Escherichia coli

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 with two haem groups . The products encoded by H . pylori frdA, frdB and frdC were overproduced in maxicells and H . pylori FrdA was characterized using an anti-E . coli FrdA serum . H . pylori FRD activity, which was measured as fumarate-dependent benzyl viologen oxidation, is membrane-associated . Inactivation of frdA led to the loss of such activity and the mutant H . pylori cells were delayed (approx . 10-20 h behind their parent cells) in entering the mid-log phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H . pylori cells in vitro . H . pylori FRD contains three subunits, of which FrdA and FrdB appear to form the catalytic dimer, whereas FrdC serves as a membrane anchor.

Gene, 1997 Dec 19, 204(1-2), 177 - 84
Display of functional thrombin inhibitor hirudin on the surface of phage M13; Wirsching F et al.; A synthetic gene for hirudin was ligated into phagemid pCANTAB5E . This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface . Similarly, hirudin variants with extensions either at their N- or C-terminus were generated . The genes were expressed in their soluble form in a non-suppressor strain of E . coli . Periplasmatic fractions were evaluated in standard thrombin inhibition assays . Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude . This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here . Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin . The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII) . The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis . Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner . Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically . Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity . These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity . Beyond that, active hirudin can be displayed on the surface of M13 phage . As a conclusion, applied molecular evolution, i.e . the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.

Gene, 1997 Dec 19, 204(1-2), 153 - 8
Cloning and characterisation of a thermostable alpha-DNA polymerase from the hyperthermophilic archaeon Thermococcus sp . TY; Niehaus F et al.; The gene for an extremely thermostable DNA polymerase has been cloned from chromosomal DNA of the recently characterised hyperthermophilic archaeon Thermococcus sp . TY by using degenerate primers derived from consensus sequences of known archaeal enzymes . The corresponding enzyme was overexpressed in Escherichia coli . Sequence comparison of the gene with related DNA polymerase genes revealed that it is interrupted by three regions showing high similarities to self-splicing protein elements, so-called "inteins" . This is the first DNA polymerase containing such a large number of self-splicing elements . To ensure an efficient expression, these regions were deleted on the DNA level . The resulting protein showed DNA polymerase and 3'-5' exonuclease activity at high temperatures, being a promising candidate for use in the polymerase chain reaction (PCR).

Biochim Biophys Acta, 1997 Dec 5, 1343(2), 278 - 86
Functional implications of disulfide bond, Cys45-Cys50, in recombinant prochymosin; Zhang Y et al.; Bovine prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210 and Cys250-Cys283 (pepsin numbering) . We have demonstrated that Cys250-Cys283 is not intimately involved in the catalytic mechanism of chymosin but is required for the correct refolding of prochymosin . To elucidate the functional implications of another disulfide bond of Cys45-Cys50, these two cysteine residues were replaced separately or simultaneously by site-directed mutagenesis . Like the wild-type prochymosin all the seven mutants generated (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C50S, C45A/C50S) exhibit the activity of autocatalytic activation after refolding, indicating that Cys45-Cys50 is dispensable in prochymosin refolding . Spectroscopic analyses and urea-induced denaturation studies of prochymosin and four mutants tested (C45A, C50A, C45A/C50A, C45D/C50S) show that: (1) they share similar far-UV CD spectra and similar fluorescence emission spectra; (2) mutation results in a perturbance of tryptophan environment and somewhat destabilization of prochymosin conformation . However, quenching studies reveal that the only one tryptophan residue inaccessible to acrylamide is still buried in the mutated molecules . All these results suggest that the overall conformation of prochymosin is maintained after mutation . As for the enzymatic properties of pseudochymosin, the activation product of prochymosin, it has been found that elimination of Cys45-Cys50 causes a marked drop of thermostability and an alteration of substrate specificity.

Biochim Biophys Acta, 1997 Dec 5, 1343(2), 139 - 43
Ion-binding properties of recombinant S100beta and two derivatives with either an inactivated Ca2+ site II or a normalized Ca2+ site I; Durussel I et al.; S100beta contains one unusual and one canonical Ca2+-binding motif . In this study, we measured Ca2+-binding and ensuing conformational changes of recombinant S100beta (rS100beta) and of two mutant forms in which either the canonical loop was inactivated (NoEF) or the unusual one replaced by a canonical one (Caloops) . Caloops binds two Ca2+ per monomer with a 3-fold higher affinity than rS100beta; the affinity of NoEF was too low for accurate direct determination . All three proteins bind 3-4 Zn2+ per monomer . Tyrosine 17 fluorescence spectra showed a decrease of intensity upon binding of Ca2+ to the three proteins and an increase upon binding of Zn2+ to rS100beta and NoEF but not in Caloops . The fluorescence change as a function of the Ca2+ concentration yielded half-maximal changes ({Ca2+}0.5) at 1.7, 11.3 and 0.55 mM free Ca2+ for rS100beta NoEF and Caloops, respectively . Our data demonstrate that in S100beta alterations in one site can affect the Ca2+ binding properties of the other site.

Nephron, 1997, 77(4), 412 - 6
Renal scarring by mannose-sensitive adhesin of Escherichia coli type 1 pili; Mizunoe Y et al.; Most Escherichia coli isolates from patients with pyelonephritis possess both pap (mannose-resistant) pili and type 1 (mannose-sensitive) pili . In the experimental pyelonephritis model of rats, the mannose-sensitive-piliated strain caused severe renal scarring, whereas the mannose-resistant or nonpiliated strain did not . Type 1 pili consist of several subunits; one major subunit and other minor subunits . One of the minor subunits, adhesin, is responsible for mannose-sensitive adhesion to eukaryotic cells . The role of adhesin was examined in scar formation after infection with a newly constructed adhesin-deficient mutant which has pilus structure but cannot agglutinate guinea pig erythrocytes . A mutant plasmid, pYMZ84, containing a deletion in the adhesin gene of type 1 pili, failed to agglutinate guinea pig erythrocytes even though the bacteria expressed pili morphologically indistinguishable from those produced by plasmid pSH2, carrying the intact genes for the type 1 pili . E . coli harboring pYMZ84 caused negligible or minimal renal scarring, whereas E . coli harboring pSH2 caused severe renal scarring in rats . These data suggest that the mannose-sensitive adhesin of type 1 pili stimulates renal scarring.

Free Radic Biol Med, 1998 Jan 15, 24(2), 370 - 6
Purification of mitochondrial thioredoxin reductase and its involvement in the redox regulation of membrane permeability; Rigobello MP et al.; The isolation to purity of a rat liver mitochondrial thioredoxin reductase is reported . The mitochondrial enzyme shows a chromatographic behavior different from that of the cytosolic enzyme . The purified enzyme, after sodium dodecylsulfate-polyacrylamide gel electrophoresis, yields a single band with a molecular weight of approximately 54 kDa . The apparent Km for E . coli thioredoxin is about 13 microM, while the apparent Km for 5,5'-dithiobis (2-nitrobenzoic acid) is 530 microM, values comparable to those reported for the cytosolic enzyme . Mitochondrial thioredoxin reductase, in addition to its natural substrate thioredoxin, is also able to reduce chemically unrelated compounds such as 5,5 '-dithiobis (2-nitrobenzoic acid), selenite, and alloxan; the enzyme is inhibited by classical inhibitors of the cytosolic enzyme such as 1-chloro-2,4-dinitrobenzene and 13-cis-retinoic acid . A strong inhibitory action is also elicited by Mn2+ and Zn2+ ions . Thiol status appears critically involved in the control of membrane permeability and, therefore, a thiol/disulfide transition involving reduced pyridine nucleotides, matrix soluble thiols, and inner membrane thiols appears to play a fundamental role . The potential role of thioredoxin/thioredoxin reductase system in the control and redox regulation of the mitochondrial membrane permeability, is discussed.

Nutrition, 1997 Nov-Dec, 13(11-12), 959 - 64
Recombinant human tumor necrosis factor and recombinant murine interleukin-1 alter the binding of Escherichia coli to intestine, mucin glycoprotein, and the HT29-C1 intestinal cell line; Wanke CA et al.; Little is known about the effects of cytokines at the intestinal mucosal surface on the adherence of bacteria . We examined the effects of recombinant tumor necrosis factor (TNF) and interleukin-1 (IL-1) on the adherence of various strains of Escherichia coli to intestinal mucosa in vivo and in in vitro models . We studied the effects of TNF or IL-1 injected intraperitoneally on the ability of a rabbit enteric pathogen (RDEC-1) and a nonpathogenic E . coli (1392-) to colonize rabbit small bowel and found that there was a trend toward increased colonization by the RDEC-1 organisms in the TNF-treated rabbits, and a significant increase in colonization by the RDEC-1 organisms in the IL-1-treated animals (P < 0.01) . Both TNF and IL-1 altered the density and the level of glycosylation of the small bowel mucus glcoprotein purified from the treated and untreated rabbits, and TNF treatment significantly increased the number of bacteria bound by this purified mucin (P < 0.01 for all strains) . HT29-C1 intestinal cells in tissue culture were also grown in media with TNF or IL-1 and used in bacterial binding assays . The cells provided with media with 50 pg/mL of either cytokine bound significantly more of the three bacterial strains than cells in untreated media (P < 0.01 for all strains) . The cytokines TNF and IL-1 have the potential to alter bacterial adherence to intestinal mucosa in vivo and in vitro; additional studies to clarify the role that these alterations in adherence may play in the clinical syndromes characterized by increased levels of intestinal cytokines are warranted.

