had no such inhibitory effects . Further studies revealed that mRNA for inducible NO synthase was not inhibited by urea . We thus conclude that urea inhibits inducible NO synthesis in macrophages by a posttranscriptional mechanism and that this may be important in macrophage dysfunction of uremia. Anal Chem, 1998 Jan 1, 70(1), 136 - 43 Determination of disulfide bonds in highly bridged disulfide-linked peptides by matrix-assisted laser desorption/ionization mass spectrometry with postsource decay; Jones MD et al.; Matrix-assisted laser desorption/ionization mass spectrometry with postsource decay was used to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds . Postsource decay analysis of these peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides . The assignment was made possible by fragmentation at peptide bonds between two Cys residues in a peptide that constitutes the highly bridged fragment, while retaining the disulfide linkage to the other peptide . Fragmentation using other types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fragment ions . The data obtained from postsource decay also provide fragment ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds . The present method is a highly sensitive technique which requires no further sample handling and should be complementary to other classical chemical methods . The method proved useful in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al . Biochemistry, in press) . Postsource decay analysis of large disulfide-containing peptides usually produces no fragmentation but generates a series of high-intensity ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 554 - 9 Modulation of cell death by Bcl-XL through caspase interaction; Clem RJ et al.; The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans . Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells . Here, we report a direct interaction between caspases and Bcl-XL . The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal . Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death . However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis . Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein. Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 531 - 6 An ERp60-like protein from the filarial parasite Dirofilaria immitis has both transglutaminase and protein disulfide isomerase activity; Chandrashekar R et al.; Transglutaminases (TGases; EC 2.3.2.13) are a family of enzymes that catalyze calcium-dependent covalent cross-linking of cellular proteins by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds . These covalent isopeptide bonds are of great physiological significance because they are highly resistant to proteolysis, denaturants, and reducing agents . Prior studies have demonstrated the presence of isopeptide bonds in the sheath and cuticle of filarial parasites, suggesting an important role for TGase-catalyzed reactions during the growth and development of filarial nematodes . Herein we report the identification and cloning of a cDNA encoding a TGase from the dog heartworm Dirofilaria immitis (DiTG) . The DiTG expressed in Escherichia coli (recombinant DiTG) was able to catalyze calcium-dependent cross-linking reactions . The derived amino acid sequence of the DiTG cDNA (pDiTG) predicts a protein of 57.1 kDa and includes an N-terminal hydrophobic signal peptide . The pDiTG has no sequence similarity with any of the known TGases, but it has significant homology to protein disulfide isomerase (PDI) and, particularly, to the PDI-related endoplasmic reticulum protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum . As predicted from the amino acid sequence homology, recombinant DiTG catalyzed the isomerization of intramolecular disulfide/sulfhydryl bonds in denatured RNase in vitro as effectively as did mammalian PDI . Conversely, purified PDI from bovine liver could catalyze protein cross-linking reactions in a Ca(2+)-dependent manner . This report describes the dual catalytic activity of TGase and PDI in post- and/or cotranslational modification of newly synthesized proteins . These TGase-catalyzed posttranslational modifications may play a pivotal role in the synthesis of new cuticle during the growth and maturation of filarial parasites. Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 478 - 83 Targeting of GroEL to SecA on the cytoplasmic membrane of Escherichia coli; Bochkareva ES et al.; Chaperonin GroEL has been found to interact with isolated cytoplasmic membrane of Escherichia coli . Interaction requires Mg ions, whereas MgATP inhibits, and inhibition is stronger in the presence of co-chaperonin GroES . "Heat-shock" of the membrane at 45 degrees C destroys irreversibly its ability to bind GroEL . The binding of GroEL is characterized by saturation with a maximum of about 100 pmol GroEL bound per mg of total membrane protein, indicating a limited capacity and specificity of the membrane to bind GroEL . According to results of immunoblotting analysis and cleavable photoactivable cross-linking, a membrane target of GroEL is SecA, a protein known as a central component of the translocation machinery . Moreover, in some cases GroEL could modulate a cycle of association of SecA with the membrane by stimulating release of SecA from the membrane . A physiological role of targeting of GroEL in or close to the protein-conducting membrane apparatus is discussed. Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 460 - 5 The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation; McClain WH et al.; We have identified six new aminoacylation determinants of Escherichia coli tRNAGln in a genetic and biochemical analysis of suppressor tRNA . The new determinants occupy the interior of the acceptor stem, the inside corner of the L shape, and the anticodon loop of the molecule . They supplement the primary determinants located in the anticodon and acceptor end of tRNAGln described previously . Remarkably, the three-dimensional structure of the complex between tRNAGln and glutaminyl-tRNA synthetase shows that the enzyme interacts with the phosphate-sugar backbone but not the base of every new determinant . Moreover, a small protein motif interacts with five of these determinants, and it binds proximal to the sixth . The motif also interacts with the middle base of the anticodon and with the backbones of six other nucleotides . Our results emphasize that synthetase recognition of tRNA is more elaborate than amino acid side chains of the enzyme interacting with nucleotide bases of the tRNA . Recognition also includes synthetase interaction with tRNA backbone functionalities whose distinctive locations in three-dimensional space are exquisitely determined by the tRNA sequence. J Pharmacol Exp Ther, 1998 Jan, 284(1), 189 - 95 ONO-4007, an antitumor lipid A analog, induces tumor necrosis factor-alpha production by human monocytes only under primed state: different effects of ONO-4007 and lipopolysaccharide on cytokine production; Matsumoto N et al.; ONO-4007 is a synthetic lipid A analog that exhibits strong antitumor activity in several animal models via intratumoral production of tumor necrosis factor (TNF) . In the present study the cytokine-inducing effect of ONO-4007 was investigated in human monocytes that were freshly isolated or had been incubated for 3 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor . ONO-4007 induced slight production of TNF-alpha, Interleukin (IL)-1 beta, IL-6 and IL-12 in fresh monocytes but strongly induced TNF-alpha production in GM-CSF-treated monocytes . Monocytes treated with macrophage colony-stimulating factor were also primed to produce TNF-alpha in response to ONO-4007 . In the production of IL-1 beta, IL-6 and IL-12, GM-CSF did not show a priming effect . In contrast to ONO-4007, lipopolysaccharide (LPS) induced significant amounts of all these cytokines in fresh monocytes . In whole blood, ONO-4007 failed to induce TNF-alpha, whereas LPS and LA-15-PP (Escherichia coli-type lipid A) strongly induced TNF-alpha production . In the GM-CSF-treated monocytes, both elimination of serum from the culture medium and anti-CD14 antibody treatment attenuated LPS-induced TNF-alpha production but not ONO-4007-induced TNF-alpha production . This study shows that ONO-4007 activates human monocytes/ macrophages to release TNF-alpha only in a primed state and suggests that ONO-4007 would activate these cells via different pathways from LPS . These differences could mean that ONO-4007 has potent antitumor activity with lower toxicity than LPS. J Pharmacol Exp Ther, 1998 Jan, 284(1), 103 - 10 Substantially attenuated hemodynamic responses to Escherichia coli-derived vascular endothelial growth factor given by intravenous infusion compared with bolus injection; Yang R et al.; Vascular endothelial growth factor (VEGF) produces beneficial angiogenesis in animal models of coronary and peripheral ischemia . However, intravenous bolus injection of Chinese hamster ovary cell (CHO)-derived VEGF produces adverse effects on hemodynamics . The present study examined pharmacokinetic and hemodynamic responses to Escherichia coli-derived VEGF, which will be used in clinical patients, compared with responses to CHO-derived VEGF, and tested whether intravenous infusion of E . coli-derived VEGF attenuates the hemodynamic responses compared with the responses observed with intravenous bolus injection . Hemodynamic parameters were measured before and after administration of