J Immunol Methods, 1997 Oct 13, 208(1), 35 - 42
A modified immuno-polymerase chain reaction for the detection of beta-glucuronidase from Escherichia coli; Chang TC et al.; A modified immuno-polymerase chain reaction (immuno-PCR) for the detection of E . coli beta-glucuronidase (GUD) is described . Flexible polycarbonate microtiter plates (Biozyme, Landgraaf) with 96 V-bottomed wells were used throughout all steps including the antigen-antibody reaction and polymerase chain reaction . The plates were coated with anti-GUD antibodies to capture the antigen, which was then detected using biotinylated anti-GUD antibodies . Following this, avidin was used to bridge the biotinylated antibodies and biotinylated lamda phage DNA, which was amplified by PCR to produce a product of 500 nucleotides . Following optimization, the detection limit of the immuno-PCR for GUD was 1 x 10(-17) g/ml (or 5 x 10(-19) g/well); this is equivalent to two GUD molecules in a sample solution of 50 microliters . The method was used to detect GUD in a cell extract of E . coli, and it was found that the enzyme released from a single E . coli cell in a solution of 10 l could be detected . So far, this is the most sensitive method ever published for the detection of an antigen . In addition to high sensitivity, the present protocol is capable of automation.

Invest Ophthalmol Vis Sci, 1998 Jan, 39(1), 64 - 9
Interleukin-6 does not mediate endotoxin-induced uveitis in mice: studies in gene deletion animals; Rosenbaum JT et al.; PURPOSE: Interleukin-6 (IL-6) has been strongly implicated in anterior uveitis based on its presence in aqueous humor from infected eyes and its inflammatory effects when injected intravitreally into rats . We used IL-6-deficient mice to test further the hypothesis that IL-6 contributes to the development of endotoxin-induced uveitis . METHODS: Uveitis was scored by histologic analysis of C3H/HeN mice 24 hours after intravitreal injections of up to 200 ng of recombinant murine IL-6 . Uveitis was similarly measured in IL-6-deficient mice and congenic controls 24 hours after intravitreal injection of 250 ng of Escherichia coli endotoxin . Reverse transcription-polymerase chain reaction was used to detect mRNAs for several cytokines at 3 hours postinjection . The IL-6 concentration in aqueous humor samples was determined with a bioassay using the murine B9 plasmacytoma cell line . RESULTS: Direct injection of IL-6 did not induce uveitis . Mice genetically deficient in IL-6 developed endotoxin-induced uveitis that was comparable or more severe than congenic control mice . Compensatory changes in the expression of mRNA for other cytokines were not detected in irises from the IL-6-deficient mice . In IL-6-competent mice that received bilateral endotoxin injections, no correlation was found between the number of infiltrating cells in one eye and the IL-6 concentration in the aqueous humor of the contralateral eye . CONCLUSIONS: In marked contrast to previous conclusions with rats, IL-6 was not sufficient for inducing uveitis in mice . Additionally, IL-6 was not necessary for the development of uveitis subsequent to intravitreal injection of endotoxin in mice.

Chin J Biotechnol, 1997, 13(3), 143 - 52
Cloning and sequencing of draTG genes and their downstream region of Azospirillum brasilense Yu62; Ma L et al.; An 8-kb fragment was cloned by probing the gene library of Azospirillum brasilense Yu62 with the 4.0-kb draTG fragment of A . brasilense Sp7 . DNA hybridization of this fragment demonstrated that draTG genes were located in a 3.0-kb EcoR I-Kpn I fragment, and were contiguous to the nifH gene . This 3.0-kb fragment was completely sequenced on both strands . Sequence analysis of the fragment revealed that it included the full-length draTG genes and two ORFs downstream of draG (ORF3 and incomplete ORF4) . The draTG and downstream ORF3 are presumed to be cotranscribed as a single operon . Promoter element analysis of the sequenced region showed that there were some elements of the sigma 54-dependent promoter (DPEs and UASs) in the upstream region of draG and ORF3 . This suggests that the draG and ORF3 might be transcribed independently in addition to being cotranscribed with draT . Both the DNA and amino acid sequences of the draTG genes from A . brasilense Yu62 were compared with those of other nitrogen-fixing bacteria . The results showed the draTG genes were highly conservative . There were only a few changes among strains and/or species . Homology analysis of ORF revealed that the ORF3 immediately downstream of draG was homologous not only with the ORF at the same position in Rhodospirillum rubrum and A . lipoferum, but also with the ORF14 of Azotobacter vinelandii and the ORF4 showed extensive similarity to yafJ of Escherichia coli.

J Muscle Res Cell Motil, 1997 Dec, 18(6), 631 - 41
Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants; Lescaudron L et al.; The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation . The appearance of desmin occurs at the very beginning of myogenesis . The transgenic desmin nls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase . The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced . We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice . Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments . In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant . In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19 . Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression . In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix . Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle.

FEBS Lett, 1997 Dec 1, 418(3), 297 - 300
The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution; Quintas A et al.; Amyloid fibril formation and deposition are the basis for a wide range of diseases, including spongiform encephalopathies, Alzheimer's and familial amyloidotic polyneuropathies . However, the molecular mechanisms of amyloid formation are still poorly characterised . In certain forms of familial amyloidotic polyneuropathy (FAP), the amyloid fibrils are mostly constituted by variants of transthyretin (TTR) . V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of amyloidosis . Here, we report gel filtration chromatography experiments to characterise the aggregation states of WT-, V30M-, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, in solution, at nearly physiological pH . These studies show that all four protein tetramers dissociate to monomer upon dilution, in the submicromolar range, at pH 7.0 . The amyloidogenic proteins V30M- and L55P-TTR show a complex equilibrium between monomers, tetramers and high molecular weight aggregate species . These aggregates dissociate directly to monomer upon dilution . This study shows that the tendency to form aggregates among the four studied proteins correlates with their known amyloidogenic potential . Thus, the amyloidogenic mutations could perturb the structure and/or stability of the monomeric species leading initially to the formation of soluble aggregates and at a later stage to insoluble amyloid fibrils.

Eur J Biochem, 1997 Dec 1, 250(2), 578 - 82
Structural constraints in protein engineering--the coenzyme specificity of Escherichia coli isocitrate dehydrogenase; Chen R et al.; In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD {Chen, R., Greer, A . & Dean, A . M . (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc . Natl Acad . Sci . USA 92, 11666-11670} . Here, we explore alternative means to generate NAD dependence in the NADP-dependent scaffold of Escherichia coli IDH . The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity . Mutations in the 316-325 loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 A distant . Conformational changes seen upon substituting Cys332-->Tyr are not directly involved with improving activity . Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses . We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of rational design of active-site residues based on X-ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.

Eur J Biochem, 1997 Dec 1, 250(2), 567 - 77
Kinetics of membrane-bound nitrate reductase A from Escherichia coli with analogues of physiological electron donors--different reaction sites for menadiol and duroquinol; Giordani R et al.; We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered beta subunits . To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified alpha beta gamma complex . With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant . The results as a whole are consistent with a substituted-enzyme mechanism for the reduction of nitrate by the quinols . Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition . Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo . To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with beta subunits that lack an Fe-S centre . The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity . The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity . The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of +80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical.

Eur J Biochem, 1997 Dec 1, 250(2), 403 - 10
Dof DNA-binding domains of plant transcription factors contribute to multiple protein-protein interactions; Yanagisawa S; Dof proteins are a family of plant transcription factors that have a strongly conserved DNA-binding domain, designated the Dof domain . This domain has the potential to form a single zinc finger . This report describes the self-association of a maize Dof protein, Dof1 (previously designated MNB1a) . Affinity chromatography revealed that Dof1 also interacted with another maize Dof protein, Dof2, as well as with high-mobility-group (HMG) protein 1 . Results of mapping of the region required for the protein-protein interactions of Dof1 suggested that these interactions may be mediated by the Dof domain . When gel mobility shift assays were performed with purified recombinant Dof proteins, homomeric and heteromeric complexes of Dof proteins on DNA were detected . It seems possible that formation of complexes of different Dof proteins through direct protein-protein interactions might be involved in the regulation of transcription . Evidence is also presented that HMG1 has an effect on the binding of Dof1 to DNA . Therefore, it appears that the Dof domain is a multifunctional domain that is involved not merely in binding to DNA but also in multiple protein-protein interactions.

Eur J Biochem, 1997 Dec 1, 250(2), 296 - 302
Information transfer in multienzyme complexes--2 . The role of Arg64 of Chlamydomonas reinhardtii phosphoribulokinase in the information transfer between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase; Avilan L et al.; A mutant phosphoribulokinase has been isolated from the 12-2B mutant of Chlamydomonas reinhardtii . In this mutant, Arg64 has been replaced by Cys . The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity . Neither of these enzymes is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase . The phosphoribulokinase gene has been expressed in Escherichia coli . The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast . Three mutants have been generated by site directed mutagenesis . Arg64 has been replaced by Ala, Lys or Glu . With the exception of the latter, the two other mutants, {A64}phosphoribulokinase and {K64}phosphoribulokinase, are active when they are reduced, and nearly totally inactive in their oxidized state . Their activity, however, is decreased relative to that of the native, or to that of the wild-type recombinant phosphoribulokinase . Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding values obtained for the wild-type, the native or the recombinant enzyme . Whereas the {A64}phosphoribulokinase is unable to form a complex with glyceraldehyde-3-phosphate dehydrogenase, {K64}phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex . The oxidized mutant {K64}phosphoribulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the corresponding wild-type enzyme . Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase . As this residue also appears to be important for catalytic activity, it may be tempting to consider that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.

Eur J Biochem, 1997 Dec 1, 250(2), 260 - 8
Expression and characterisation of the homodimeric E1 component of the Azotobacter vinelandii pyruvate dehydrogenase complex; Hengeveld AF et al.; We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli . Cloned E1p can be used to fully reconstitute complex activity . The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly . This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH . Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors . These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component . Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed . The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity . E1p was protected against proteolysis by E2p . The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.

FEBS Lett, 1997 Dec 15, 419(2-3), 206 - 10
Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells; Baldet P et al.; Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli . To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E . coli overexpression strain . The purified A . thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with {2Fe-2S} clusters . Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.

Crit Care Med, 1998 Jan, 26(1), 138 - 41
ONO-5046, an elastase inhibitor, attenuates liver mitochondrial dysfunction after endotoxin; Kudoh A et al.; OBJECTIVE: To investigate the effect of ONO-5046, an elastase inhibitor, on liver mitochondrial dysfunction after endotoxin administration . DESIGN: Prospective, randomized, controlled animal study . SETTING: Research laboratory . SUBJECTS: Male Hartley guinea pigs . INTERVENTIONS: Endotoxin shock was induced by intravenous infusion of Escherichia coli lipopolysaccharide endotoxin (50 mg/kg) . Six guinea pigs were treated with endotoxin and saline . Twenty-four guinea pigs received 5, 10, and 30 mg/kg/hr of ONO-5046 after endotoxin administration . Six guinea pigs received only saline . MEASUREMENTS AND MAIN RESULTS: We measured oxygen uptake in state 3 (substrate and adenosine 5'-diphosphate {ADP}) and state 4 (excess substrate, no ADP), as well as the respiratory control ratio (state 3/state 4), adenosine 5'-diphosphate/oxygen ratio (ADP/O), and arterial ketone body ratio . ONO-5046 was dose dependently effective in liver mitochondrial oxidative phosphorylation, such as oxygen uptake in stage 4, respiratory control ratio, adenosine triphosphate synthesis, ADP/O, and arterial ketone body ratio when ONO-5046 was started 30 mins after endotoxin . The administration of 30 mg/kg/hr of ONO-5046 improved mean blood pressure, which had decreased after endotoxin . CONCLUSION: ONO-5046 attenuates the endotoxin-induced liver mitochondrial dysfunctions that may be related to increased liver blood flow.

Cell, 1997 Dec 26, 91(7), 995 - 1005
The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch; Gradia S et al.; The mechanism of DNA mismatch repair has been modeled upon biochemical studies of the E . coli DNA adenine methylation-instructed pathway where the initial recognition of mismatched nucleotides is performed by the MutS protein . MutS homologs (MSH) have been identified based on a highly conserved region containing a Walker-A adenine nucleotide binding motif . Here we show that adenine nucleotide binding and hydrolysis by the human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch . The hMSH2-hMSH6 complex is ON (binds mismatched nucleotides) in the ADP-bound form and OFF in the ATP-bound form . These results suggest a new model for the function of MutS proteins during mismatch repair in which the switch determines the timing of downstream events.

Cell, 1997 Dec 26, 91(7), 939 - 47
PDZ-like domains mediate binding specificity in the Clp/Hsp100 family of chaperones and protease regulatory subunits; Levchenko I et al.; ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity . Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins . The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions . Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.

Gene, 1997 Nov 20, 202(1-2), 69 - 74
Molecular cloning, sequencing and expression of cDNA encoding human trehalase; Ishihara R et al.; A complete cDNA clone encoding human trehalase, a glycoprotein of brush-border membranes, has been isolated from a human kidney library . The cDNA encodes a protein of 583 amino acids with a calculated molecular weight of 66,595 . Human enzyme contains a typical cleavable signal peptide at amino terminus, five potential glycosylation sites, and a hydrophobic region at carboxyl terminus where the protein is anchored to plasma membranes via glycosylphosphatidylinositol . The deduced amino acid sequence of the human enzyme showed similarity to sequences of the enzyme from rabbit, silk worm, Tenebrio molitor, Escherichia coli and yeast . Northern blots revealed that human trehalase mRNA of approx . 2.0 kb was found mainly in the kidney, liver and small intestine . Expression of the recombinant trehalase in E . coli provided a high level of the enzyme activity . The isolation and expression of cDNA for human trehalase should facilitate studies of the structure of the gene, as well as a basis for a better understanding of the catalytic mechanism.

Biochim Biophys Acta, 1997 Nov 20, 1354(3), 211 - 8
Cleavage effect of oligoribonucleotides substituted at the cleavage sites with modified pyrimidine- and purine-nucleosides; Hosono K et al.; The precursor of an RNA molecule from T4-infected E . coli cells (p2Spl RNA) has the capacity to cleave itself at specific positions {UpA (139-140) and CpA (170-171)}, within a putative loop and stem structure . This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents . In order to determine the influence of the pyrimidine and purine bases on these sequence-specific cleavage reactions, we studied the cleavage reactions of hairpin loop RNAs substituted at the cleavage sites with modified pyrimidine- and purine-nucleosides . The cleavage was affected by the 2'-hydroxyl groups and the bases of the pyrimidines, and the 6-amino group of the purine.

Brain Res Mol Brain Res, 1997 Nov, 51(1-2), 179 - 87
In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis; Grifman M et al.; Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro . This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors . Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo . Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation . Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites . In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents . Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Brain Res Mol Brain Res, 1997 Nov, 51(1-2), 170 - 8
Induction of interleukin-1 beta-converting enzyme (ICE) in murine microglia by lipopolysaccharide; Yao J et al.; Interleukin-1beta (IL-1beta) synthesized in the brain is thought to be involved in the behavioral response to LPS . In this study, we examined in microglia the ability of LPS to induce interleukin-1 beta-converting enzyme (ICE), the protease responsible for processing proIL-1beta to its mature biologically active form . The murine microglial cell line, N13, and primary microglia which had been previously isolated from brains of 2-d-old endotoxin-responsive C3H/HeOuJ mice and endotoxin-resistant C3H/HeJ mice, were cultured in the presence of various concentrations of LPS . Oligonucleotide primers for ICE and GAPDH were used in RT-PCR to determine the levels of ICE mRNA in microglia . ICE mRNA was constitutively expressed in N13 cells and primary microglia from both C3H/HeOuJ and C3H/HeJ mice . Upon exposure to LPS, ICE mRNA levels were markedly elevated in N13 cells and in microglia from C3H/HeOuJ mice, but not in microglia from endotoxin-resistant C3H/HeJ mice . Western immunoblotting was used to determine if the increase in ICE mRNA induced by LPS was accompanied by an increase in ICE protein . A modest increase in immunoreactivity for the p45 ICE precursor protein was evident in N13 cells and primary microglia from C3H/HeOuJ mice following exposure to LPS . Because corticosteroids inhibit the synthesis and secretion of IL-1beta, in a second experiment microglia were treated with LPS in the presence of dexamethasone (0, 10 and 100 microM) . Dexamethasone inhibited the LPS-induced increase in ICE mRNA and protein in microglia from C3H/HeOuJ mice . These results indicate that LPS stimulates microglia to express ICE . That dexamethasone inhibited the expression of ICE, suggests that corticosteroids regulate the secretion of IL-1beta by microglial cells, in part, by regulating the expression of ICE.

Mol Microbiol, 1997 Nov, 26(4), 821 - 31
Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli; Mourez M et al.; We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli . We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses . The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane . Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter . Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF . These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery . Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.

Mol Microbiol, 1997 Nov, 26(4), 799 - 808
Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein; Escolar L et al.; The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+-responsive Fur (ferric uptake regulation) protein has been investigated . In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences . However, binding to the primary target site suffices for the entire repression effect . Comparison of transcription patterns generated with run-off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers of the promoter region was fully prevented by Fur-Mn2+ bound to its primary site . Similarly, promoter-bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur-Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription . The high affinities of either protein for the promoter produce, in practice, a first-come, first-served effect that helps the system to respond instantly to changes in the iron status of the cells.

Mol Microbiol, 1997 Nov, 26(4), 767 - 77
In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site; Espeli O et al.; The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs) . These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs) . Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site . Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated . In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements . The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs . A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions . The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs . Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase . DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

Mol Microbiol, 1997 Nov, 26(4), 747 - 53
Secondary structures and starvation-induced frameshifting; Atkinson J et al.; We have examined the effect of a downstream secondary structure (the stem-loop sequence found downstream on the MMTV gag-pro frameshift site) on frameshifting at a bacterial shifty site (U UUC AUA) that responds strongly to a isoleucine-tRNA limitation . Our findings are as follows: (i) the downstream stem-loop has little effect on frameshifting in growing, unstarved cells; (ii) the stem-loop increases the frameshifting effect of isoleucine-tRNA limitation about fourfold, and this synergism is maximal with a distance of 5-9 nucleotides between the 'hungry' AUA codon and the stem-loop; and (iii) a stem-loop of different sequence at the same position has the same effect.

Mol Microbiol, 1997 Nov, 26(4), 687 - 97
CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over-replication; Katayama T et al.; We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli . At 30 degrees C, the dnaAcos mutant causes over-replication of the chromosome, and colony formation is inhibited . We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited . This inhibition was independent of SfiA, an inhibitor of the septum-forming protein, FtsZ . To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold-sensitive phenotype of the dnaAcos mutant . One of these proved to be a previously unknown gene, which we named cedA . This gene encoded a 12 kDa protein and resided at 38.9min on the E . coli genome map . A multicopy supply of the cedA gene to the dnaAcos cells did not repress over-replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number . Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30 degrees C . We propose that over-replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control . In the dnaA+ background, cedA also seems to affect cell division.

Mol Microbiol, 1997 Nov, 26(4), 655 - 64
DNA-binding domain mutants of sigma-N (sigmaN, sigma54) defective between closed and stable open promoter complex formation; Oguiza JA et al.; The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form . Activation by enhancer binding positive control proteins results in the formation of an open promoter complex . In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain . Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function . Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained . The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro . However, they were defective in response to activator in vitro . They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes . The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition . The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.

Mol Microbiol, 1997 Nov, 26(4), 631 - 41
Transcriptional control of the yeast acetyl-CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and the pleiotropic repressor UME6; Kratzer S et al.; The ACS1 gene, encoding one out of two acetyl-CoA synthetase isoenzymes of Saccharomyces cerevisiae, is strictly regulated at the transcriptional level by the carbon source of the medium . While ACS1 is poorly expressed in the presence of a high glucose concentration, a several hundred-fold derepression occurs with ethanol as the sole carbon source or under conditions of sugar limitation . The molecular mechanism responsible for the carbon source control of ACS1 turned out to be highly complex . A carbon source-responsive element (CSRE), previously identified upstream of gluconeogenic structural genes, and a binding site of the alcohol dehydrogenase regulator, Adr1p, together mediate about 80% of the derepressed gene activity . Binding of Adr1p synthesized by Escherichia coli to the ACS1 control region was shown by an electrophoretic mobility shift assay . In addition to these activating elements, two URS1 motifs confer negative control on the ACS1 promoter . The URS1 element was found to be a constitutive repression site, which is most effective from a downstream position with respect to an upstream activation site (UAS) . In a mutant lacking the URS1-binding factor, Ume6p, ACS1 expression was partially glucose insensitive . Ume6p must counteract transcription factors that are constitutively active . Site-directed mutagenesis of Abf1p binding sites in the ACS1 promoter significantly reduced gene expression in the ume6 mutant, grown under repressing conditions . Thus, a functional balance of the pleiotropic positive factor Abf1p and the negative factor Ume6p is in part responsible for glucose repression of ACS1 . The combined influence of the regulated UAS elements, CSRE and Adr1p binding site, mediates a strong increase in ACS1 expression under derepressing conditions.