VEGF in conscious, instrumented rats . Intravenous injection of both CHO- and E . coli-derived VEGF produced a similar maximal reduction in arterial pressure, although E . coli-derived VEGF exhibited less of a depressor effect in the initial phase after injection . Either infusion or injection of E . coli-derived VEGF caused hypotension, tachycardia and reduced cardiac output and stroke volume, which were significantly attenuated when given by infusion compared with injection . The maximal hypotensive and tachycardiac responses to infusion were decreased by 50 to 60% compared with those responses observed after injection . Cardiac output was maximally reduced by 34% after injection, but only 18% after infusion . A sustained elevation in systemic vascular resistance observed after injection was avoided after infusion . Thus, the hemodynamic side effects of VEGF administration can be substantially attenuated by controlling the rate of VEGF infusion . The data indicate that infusion, instead of bolus injection, is a more appropriate regimen for VEGF administration. Appl Environ Microbiol, 1998 Jan, 64(1), 147 - 52 Detection of verotoxigenic Escherichia coli by magnetic capture-hybridization PCR; Chen J et al.; Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin {VT}-producing) Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures . The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization . The hybrids formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR . The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products . With 5, 7, or 10 h of enrichment, the limits of detection were 10(3), 10(2), and 10(6) CFU/ml, respectively, by agarose gel electrophoresis . Southern hybridization did not seem to improve the limit of the detection . When applied to food, MCH-PCR was capable of detecting 10(0) CFU of VTEC per g of ground beef with 15 h of nonselective enrichment . The results of MCH-PCR for pure cultures of VT1- and/or VT2-producing E . coli cells were in total agreement with toxin production as measured by a VT enzyme-linked immunosorbent assay. Scand J Infect Dis, 1997, 29(5), 524 - 5 Botryomycosis mimicking a liposarcoma; Slootmaekers V et al.; Botryomycosis is an atypical reaction of the host to a common bacterial infection . A case of botryomycosis mimicking a liposarcoma as suspected on clinical and radiological grounds is described . The diagnosis was made by biopsy and culture of the lesion . A review of the literature is given. Plasmid, 1997, 38(3), 188 - 201 Expression in Escherichia coli of Y5-mutant and N-terminal domain-deleted DNA gyrase B proteins affects strongly plasmid maintenance; Brino L et al.; Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA) . Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F) . Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid . This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used . Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number . By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria . Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins . In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions . Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities . Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo . This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits . This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo . Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions. Plasmid, 1997, 38(3), 174 - 9 Effect of Escherichia coli IHF mutations on plasmid p15A copy number; Hiszczynska-Sawicka E et al.; Four isogenic strains (himAhimD double mutant, himA and himD single mutants, and their wild type counterpart) harboring orip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation . The copy number of orip15A plasmid increases about four times in the himAhimD double (65-70 copies per cell) and himD single mutant cells (50-56 copies per cell) and was almost the same in himA mutant (17-18 copies per cell) and wild type cells (14-16 copies per cell) . The results suggest that HimD can form homodimers, which are functionally competent for the regulation of orip15A plasmid copy number . Complementation experiments of himAhimD double mutant cells using plasmid carrying himA and himD genes (pPLhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number of orip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant) . The absence of IHF (using himAhimD double mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid. Mamm Genome, 1998 Jan, 9(1), 32 - 7 Genomic structure and chromosomal localization of the mouse Ogg1 gene that is involved in the repair of 8-hydroxyguanine in DNA damage; Tani M et al.; 8-Hydroxyguanine (7,8-dihydro-8-oxoguanine: oh8Gua) is a damaged