Fold Des, 1997, 2(6), 357 - 61
X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain; Uson I et al.; BACKGROUND: Immunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation . Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation . Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts . RESULTS: Immunoglobulin REIv is the variable domain of a human kappa light chain . The disulfide-free variant REIv-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 A resolution) . The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REIv-T39K . This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge . The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond . There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant . CONCLUSIONS: Immunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach . First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines . Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E . coli cell . Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.

Curr Eye Res, 1997 Dec, 16(12), 1239 - 44
Cloning and expression of bovine corneal antigen cDNA; Gottsch JD et al.; PURPOSE: A cornea-associated antigen (CO-Ag) has been found to be the target for autoantibodies in patients with Mooren's ulcer . The study goals were to isolate a full-length clone encoding CO-Ag from a bovine corneal cDNA library and to express this clone in Escherichia coli (E . coli) . METHODS: A DNA fragment of CO-Ag was generated, using unique oligonucleotide primers and reverse transcription polymerase chain reaction . This fragment was used as a probe to obtain cDNA clones from a bovine corneal cDNA library . The clone with the longest cDNA insert was selected for sequence analysis . Expression of the CO-Ag protein in E . coli was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) . The bacterially-produced CO-Ag was partially purified by calcium (Ca2+)-dependent hydrophobic interaction chromatography . RESULTS: The 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 . The cDNA and deduced amino acid sequence of CO-Ag are completely identical to a S-100 protein, bovine calgranulin C . The cDNA was expressed in E . coli as a fusion protein consisting of 583 N-terminal amino acids of beta-galactosidase (beta-gal), 91 amino acids of CO-Ag, and possibly a number of additional N-terminal and C-terminal residues . The bacterially produced CO-Ag was fully functional with respect to hydrophobic interaction with phenyl-Sepharose matrix for its isolation . The fusion protein was recognized by antiserum raised against bovine CO-Ag protein on Western blots . CONCLUSIONS: The isolation and analysis of a cDNA clone containing the complete coding sequence of the CO-Ag protein and the expression of the CO-Ag protein in E . coli is reported . The availability of a CO-Ag cDNA probe and larger quantities of the CO-Ag protein should aid in elucidating the possible pathogenic role of CO-Ag in Mooren's ulcer.

Plant Mol Biol, 1997 Dec, 35(6), 763 - 75
Isolation and characterization of a dnaK genomic locus in a halotolerant cyanobacterium Aphanothece halophytica; Lee BH et al.; We cloned and characterized a genomic locus encoding a distinct member of the DnaK/Hsp70 family of molecular chaperones, dnaK1, from the halotolerant cyanobacterium Aphanothece halophytica . Co-expression of dnaK1 with a plant plastocyanin precursor in Escherichia coli resulted in a dramatic increase in the solubility of the plant protein . This indicates that A . halophytica dnaK1 encodes a functional protein possessing functions assigned to DnaK/Hsp70 chaperone members . The A . halophytica dnaK1 locus also encompasses grpE and dnaJ homologue genes in the order grpE-dnaK1-dnaJ . The transcript content of dnaK1 increased strongly upon subjecting cyanobacterial cells to heat stress . Northern analyses using specific probes indicated transcript species of 2.8, 2.2, 1.3, and 0.7 kb, which comprised grpE-dnaK1, dnaK1, dnaJ, and grpE, respectively . This indicates the presence of different terminators and/or heat stress promoters in this locus . Both dnaK1 transcript and protein levels increased in cyanobacterial cells transferred to hyperosmotic environments, suggesting a role of DnaK1 in the protection and/or recovery of A . halophytica from this particular stress.

Plant Mol Biol, 1997 Dec, 35(6), 735 - 48
Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization; Epelbaum S et al.; We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags . A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5' and 3' Race . DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming) . Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively . No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein . The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy . 5' sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box . The 3'-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA . The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize . This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.

Plant Mol Biol, 1997 Dec, 35(6), 723 - 34
Nitrogen availability and electron transport control the expression of glnB gene (encoding PII protein) in the cyanobacterium Synechocystis sp . PCC 6803; Garcia-Dominguez M et al.; The glnB gene from Synechocystis sp . PCC 6803 that encodes the PII protein has been cloned by heterologous hybridization using the corresponding glnB gene from Synechococcus sp . PCC 7942 . An ORF of 336 nucleotides appeared that potentially coded for a protein of 112 amino acid residues (M(r) 12,397) . The deduced amino acid sequence revealed a high identity (higher than 80%) with its cyanobacterial counterparts and a basal level of identity (close to 60%) with other PII proteins . A single mRNA of about 680 nucleotides was found under all growth conditions studied . glnB gene expression was specifically activated under nitrogen deprivation (a 10-fold increase respect to nitrogen-replete conditions) . No differences in glnB mRNA levels were observed when using nitrate or ammonium as nitrogen sources . Amount of glnB mRNA decreased to undetectable levels when transferring cells to the dark, but effect was avoided by adding glucose to the culture medium . Primer extension analysis and band-shift assays indicated that expression of the glnB gene, elevated under nitrogen deprivation, might lie under the control of the nitrogen transcriptional regulator NtcA, although constitutive levels of expression were also detected from a sigma 70-dependent Escherichia coli-like promoter.

Plant Mol Biol, 1997 Dec, 35(6), 701 - 9
Induction of a ribosome-inactivating protein upon environmental stress; Rippmann JF et al.; Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD) . One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress . RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase . Rip1 encodes a reading frame of 289 amino acids (molecular mass 32,652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases) . The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance . RT-DD also detected mRNA for pyruvate, orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant {16} . RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes . The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.

Virology, 1997 Dec 8, 239(1), 36 - 45
Equine herpesvirus 1 mutants devoid of glycoprotein B or M are apathogenic for mice but induce protection against challenge infection; Neubauer A et al.; Equine herpesvirus 1 (EHV-1) mutants devoid of the open reading frames (ORFs) of either glycoprotein (g) B or M were constructed and tested for their immunogenic potential in a murine model of EHV-1 infection . The mutant viruses were engineered using the virulent EHV-1 strain RacL11 or the modified live vaccine strain RacH by inserting the Escherichia coli LacZ gene into the viral ORFs . RacL11-infected mice showed signs typical of an EHV-1 infection, whereas mice infected with the EHV-1 gB- or gM-negative mutants or with RacH did not develop disease . No difference in the pathogenic potential of RacL11 gB- and gM-negative viruses was observed after application of either phenotypically completed or negative viruses . However, revertant RacL11 viruses in which the gB or gM gene had been restored caused EHV-1-related symptoms that were indistinguishable from those induced by RacL11 . Mice that had been immunized with phenotypically negative gB- and gM-deficient EHV-1 were challenged with the RacL11 virus 25 days after immunization . Mock-immunized mice developed EHV-1 disease and high virus loads in their lungs were observed . In contrast, mice developed not exhibit EHV-1-caused disease . It was concluded (i) that deletion of either gB or gM abolished the virulence of strain RacL11 and (ii) that immunization with gB- or gM-negative EHV-1 elicited a protective immunity that was reflected by both virus-neutralizing antibodies and EHV-1-specific T-cells in spleens of immunized mice.

FEBS Lett, 1997 Dec 8, 419(1), 37 - 40
Cloning and transcriptional regulation of the gene encoding the vacuolar/H+ ATPase B subunit of Dictyostelium discoideum; Bracco E et al.; The main function of vacuolar H+ ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments . We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+ ATPase (vatB) and analyzed its transcriptional regulation . The deduced protein comprises 493 amino acids with a calculated molecular mass of 54874 Da . The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences . The protein is encoded by a single gene in the Dictyostelium genome . The gene is maximally expressed during growth and it decreases during the first hours of development . Gene expression is rapidly enhanced by phagocytosis, but not by fluid-phase endocytosis . Acidic and alkaline conditions affect vatB gene expression differently.

Biol Chem, 1997 Nov, 378(11), 1381 - 5
Medium-chain acyl CoA dehydrogenase: evidence for phosphorylation; Macheroux P et al.; Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (pkMCADH) and originating from mitochondria carries a phosphate group as demonstrated by 31P-NMR-spectroscopy and chemical analysis . Two broad resonances at -6.3 and -8 ppm are observed and are assigned to the pyrophosphate group of the cofactor FAD . A third, narrow resonance at 4.65 ppm indicates the presence of a phosphomonoester residue . Chemical analysis of intact pkMCADH shows the presence of 3 +/- 0.3 phosphates, those of FAD and of an additional covalently attached phosphate . With recombinant, human wild type MCADH expressed in and purified from E . coli only the two FAD phosphates (2 +/- 0.35) are found . Similarly, pkMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 +/- 0.4 phosphates . The covalently bound phosphate can be hydrolyzed by phosphatase and subsequently removed by dialysis . The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron acceptors such as pig electron transferring flavoprotein . However, phosphorylation has a marked effect on protein solubility which is +5-fold lower for the dephosphorylated protein.

Biol Chem, 1997 Nov, 378(11), 1353 - 60
Functional activity and developmental regulation of DdRBP1, a RNA binding protein in Dictyostelium discoideum; Oberosler P et al.; In an attempt to find potential components of natural antisense mechanisms in Dictyostelium, we investigated RNA binding protein (RBD) genes of the RNP-CS family . RBD proteins can enhance hybridization of complementary RNAs and may thus mediate the interaction of sense and antisense RNA . Using the conserved RNP1 and RNP2 motifs as primers, we cloned 4 PCR fragments containing ORFs and additional homologies to known members of the RNP-CS family . We cloned a full length cDNA for one protein (DdRBP1) that showed similarities to hnRNP A1 . Recombinant protein synthesized in E . coli displayed binding to single stranded RNA and a weak annealing activity for partially complementary RNAs in vitro . Deletion of the RNP1 motif reduced RNA binding considerably but not completely . DdRBP1 is thus one of the few members of the RNP-CS family for which binding and annealing activities have been experimentally demonstrated . Polyclonal antisera directed against recombinant DdRBP1 detected a protein of approx . 40 kDa . In whole cell extracts, this protein was present in equal amounts throughout the developmental cycle of Dictyostelium while differential accumulation was observed in nuclei during early and late development.

Mol Microbiol, 1997 Dec, 26(5), 1125 - 35
Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation; Matsunaga J et al.; Control of mRNA stability is an established means of regulating gene expression . However, the detailed mechanisms by which such control is achieved are only now emerging . In particular, there remains a question about the involvement of translation . Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role . Here, we define the mechanism of this control further . Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species . Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III . In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway . Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed . Importantly, this mini-rnc transcript is regulated normally by RNase III . Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III . These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation . How rnc gene translation is optimized for efficient autoregulation will also be discussed.

Mol Microbiol, 1997 Dec, 26(5), 1013 - 21
Expression of gpsA encoding biosynthetic sn-glycerol 3-phosphate dehydrogenase suppresses both the LB- phenotype of a secB null mutant and the cold-sensitive phenotype of a secG null mutant; Shimizu H et al.; SecB maintains the structures of a subset of precursor proteins competent for translocation across the Escherichia coli cytoplasmic membrane . SecG, a membrane component of the translocation machinery, stimulates protein translocation by undergoing the cycle of membrane topology inversion . Null mutants of secB and secG are unable to form isolated colonies on rich medium and at low temperature respectively . A 3.2 kb DNA fragment carrying the secB-gpsA region on a multicopy plasmid was found to suppress the null mutation of either gene . However, subcloning of the DNA fragment revealed that secB is not involved in the suppression of either mutation . Instead, gpsA located downstream from the secB gene was found to be responsible for the suppression of both mutations . The activity of the gpsA-encoded sn-glycerol-3-phosphate dehydrogenase, which is involved in phospholipid synthesis, was significantly lower in the secB null mutant than in the wild type, presumably because of a polar effect . Suppression of the secB null mutation required the wild-type level of GpsA activity . In contrast, overexpression of the enzyme was essential for suppression of the secG null mutation . Moreover, the gpsA-dependent suppression of the secG null mutation occurred only on rich medium, i.e . not on minimal medium . These results indicate that the SecB function is dispensable even in rich medium, and further demonstrate that overexpression of enzymes involved in phospholipid synthesis partly compensates for the SecG function.

Mol Microbiol, 1997 Dec, 26(5), 911 - 25
Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K-12; Williams SB et al.; Anaerobic respiratory gene expression in Escherichia coli is differentially controlled by nitrate and nitrite through dual interacting two-component regulatory systems . The NarX sensor is one of two membrane-spanning sensor kinases that control the phosphorylation state of two DNA-binding response regulators . We have studied NarX autophosphorylation in crude membrane preparations from cells that overexpress NarX protein . The low basal autophosphorylation rate was stimulated about sixfold and threefold by nitrate and nitrite respectively . This demonstrates that nitrate and nitrite differentially activate NarX autokinase activity . We also isolated single-residue substitutions in NarX that affect its ability to respond to or discriminate between nitrate and nitrite . Most of these substitutions affect residues within the conserved P-box sequence in the periplasmic domain . We characterized several of the mutants in vivo, by monitoring ligand-regulated gene expression, and in vitro, by monitoring ligand-responsive autophosphorylation . At least one change, K491 (Lys at position 49 changed to Ile), resulted in a protein with greatly impaired ability to discriminate between nitrate and nitrite . Other changes (H45E and R59K) resulted in proteins that responded normally to nitrate but were unable to respond to nitrite . These results implicate the P-box region in discrimination between subtly different small molecules.

Mol Microbiol, 1997 Dec, 26(5), 889 - 96
DNA sequestration and transcription in the oriC region of Escherichia coli; Bogan JA et al.; In Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation . The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E . coli PC2 dnaC2(ts) aligned for initiation of chromosome replication . In both seqA- and dam- cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered . After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA+ dam+ cells than in seqA- or dam- cells, but before the release of oriC from sequestration . Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA . These findings, plus analyses of transcript levels in steady-state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC . Accordingly, a proposal for the sequence of events during the interinitiation interval in E . coli is presented based on the evident coupling of transcription to replication.

J Vasc Res, 1997 Nov-Dec, 34(6), 455 - 63
New model for the study of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane: the gelatin sponge/chorioallantoic membrane assay; Ribatti D et al.; Several methods for the in vivo study of angiogenesis are available, and each angiogenic assay presents distinct advantages and disadvantages . In this study, we present a new method for the quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM), based on the implantation of gelatin sponges on the top of growing CAM, on day 8 of incubation . After implantation, the sponges were treated with a stimulator (recombinant human basic fibroblast growth factor, FGF2) or an inhibitor (a rabbit polyclonal anti-FGF2 antibody) of blood vessel formation . Blood vessels growing vertically into the sponge and at the boundary between sponge and surrounding CAM mesenchyme were counted by a morphometric method on day 12 . In addition, to assess whether the gelatin sponge is an appropriate vehicle to deliver cultured cells and evaluate their angiogenic potential, mouse aortic endothelial cells were cotransfected with human FGF2 and the Escherichia coli beta-galactosidase (beta-GAL) reporter gene . Stable transfectants were absorbed by the sponge, and evaluation of the angiogenic response was paralleled by beta-GAL staining to visualize implanted cells . This technique may facilitate the discovery and development of agonists or antagonists of angiogenesis.

J Egypt Soc Parasitol, 1997 Dec, 27(3), 817 - 23
Blastocystis hominis among apparently healthy food handlers in Jeddah, Saudi Arabia; Amin AM; A prospective study was carried out to investigate the prevalence of Blastocystis hominis among random sample of apparently healthy food handlers . A total of 250 non Saudi males over 21 years of age were examined . Ninety (36%) had pathogenic and non pathogenic intestinal parasites . A total of 143 parasites were detected in their stool specimens . Twenty were B . hominis (13.99%) while other parasites were 123 (86.01%) . B . hominis was found in 20 positive cases (22.22%) with an overall rate of 8% . Of these twenty cases, two had B . hominis as a sole parasite, twelve as a double infection and six as a triple infection . Other intestinal parasites were found, Giardia lamblia (16.8%), Entamoeba histolytica (10%), E . coli (6.4%), Chilomastix mesnili (5.6%), Trichomonas hominis (1.2%), and Endolimax nana (0.8%) . The helminths were represented by Ascaris lumbricoides (4%), Hymenolepis nana (3.2%), Enterobius vermicularis (1.2%) and Trichocephalus trichiura (0.8%) . No doubt, B . hominis should be in mind of parasitologists and physicians when dealing with patients with gastrointestinal troubles.

Protein Expr Purif, 1997 Dec, 11(3), 297 - 303
High level expression in soluble form, one step purification, and characterization of the DNA binding domain of MEF-2C; Meierhans D et al.; Members of the MEF-2 family of transcription factors act as coregulators of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains . To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed . A T7 expression system was used to produce in high yield in Escherichia coli an N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain . Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein . Recombinant MEF-2C (1-117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG) . Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1-117) . The introduction of G-C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1-117) needed for half maximal DNA binding . We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.

Protein Expr Purif, 1997 Dec, 11(3), 279 - 83
An efficient system for production of recombinant urokinase-type plasminogen activator; Tang W et al.; A system was developed to produce recombinant urokinase-type plasminogen activator in Escherichia coli . The urokinase-type plasminogen activator was produced with a 6 x His-tag at the C-terminus which was shown to have the same activity, after refolding, as the wild-type protein . Purification of the recombinant protein to homogeneity (95%) was possible by one-step affinity chromatography under denaturing conditions . As a result, proteolysis by bacterial proteases during purification was avoided . A higher refolding efficiency (40%) and a higher total recovery yield (25%) of the recombinant protein were obtained by this method.

Protein Expr Purif, 1997 Dec, 11(3), 271 - 8
Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66; Binnie C et al.; A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes . The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S . fradiae aph gene, a signal peptide coding region derived from the protease B gene of S . griseus, and a transcription terminator sequence also derived from the S . fradiae aph gene . Extracellular expression of authentic EPO-R by S . lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product . Specific binding of S . lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

Protein Expr Purif, 1997 Dec, 11(3), 241 - 9
Purification and characterization of an active form of the p78Rep protein of adeno-associated virus type 2 expressed in Escherichia coli; Wollscheid V et al.; The 78-kDa product (p78Rep) of the rep gene of AAV-2 was expressed with an amino-terminal histidine-tag in Escherichia coli and was purified under denaturing conditions . After renaturation of the p78Rep protein by serial steps of dialysis, the biochemical activities of the p78Rep protein were demonstrated, which include the ATP-dependent endonuclease and helicase activity as well as sequence-specific binding to the AAV-2 terminal repeat . These activities were retained when the protein was purified under denaturing conditions followed by renaturation . When compared with published data for p68Rep, the helicase activity of p78Rep was stronger and the endonuclease activity was weaker . The p78Rep protein was able to inhibit HIV-1 replication after co-microinjection together with infectious proviral HIV-1 DNA into the nuclei of human cells, suggesting that p78Rep is necessary for inhibition of HIV-1 in vivo.