form of guanine induced by oxygen-free radicals and causes GC to TA transversions . Previously we isolated the hOGG1 gene, a human homolog of the yeast OGG1 gene, which encodes a DNA glycosylase and lyase to excise oh8Gua in DNA . In this study, we isolated a mouse homolog (Ogg1) of the OGG1 gene, characterized oh8Gua-specific DNA glycosylase/AP lyase activities of its product, and determined chromosomal localization and exon-intron organization of this gene . A predicted protein possessed five domains homologous to human and yeast OGG1 proteins . Helix-hairpin-helix and C2H2 zinc finger-like DNA-binding motifs found in human and yeast OGG1 proteins were also retained in mouse Ogg1 protein . The properties of a GST fusion protein were identical to human and yeast OGG1 proteins in glycosylase/lyase activities, their substrate specificities, and suppressive activities against the spontaneous mutagenesis of an Escherichia coli mutM mutY double mutant . The mouse Ogg1 gene was mapped to Chromosome (Chr) 6, and consisted of 7 exons approximately 6 kb long . Two DNA-binding motifs were encoded in exons 4 through 5 . These data will facilitate the investigation of the OGG1 gene to elucidate the relationship between oxidative DNA damage and carcinogenesis. Curr Opin Struct Biol, 1997 Dec, 7(6), 881 - 9 Aminoacyl-tRNA synthetases; Cusack S; Crystallographic studies of a number of aminoacyl-tRNA synthetases and their complexes with ATP, amino acid and cognate tRNA are leading to an increasingly detailed picture of how these sophisticated enzymes function . Within the two distinct structural classes of ten synthetases, many common features are apparent, although evolution has led to many interesting idiosyncrasies in certain enzymes . Recent advances, specifically concerning class II enzymes, have increased our knowledge of both the role of electrophiles in the mechanism of amino acid activation and cross-subunit tRNA recognition and help solve the evolutionary puzzles that have emerged from the extension of the aminoacyl-tRNA synthetase database to include Archae. Curr Opin Struct Biol, 1997 Dec, 7(6), 798 - 803 Insights into the mechanisms of homologous recombination from the structure of RuvA; Rice DW et al.; The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA . Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target . This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination. Mutat Res, 1997 Nov 27, 394(1-3), 141 - 51 Characterization of the pUC19-lacZC141 reversion system for assaying chemical mutagenesis; Ogawa H et al.; pUC19-lacZC141 DNA contains a proline codon at positions 141 to 143, where an alanine codon normally appears in the original lacZ gene . pUC19-lacZC141 DNA was produced using site-directed mutagenesis . After transfection of pUC19-lacZC141 DNA into lacZ hosts, the transformants produce white colonies on an agar plate containing X-gal and IPTG . lacZ+ revertants can be identified by their dark- and light-blue colony color against a background of non-mutant white colonies, indicating restoration of beta-galactosidase activity . N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) were used to characterize the pUC19-lacZC141 DNA reversion assay . Mutagenesis resulting from methylated DNA was examined in Escherichia coli strains JM109, BMH71-18mutS, and SURE, which differ in their repair systems for DNA damage . In JM109 and BMH71-18mutS, mostly G:C-->A:T transitions and some G:C-->C:G or G:C-->T:A transversions were observed . E . coli SURE produced, in addition, frameshift mutations (approximately 10%) . The DNA sequence analysis of 174 induced mutants indicated that the major effect of methylation is on single base-pair substitutions with a slight effect on deletion frameshifts . All mutations are consistent with miscoding of guanine or cytosine adducts or lesions . Transitions account for 158 of 165 (96%) induced base substitutions . Approximately 93% of the base-substitution mutations occurred at the expected positions 141 to 143 in the lacZ gene . The pUC19-lacZC141 assay was sufficiently sensitive to allow the detection of mutations in lacZ- hosts with different repair mechanisms . The pUC19-lacZC141 DNA reversion system will permit the assaying of other chemicals not otherwise amenable to mutagenesis studies. Biochem Biophys Res Commun, 1997 Dec 29, 241(3), 646 - 52 Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon; Abd Rahman RN et al.; Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp . KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli . Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms . Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active . Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other . These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II) . Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold . Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form . These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH. Arch Biochem Biophys, 1997 Dec 15, 348(2), 337 - 46 Turnover of recombinant human hemoglobin in Escherichia coli occurs rapidly for insoluble and slowly for soluble globin; Weickert MJ et al.; Co-expression of di-alpha-globin and beta-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1) and, under certain conditions, large amounts of insoluble globin protein . Insoluble rHb1.1 accumulated in large, amorphous inclusion bodies visible by electron microscopy . The half-life of soluble rHb1.1 in E . coli, measured by pulse-chase experiments, was at least 11 h for each globin subunit . The in vivo half-life for insoluble globin was about fivefold shorter than that for soluble rHb1.1 . We expressed significant amounts of each subunit, di-alpha-globin and beta-globin, independently with exogenous heme . The half-life of the soluble subunits alone was approximately 1 and 4 h, respectively, shorter than when they were expressed together as rHb1.1 . Individually, the insoluble di-alpha-globin subunit had a half-life of just under 1 h when exogenous heme was added, but under 20 min when exogenous heme was not provided . The greater persistence of insoluble subunits in the presence of heme indicated that heme may stabilize the insoluble globin protein . The soluble rHb1.1 persistence in the E . coli cytoplasm during long periods of stationary phase growth indicated that once assembled, rHb1.1 is extremely resistant to proteolysis. Arch Biochem Biophys, 1997 Dec 15, 348(2), 329 - 36 Molecular evolution of amphioxus fructose-1,6-bisphosphate aldolase; Kuba M et al.; The cDNA for amphioxus fructose-1,6-bisphosphate (FBP)-aldolase was isolated and its nucleotide sequence was determined . In the cDNA, there existed a probable open reading frame comprising 1080 bp; hence, 359 amino acid residues were deduced . The amino acid sequence indicates the deletion of 4 residues from N-terminus, in comparison with the sequence of FBP-aldolase isozymes from other sources . There was only one FBP-aldolase gene, and one enzyme species corresponding, in the amphioxus; this is the first report of the existence of a single FBP-aldolase species in animals . Enzymatic studies of both native and the recombinant FBP-aldolase suggest that the amphioxus enzyme belongs to an ancestral class I type which is not discovered among vertebrate aldolase isozymes. Arch Biochem Biophys, 1997 Dec 15, 348(2), 295 - 302 Expression and characterization of the catalytic domain of human phenylalanine hydroxylase; Daubner SC et al.; A truncated version of human phenylalanine hydroxylase which contains the carboxy terminal 336 amino acids was produced in Escherichia coli . It was purified by ammonium sulfate precipitation, Q-Sepharose chromatography, and hydroxyapatite chromatography . The K(m) values of the truncated enzyme for tetrahydropterin substrates are not different from those of the full-length enzyme, nor are the Vmax values . The KM value for phenylalanine is 2-fold lower for the truncate than for the full-length enzyme . The metal content of the enzyme is 0.27 mol Fe per mole enzyme subunit, and it is activated 2.3-fold by addition of ferrous ion to assays; it is not activated by addition of copper . The truncated enzyme shows no lag in activity when an assay is started with phenylalanine, while the full-length enzyme shows a marked lag. Arch Biochem Biophys, 1997 Dec 15, 348(2), 247 - 54 Kinetic characterization of recombinant human glutathione transferase T1-1, a polymorphic detoxication enzyme; Jemth P et al.; Recombinant human theta class glutathione transferase T1-1 has been heterologously expressed in Escherichia coli and a simple purification method involving immobilized ferric ion affinity chromatography and Orange A dye chromatography is described . The catalytic properties of the enzyme differ significantly from those of other glutathione transferases, also within the theta class, with respect to both substrate selectivity and kinetic parameters . In addition to 1,2-epoxy-3-(4-nitrophenoxy)propane, the substrate used previously to monitor the enzyme, human glutathione transferase T1-1 has activity with the naturally occurring phenethylisothiocyanate and also displays glutathione peroxidase activity with cumene hydroperoxide . Further, the enzyme is active with 4-nitrobenzyl chloride and 4-nitrophenethyl bromide, but shows no detectable activity with the more chemically reactive 1-chloro-2,4-dinitrobenzene . The Michaelis constant for glutathione, K(m)GSH, with 