Protein Expr Purif, 1997 Dec, 11(3), 227 - 32
Expression of the protein-tyrosine kinase p56lck by the pTRX vector yields a highly soluble protein recovered by mild sonication; Hlavac F et al.; We have expressed the protein-tyrosine kinase p56lck as a fusion with glutathione-S-transferase (GST) or thioredoxin (TRX) . While the GST-Lck fusion protein was found to be poorly soluble and solubilization techniques led to a high degree of degradation, the TRX-Lck fusion protein was found to be highly soluble . This solubilization was achieved by mild sonication in a simple low ionic strength buffer which makes the fusion protein immediately available for further use . Even in the absence of protease inhibitors, the TRX-Lck fusion protein exhibited no trace of degradation . We produced 40-60 mg of TRX-Lck/l of culture which was efficiently cleaved at the endopeptidase site . The TRX-Lck and GST-Lck fusion proteins exhibited similar phosphorylation activity.

Gene Ther, 1997 Nov, 4(11), 1270 - 7
Foamy virus vectors for suicide gene therapy; Nestler U et al.; To improve the delivery of so-called suicide genes into tumors, recombinant retroviruses were constructed by inserting the herpes virus type 1 (HSV-1) thymidine kinase (tk), the E . coli cytosine deaminase (cd) and polynucleoside phosphorylase (pnp), or the jellyfish gene for the green fluorescent protein (gfp) into a foamy virus (FV)-derived replication-competent vector (pFOV-7) . Expression and stability of the inserted foreign gene was analyzed by immunoblot and polymerase chain reaction (PCR) . The functionality of the suicide genes was determined by a metabolic assay on virus vector infected cells and treatment with the respective prodrugs . In terms of vector stability and effectiveness of specific cell killing a virus transducing the pnp gene (FOV-7/pnp) was superior to those using the other two suicide genes . FOV-7/pnp is a candidate virus for suicide gene delivery into solid tumors.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 570 - 3
Interaction of P1 RepA with replication origin of plasmid Rts1: capability of an initiator protein inducing replication from a foreign origin; Li YF et al.; Rts1 RepA and P1 RepA are trans-acting proteins essential for the initiation of replication of plasmid Rts1 and prophage P1, respectively . In this study, we found that, in vitro, P1 RepA bound to the Rts1 ori fragment and Rts1 incI fragment as strongly as Rts1 RepA . In addition P1 RepA, in trans, activated the Rts1 replication origin that was cloned in pBR322, thus allowing the ori plasmid to be maintained in a polA E . coli host . Under these conditions, however, the ori plasmid was unstable as compared with that when activated by Rts1 RepA . In addition, we found that Rts1 RepA showed no interaction with the P1 replication origin.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 553 - 7
Purification and crystallization of the oxygenase component of naphthalene dioxygenase in native and selenomethionine-derivatized forms; Lee K et al.; A new procedure was developed for the purification of the terminal oxygenase component (ISPNAP) of naphthalene dioxygenase . From a five liter culture of Escherichia coli JM109(DE3)(pDTG121), 91 mg of pure protein were obtained with a specific activity of 2.48 mumol/ min/mg protein . ISPNAP was crystallized in the rhombohedral space group R32 with cell dimensions of a = b = 179.2 A; c = 322.5 A in the hexagonal setting . The crystals are brown, indicating the presence of an intact Rieske iron-sulfur center . Problems with non-isomorphism between native data sets necessitated the preparation of a selenomethionine-substituted protein . Complete replacement of methionine with selenomethionine was achieved and the purified protein had a specific activity almost identical to native ISPNAP . Crystals from this preparation belong to the same space group and have similar cell dimensions to native ISPNAP.

Biochemistry, 1997 Dec 9, 36(49), 15089 - 100
Structure of the carboxy-terminal fragment of the apo-biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase; Yao X et al.; The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis . In its functional cycle the biotin carboxyl carrier protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of posttranslational modification . Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the posttranslational attachment of biotin to an essential lysine residue on BCCP . Holo-BCCP then interacts with the biotin carboxylase subunit, which leads to the addition of the carboxylate group of bicarbonate to biotin . Finally, the carboxybiotinylated form of BCCP interacts with transcarboxylase in the conversion of acetyl-CoA to malonyl-CoA . The determinants of protein-protein interaction specificity in this system are unknown . One hypothesis is that posttranslational modification of BCCP may result in conformational changes that regulate specific protein-protein interactions . To test this hypothesis, we have determined the NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment of BCCP (apoBCCP87) from Escherichia coli acetyl-CoA carboxylase and compared this structure with the high-resolution structure of the biotinylated form that was recently solved by X-ray crystallographic techniques . Although the overall folding of the two proteins is highly similar, small structural differences are apparent for residues of the biotin-binding loop that may be important for mediating specific protein-protein interactions.

Vopr Virusol, 1997 Sep-Oct, 42(5), 208 - 12
{Preparation and purification of a polypeptide containing antigenic determinants of hepatitis C core protein}; Petrakova NV et al.; A method has been developed for preparing and purifying recombinant polypeptide--hepatitis C virus core protein (HCcoreAg) expressed in E . coli from pFC105-302 plasmid coding for 150 N-terminal amino acids of HCcoreAg in the hybrid from the C-terminal with beta-galactosidase . HCcoreAg was purified by ion-exchange chromatography on P-11 phosphocellulose . The bulk of protein carrying HCcoreAg antigenic determinants was found in two polypeptides: with molecular weights 26 and 136 kD . Antigenic and immunogenic properties of the resultant polypeptide were studied . The 26 kD protein can be used in enzyme immunoassay and immunoblotting as antigen for detecting antibodies to the HCV core protein . The results of immunization of rabbits indicate a high immunogenic activity of the protein . High diagnostic value of HCcoreAg preparation has been demonstrated, for the rapid variant of indirect solid-phase enzyme immunoassay among other tests.

Neuropharmacology, 1997 Oct, 36(10), 1377 - 85
Conserved phosphorylation of the intracellular domains of GABA(A) receptor beta2 and beta3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase; McDonald BJ et al.; All mammalian GABA(A) receptor beta subunits contain a conserved consensus site for phosphorylation by a number of serine/threonine protein kinases . This site corresponds to Serine 410 of the beta2 subunit and Serine 409 of the beta3 subunit, each of which lies within the conserved sequence R-R-R-X-S-L-Q-K, where X = A (beta1, beta2 and beta4) or S (beta3) . We have analysed the phosphorylation of the beta2 and beta3 subunits of the murine GABA(A) receptor by expressing the large intracellular domains of these subunits as soluble fusion proteins in E . coli . The intracellular domain of the beta2 subunit was phosphorylated to high stoichiometry by both cAMP- and cGMP-dependent protein kinases, protein kinase C and Ca2+/calmodulin type II-dependent protein kinase in vitro . Site-directed mutagenesis identified Serine 410 as the single site within the beta2 subunit phosphorylated by these four protein kinases . Using similar methodologies, Serine 409 of the beta3 subunit was shown to be a substrate for phosphorylation by these protein kinases . Serine 408 was also seen to be phosphorylated by protein kinase C and Serine 383 was phosphorylated by Ca2+/calmodulin type II-dependent protein kinase . Since beta subunits are believed to be essential for robust GABA(A) receptor expression, these results suggest a critical role for conserved phosphorylated amino acids within the beta subunits in coordinating cellular regulation of GABA(A) receptors via multiple protein kinases.

Infect Immun, 1998 Jan, 66(1), 369 - 72
Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family; White PA et al.; The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability . The gene encoding this protein was isolated from a genomic library of A . actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis . Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa . The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins . We have named the major OMP of A . actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.

Infect Immun, 1998 Jan, 66(1), 347 - 52
Characterization of the immunostimulatory properties of Leishmania infantum HSP70 by fusion to the Escherichia coli maltose-binding protein in normal and nu/nu BALB/c mice; Rico AI et al.; Leishmania infantum HSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis . In this study, we used L . infantum HSP70 fused to Escherichia coli maltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice . Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP, L . infantum HSP70, and MBP-HSP70, which consists of MBP fused to the L . infantum HSP70 amino terminus . Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70 . Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response . Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response . Immunization of athymic nu/nu mice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response . Thus, we present evidence that L . infantum HSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.

Infect Immun, 1998 Jan, 66(1), 107 - 14
Identification of a genetic locus essential for serotype b-specific antigen synthesis in Actinobacillus actinomycetemcomitans; Yoshida Y et al.; A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized . Western blot analysis showed that Escherichia coli DH5alpha, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A . actinomycetemcomitans Y4 . High-performance liquid chromatography analysis indicated that the polysaccharide produced by an E . coli transformant, as well as A . actinomycetemcomitans Y4 SPA, was composed of rhamnose and fucose . Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kb BssHII-BspHI fragment was indispensable for SPA synthesis in E . coli DH5alpha . The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined . In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found . Most of these were located sequentially within a short distance of each other . Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria . The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of A . actinomycetemcomitans Y4 . It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kb BssHII-BspHI fragment indispensable for SPA synthesis in E . coli was found to be especially low (27.0%).

Infect Immun, 1998 Jan, 66(1), 52 - 8
Effect of prior experimental human enteropathogenic Escherichia coli infection on illness following homologous and heterologous rechallenge; Donnenberg MS et al.; Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge . In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain . In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain . There was no significant effect of prior infection on the incidence of diarrhea in either study . However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain . Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide . These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge . Further studies may require the development of new model systems.

J Physiol, 1997 Dec 1, 505 ( Pt 2), 411 - 23
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca(2+)-dependent HCO3- secretion; Seidler U et al.; 1 . Most segments of the gastrointestinal tract secrete HCO3-, but the molecular nature of the secretory mechanisms has not been identified . We had previously speculated that the regulator for intestinal electrogenic HCO3- secretion is the cystic fibrosis transmembrane regulator (CFTR) channel . To prove this hypothesis, we have now measured HCO3- secretion by pH-stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein ('CF-mice') and their normal littermates . 2 . Basal HCO3- secretory rates were reduced in all small intestinal segments of CF mice . Forskolin, PGE2, 8-bromo-cAMP and VIP (cAMP-dependent agonists), heat-stable enterotoxin of Escherichia coli (STa), guanylin and 8-bromo-cGMP (cGMP-dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short-circuit current (Isc) and the HCO3- secretory rate (JHCO3-) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3- in the intestine of CF mice . 3 . To investigate whether Cl(-)-HCO3- exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied Cl- gradient-driven 36Cl- uptake into brush-border membrane (BBM) vesicles isolated from normal and CF mouse small intestine . Both the time course and the peak value for 4,4'-diisothiocyanostilbene-2',2-disulphonic acid (DIDS)-inhibited 36Cl- uptake was similar in normal and CF mice BBM vesicles . 4 . In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist-induced stimulation of electrogenic HCO3- secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3- secretion exclusively via activation of the CFTR channel.

J Mol Biol, 1997 Nov 21, 274(1), 27 - 38
The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control; Matsunaga F et al.; The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication . Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription . Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers . To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function . Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208) . The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135) . Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host . One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-a-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers . Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers . On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization . Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication .

J Mol Biol, 1997 Nov 21, 274(1), 8 - 15
Pactamycin resistance mutations in functional sites of 16 S rRNA; Mankin AS; Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated . Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H . halobium single rRNA operon . Three types of mutations were found in pactamycin resistant cells, A694G, C795U and C796U (Escherichia coli 16 S rRNA numeration) located distantly in rRNA primary structure but probably neighboring each other in the three-dimensional structure . Pactamycin resistance mutations either overlapped (C795U) or were located in the immediate vicinity of nucleotides protected by the drug in E . coli and H . halobium 16 S rRNA indicating that corresponding rRNA sites might be directly involved in pactamycin binding . Ribosomal functions were not affected significantly either by mutation of C795 (one of the positions protected by the P-site-bound tRNA), or by mutations of A694 and C796 (which neighbor nucleotides protected by tRNA) suggesting that tRNA-dependent protections of C795 and G693 are explained by a conformational change in the ribosome induced by the P-site-bound tRNA . A novel mode of pactamycin action is proposed suggesting that pactamycin restricts structural transitions in 16 S rRNA preventing the ribosome from adopting a functional conformation induced by tRNA binding .

Biochemistry, 1997 Dec 9, 36(49), 15285 - 93
Cross-linking and N-(1-pyrenyl)maleimide labeling of cysteine mutants of proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli; Sedgwick EG et al.; The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed of two subunits (alpha and beta) organized as an alpha2beta2 tetramer . The enzyme contains seven cysteine residues, five in the alpha-subunit and two in the beta-subunit . The reaction of these residues with the cross-linking agent cupric 1, 10-phenanthrolinate and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was investigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues . Mutation of alphaCys395 and alphaCys397 prevented disulfide bond formation to give the cross-linked alpha2 dimer . We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues . Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show characteristic fluorescence emission bands at 378-379, 397-398, and 419-420 nm . At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) and longer times of reaction, an eximer band at 470 nm was formed . This was attributed to interaction between noncovalently bound molecules of pyrenylmaleimide . The cysteine residues of the beta-subunit (betaCys147 and betaCys260) were covalently modified by pyrenylmaleimide . betaCys147 reacted more strongly than betaCys260 with the fluorophore, and the pyrene derivative of betaCys147 was more accessible to quenching by 5-doxylstearate, suggesting a proximity to the surface of the membrane . Covalent modification of betaCys260 resulted in inhibition of enzyme activity . The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.

Biochemistry, 1997 Dec 9, 36(49), 15140 - 6
The conformation of the alpha-helical coiled coil domain of macrophage scavenger receptor is pH dependent; Suzuki K et al.; Macrophage scavenger receptor is a trimerized membrane protein that binds ligands and undergoes internalization by endocytosis . The receptor releases the ligands in the endosome, and then is recycled . The mechanisms of the ligand release and the recycling of the receptor have not been clearly determined . We analyzed the structure of the alpha-helical coiled coil domain considered to be responsible for acid-mediated ligand dissociation, by chemical cross-linking, sedimentation equilibrium, Western blot, and circular dichroism analyses . This domain has 22 heptad repeats, which are characteristic of the sequence of an alpha-helical coiled coil structure, with a discontinuity in the middle . We prepared three peptides, corresponding to the entire alpha-helical coiled coil domain (alpha), its N-terminal half (alpha-N), and its C-terminal half (alpha-C), by expression of each gene in Escherichia coli . The alpha and alpha-N peptides show triple-stranded alpha-helical coiled coil structures, but in contrast, the alpha-C peptide shows a random structure . When connected to the N-terminus by a chemical ligation method, the alpha-C peptide also shows an alpha-helical coiled coil structure, but only at an acidic pH . These results suggest that the N-terminus of the alpha-helical coiled coil domain is responsible for the formation of a stable trimer and the C-terminus exhibits the pH-dependent conformational change that might be involved in the ligand release by the macrophage scavenger receptor.

Biochemistry, 1997 Dec 9, 36(49), 15101 - 8
Identification and structural and functional characterization of human enamelysin (MMP-20); Llano E et al.; A cDNA encoding a new human matrix metalloproteinase (MMP) has been cloned from RNA prepared from odontoblastic cells . The open reading frame of the cloned cDNA codes for a polypeptide of 483 amino acids and is extensively similar to the sequence of recently described porcine enamelysin, suggesting that the isolated cDNA codes for the human homologue of this enzyme . Human enamelysin (MMP-20) has a domain organization similar to other MMPs, including a signal peptide, a prodomain with the conserved motif PRCGVPD involved in maintaining enzyme latency, a catalytic domain with a Zn-binding site, and a COOH-terminal fragment similar to the sequence of hemopexin . The calculated molecular mass of human enamelysin is about 54 kDa, which is similar to that of collagenases or stromelysins . However, this human MMP lacks a series of structural features distinctive of these subfamilies of MMPs . The full-length human enamelysin cDNA has been expressed in Escherichia coli, and the purified and refolded recombinant protein is able to degrade synthetic peptides used as substrates of MMPs, confirming that human enamelysin belongs to this family of proteases . Furthermore, the recombinant human enamelysin is able to degrade amelogenin, the major protein component of the enamel matrix . On the basis of its degrading activity on amelogenin, and its highly restricted expression to dental tissues, we suggest that human enamelysin plays a central role in the process of tooth enamel formation . Finally, we have found that the human enamelysin gene (MMP-20) maps to chromosome 11q22, clustered to at least seven other members of the MMP gene family.

Biochemistry, 1997 Dec 9, 36(49), 15055 - 61
Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli; Voss J et al.; Functional lactose permease mutants containing single Cys residues at positions 233-255 and a biotin acceptor domain at the C terminus were solubilized in dodecyl beta-d-maltopyranoside and purified by avidin affinity chromatography . Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy (EPR) . The EPR spectral line shapes and the influence of nonpolar O2 or polar potassium chromium oxalate relaxation agents on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to the relaxation agents, respectively . The results provide evidence that residues Ser233-Asn246 are within the hydrophobic core of the membrane and that Phe247 is at the lipid headgroup-solvent interface . Along with Phe247, Phe250 and Gly254 are also surface-exposed, as indicated by studies on the epitope for monoclonal antibody 4B1 {Sun, J., Wu, J., Carasco, N., and Kaback, H . R . (1996) Biochemistry 35, 990-998} . Furthermore, the nitroxide-labeled intramembrane Cys replacements exhibit variations in mobility and accessibility that are consistent with the conclusion that TM VII is an alpha-helix in contact with surrounding helices in the tertiary structure of the permease.

Biochemistry, 1997 Dec 9, 36(49), 15041 - 8
Reconstruction of quaternary structures of class II tRNA synthetases by rational mutagenensis of a conserved domain; Ribas de Pouplana L et al.; Class II tRNA synthetases have long been known to have quaternary structures of alpha, alpha2, alpha2beta2, and alpha4, depending on the amino acid specificity and the organism from which the synthetase was isolated . Even the quaternary structures of enzymes for the same amino acid show variations in evolution . The basis for these variations has not been understood . We report here that sequence manipulations of a structural motif (motif 1) characteristic of all class II tRNA synthetases can generate most of the evolutionary diversity of quaternary forms of class II synthetases . Thus, the principles elucidated here for quaternary structure assembly may be general.

J Biol Chem, 1997 Dec 5, 272(49), 31190 - 5
Footprinting of yeast DNA topoisomerase II lysyl side chains involved in substrate binding and interdomainal interactions; Li W et al.; Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule . Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit . First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves . Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide . Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding . Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.

J Biol Chem, 1997 Dec 5, 272(49), 31058 - 64
The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase . The B subunits interact with F1 as a dimer and through the delta subunit; Rodgers AJ et al.; The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0) . The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here . Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments . On F1, i.e . in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis . Both delta subunit and bsol are protected from trypsin cleavage in this complex . In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent . The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments . As purified, bsol is a stable dimer with 80% alpha helix . A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S . M., Rodgers, A . J.,W., Jeffrey, P . D., and Cox, G . B . (1996) J . Biol . Chem . 271, 7038-7042) . Monomeric bsol has less alpha helix, i.e . only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure . The bsol dimer, but not monomer, binds to delta in ECF1 . To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C . With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants . Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity . These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

J Biol Chem, 1997 Dec 5, 272(49), 30848 - 51
Activation of ADP-ribosylation factor 1 GTPase-activating protein by phosphatidylcholine-derived diacylglycerols; Antonny B et al.; Disassembly of the coatomer from Golgi vesicles requires that the small GTP-binding protein ADP-ribosylation factor 1 (ARF1) hydrolyzes its bound GTP by the action of a GTPase-activating protein . In vitro, the binding of the ARF1 GTPase-activating protein to lipid vesicles and its activity on membrane-bound ARF1GTP are increased by diacylglycerols with monounsaturated acyl chains, such as those arising in vivo as secondary products from the hydrolysis of phosphatidylcholine by ARF-activated phospholipase D . Thus, the phospholipase D pathway may provide a feedback mechanism that promotes GTP hydrolysis on ARF1 and the consequent uncoating of vesicles.