1,2-epoxy-3-(4-nitrophenoxy)propane as second substrate, is high at low pH values but decreases at higher pH values . This is mirrored in kcat/K(m)GSH which increases with an apparent pKa value of 9.0, reflecting the ionization of the thiol group of glutathione in solution . The same results are obtained with 4-nitrophenethyl bromide as electrophilic substrate, although the K(m)GSH value (0.72 mM at pH 7.5), as well as the pKa (8.1) derived from the pH dependence of kcat/K(m)GSH, are lower with this substrate . In contrast, kcat and kcat/K(m)electrophile display either a maximum or a plateau at pH 7.0-7.5, and an apparent pKa value of 5.7 was determined for the pH dependence of kcat with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates . This pKa value reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pKa value thus is lowered by the enzyme . Three differences in the cDNA as compared to the sequence previously published were found . One of these differences causes a change in the deduced amino acid sequence and involves the nucleotide triplet encoding amino acid 126, which was determined as GAG (Glu), instead of the published GGG (Gly). Virology, 1997 Dec 22, 239(2), 360 - 77 The myxoma virus M-T4 gene encodes a novel RDEL-containing protein that is retained within the endoplasmic reticulum and is important for the productive infection of lymphocytes; Barry M et al.; To investigate the contribution of the myxoma virus M-T4 gene to viral virulence, both copies of the M-T4 gene were inactivated by disruption and insertion of the Escherichia coli guanosine phosphoribosyltransferase gene . Infection of European rabbits with the recombinant M-T4-deleted virus, vMyxlacT4, resulted in disease attenuation . In contrast, infection of rabbits with vMyxlac elicited the classical features of lethal myxomatosis . A notable decrease in the number of secondary lesions in animals infected with vMyxlacT4 suggested an inability of the virus to disseminate in vivo . Infection of either a rabbit CD4+ T cell line, RL-5, or primary rabbit peripheral blood lymphocytes with vMyxlacT4- resulted in the rapid induction of apoptosis . Sequence analysis of M-T4 revealed both an N-terminal signal sequence and a C-terminal -RDEL sequence, suggesting that M-T4 resides in the endoplasmic reticulum . The M-T4 protein was found to be sensitive to endo H digestion and confocal fluorescence microscopy demonstrated that M-T4 colocalized with calreticulin, indicating that M-T4 is retained within the endoplasmic reticulum . Our results indicate that M-T4 is the first example of an intracellular virulence factor in myxoma virus that functions from within the endoplasmic reticulum and is necessary for the productive infection of lymphocytes. Virology, 1997 Dec 22, 239(2), 352 - 9 A short basic domain supports a nucleic acid-binding activity in the rice tungro bacilliform virus open reading frame 2 product; Jacquot E et al.; Little is known about the features of badnavirus open reading frame 2 products (P2) . So far, no consensus functional domain has been found in these proteins . However, they all have in common at their C-terminus amino acids which may have the capacity to bind nucleic acids . Such capacity has already been established for cacao swollen shoot virus protein P2 . We have looked for such a binding capacity of rice tungro bacilliform virus (RTBV) ORF 2 product . For this purpose, the protein was expressed as full-length or truncated versions in Escherichia coli . When used in nucleic acid-binding assays, complete RTBV P2 was shown to bind both DNA and RNA . This property may be related to a basic sequence, PPKKGIKRKYPA, localized at its C-terminus . Mutations were introduced into this sequence and revealed that four of the five basic residues, including a crucial lysine, are required for the binding to nucleic acids . Moreover, this sequence can confer binding capacity when it is fused to the N-terminus of nonbinding proteins. Gene, 1997 Dec 19, 204(1-2), 227 - 34 Cloning and functional characterization of Helicobacter pylori fumarate reductase operon comprising three structural genes coding for subunits C, A and B; Ge Z et al.; In this study, we cloned and sequenced the Helicobacter pylori genes encoding fumarate reductase (FRD) . H . pylori frdA, frdB and frdC specify polypeptides of 715, 245 and 254 aa, respectively . The deduced aa sequences of FrdA and FrdB are highly homologous to those of the corresponding subunits of Wolinella succinogenes FRD and also exhibit a significant sequence identity with other bacterial FRD and succinate dehydrogenase subunits A and B . However, H . pylori FrdC shares a striking degree of sequence identity only with W . succinogenes FrdC, which is a cytochrome b wi