J Biol Chem, 1997 Dec 5, 272(49), 30780 - 6
Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria; Aslund F et al.; Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C) . Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides . To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein . This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms . Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values . Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively . These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation . Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E . coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV . Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

J Biol Chem, 1997 Dec 5, 272(49), 30774 - 9
Further characterization of Escherichia coli endonuclease V . Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA; Yao M et al.; Endonuclease V from Escherichia coli has a wide substrate spectrum . In addition to deoxyinosine-containing DNA, the enzyme cleaves DNA containing urea residues, AP sites, base mismatches, insertion/deletion mismatches, flaps, and pseudo-Y structures . The gene coding for the enzyme was identified to be orf 225 or nfi (endonuclease five) . Using enzyme purified from an overproducing strain, the deoxyinosine- and mismatch-specific activities of endonuclease V was found to have different divalent metal requirements . The affinity of the enzyme is greater than 20-fold higher for DNA containing deoxyinosine than deoxynebularine or base mismatches . Under optimal cleavage conditions, endonuclease V forms two stable complexes with DNA containing deoxyinosine, but not with DNA containing base mismatches or deoxynebularine, suggesting that the 6-keto group of hypoxanthine in DNA is critical for stable interactions with the protein . The enzyme recognizes deoxyuridine in DNA but exhibits a much lower affinity to DNA containing deoxyuridine compared with DNA containing deoxyinosine . Interestingly, deoxyuridine-specific endonuclease activity of endonuclease V has a divalent metal requirement similar to the mismatch activity . A model for the mechanism of substrate recognition is proposed to explain these different activities.

J Biol Chem, 1997 Dec 5, 272(49), 30735 - 40
Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase; Pechatnikova E et al.; The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules . The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site . Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding . The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C) . The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant . The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1 . The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements . Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate . Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J . Biol . Chem . 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd . There are two differences that may affect the reaction pathway . The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis . The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release . The monomeric Mt.Ncd complex is not processive.

J Biol Chem, 1997 Dec 5, 272(49), 30651 - 61
The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter; Leveillard T et al.; MDM2 proto-oncogene expression is aberrant in many human tumors . Its normal role is to modulate the functions of p53 . The N terminus of MDM2 interacts with p53, whereas the properties of the rest of the molecule are poorly understood . We show that MDM2 binds to the general transcription factor TFIID in vivo . The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic domain interacts with TBP . Expression of MDM2 activates the cyclin A gene promoter but not c-fos, showing that the effects of MDM2 are specific . Deletion of the C-terminal region of MDM2 abolishes activation, showing that the C-terminal domain of MDM2 is functionally important . We found that increasing MDM2 expression to higher levels inhibits the cyclin A promoter . Inhibition appears to result from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as other promoters in vivo and basal transcription in vitro . The mechanisms of repression of the cyclin A and fos promoters appear to be different . Cyclin A repression is lost by deleting the C terminus, whereas that of c-fos is lost by removal of the acidic domain . These results reinforce the conclusion that the C terminus of MDM2 mediates effects on the cyclin A promoter . MDM2 transformed cells contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological conditions . There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation of the cyclin A promoter . The C-terminal region of MDM2, which contains the Ring finger, interacts with TAFII250 and is required for regulation of the cyclin A promoter by MDM2 . Our results link the activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator of the cell cycle.

J Biol Chem, 1997 Dec 5, 272(49), 30645 - 50
Characterization of the fatty acid-responsive transcription factor FadR . Biochemical and genetic analyses of the native conformation and functional domains; Raman N et al.; In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the level of transcription by FadR . FadR represses transcription of at least eight genes required for fatty acid transport and beta-oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encoding the transcriptional regulator of the aceBAK operon encoding the glyoxylate shunt enzymes, IclR . FadR-dependent DNA binding and transcriptional activation is prevented by long chain fatty acyl-CoA . In the present work, we provide physical and genetic evidence that FadR exists as a homodimer in solution and in vivo . Native polyacrylamide gel electrophoresis and glycerol gradient ultracentrifugation of the purified protein show that native FadR was a homodimer in solution with an apparent molecular mass of 53.5 and 57.8 kDa, respectively . Dominant negative mutations in fadR were generated by random and site-directed mutagenesis . Each mutation mapped to the amino terminus of the protein (residues 1-66) and resulted in a decrease in DNA binding in vitro . In an effort to separate domains of FadR required for DNA binding, dimerization, and ligand binding, chimeric protein fusions between the DNA binding domain of LexA and different regions of FadR were constructed . One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter sulA-lacZ, and beta-galactosidase activities were derepressed by fatty acids, suggesting that the fusion protein had determinants both for dimerization and ligand binding . These studies support the conclusion that native FadR exists as a stable homo-dimer in solution and that determinants for DNA binding and acyl-CoA binding are found within the amino terminus and carboxyl terminus, respectively.

J Biol Chem, 1997 Dec 5, 272(49), 30611 - 4
The Bloom's syndrome gene product is a 3'-5' DNA helicase; Karow JK et al.; Bloom's syndrome (BS) is an autosomal recessive condition characterized by short stature, immunodeficiency, and a greatly elevated frequency of many types of cancer . The gene mutated in BS, BLM, encodes a protein containing seven "signature" motifs conserved in a wide range of DNA and RNA helicases . BLM is most closely related to the subfamily of DEXH box-containing DNA helicases of which the prototypical member is Escherichia coli RecQ . To analyze its biochemical properties, we have overexpressed an oligohistidine-tagged version of the BLM gene product in Saccharomyces cerevisiae and purified the protein to apparent homogeneity using nickel chelate affinity chromatography . The recombinant BLM protein possesses an ATPase activity that is strongly stimulated by either single- or double-stranded DNA . Moreover, BLM exhibits ATP- and Mg2+-dependent DNA helicase activity that displays 3'-5' directionality . Because many of the mutations in BS individuals are predicted to truncate the BLM protein and thus eliminate the "helicase" motifs or map to conserved positions within these motifs, our data strongly suggest that these mutations will disable the 3'-5' helicase function of the BLM protein.

Clin Diagn Lab Immunol, 1997 Nov, 4(6), 648 - 52
Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus; Kheyar A et al.; To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M) . The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV . Some horse sera, however, showed immune reactivity to the MBP fusion partner protein . Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays . Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure . The reactivity continued to the end of the experiment at day 145 after infection . This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples . Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.

EMBO J, 1997 Nov 17, 16(22), 6823 - 34
Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition; Stellwagen AE et al.; The bacterial transposon Tn7 exhibits target immunity, a process that prevents Tn7 from transposing into target DNAs that already contain a copy of the transposon . This work investigates the mechanism of target immunity in vitro . We demonstrate that two Tn7-encoded proteins_TnsB, which binds specifically to the ends of Tn7, and TnsC, the ATP-dependent DNA binding protein_act as a molecular switch to impose immunity on target DNAs containing Tn7 (or just Tn7 ends) . TnsC binds to target DNA molecules and communicates with the Tn7 transposition machinery; here we show that target DNAs containing Tn7 ends are also bound and subsequently inactivated by TnsB . Protein-protein interactions between TnsB and TnsC appear to be responsible for this inactivation; the target DNA promotes these interactions by tethering TnsB and TnsC in high local concentration . An attractive model that emerges from this work is that TnsB triggers the dissociation of TnsC from the Tn7 end-containing target DNA; that dissociation depends on TnsC's ability to hydrolyze ATP . We propose that these interactions between TnsB and TnsC not only prevent Tn7 from inserting into itself, but also facilitate the selection of preferred target sites that is the hallmark of Tn7 transposition.

EMBO J, 1997 Nov 17, 16(22), 6626 - 35
Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants; Hopfner KP et al.; The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface . However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa . To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli . The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a . Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a . Exchanging Ile213 by Val only moderately increased reactivity of rf9a . Exchanging the 99 loop, however, dramatically increased reactivity . Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity . The results suggest a 2-fold origin of fIXa's poor reactivity . A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond . Both defects could be repaired by introducing fXa residues . Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.

Biochem J, 1997 Nov 15, 328 ( Pt 1), 231 - 5
Mutagenesis of residue 157 in the active site of human glyoxalase I; Ridderstrom M et al.; Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis . The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel . The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme . The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx . 20% of wild-type activity . The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value . This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant . With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I . Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values . The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I . The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.

Biochem J, 1997 Nov 15, 328 ( Pt 1), 159 - 63
Expression, purification, and characterization of recombinant human glutamine synthetase; Listrom CD et al.; A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx . 8 mg of purified enzyme per litre of culture . The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized . It has a subunit molecular mass of approx . 45000 Da . The Vmax value obtained using a radioactive assay with ammonia and l-{G-3H}glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate . Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2 . 9 mM for the radioactive and spectrophotometric assays respectively . The Km for ammonia in the radioactive assay was 0.15 mM . The midpoint of thermal inactivation was 49.7 degrees C . Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation . The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.

Biochem J, 1997 Nov 15, 328 ( Pt 1), 75 - 81
Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase; Craxton A et al.; The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates . We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP . The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family . A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities . Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the beta-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate . These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities . A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver . The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity {Ali, Craxton and Shears (1993) J . Biol . Chem . 268, 6161-6167}.

Nature, 1998 Jan 1, 391(6662), 96 - 9
Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis; Sakahira H et al.; Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death), which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation . Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin . Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA . In the accompanying Article, we have identified and molecularly cloned a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) . Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect . We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis . When human Jurkat cells were transformed with ICAD-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation . The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase . Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.

Nature, 1998 Jan 1, 391(6662), 43 - 50
A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD; Enari M et al.; The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis . Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA . A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells . CAD is a protein of 343 amino acids which carries a nuclear-localization signal; ICAD exists in a long and a short form . Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity . When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity which causes DNA fragmentation in nuclei . ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA.

Tumour Biol, 1998, 19 Suppl 1, 67 - 70
Monoclonal antibodies against the nonmucin domain of MUC1/episialin; Hilkens J et al.; We have tested the reactivity of the monoclonal antibodies submitted to the ISOBM TD-4 Workshop for reactivity with a hybrid molecule consisting of the bacterial beta-galactosidase and most of the extracellular nonrepeat domain of MUC1/episialin . Two monoclonal antibodies submitted to the Workshop, 232A1 and M29, were directed against the protein moiety of this domain as shown by immunoblotting and immunoprecipitation.

J Bacteriol, 1998 Jan, 180(1), 190 - 3
The leuO gene product has a latent ability to relieve bgl silencing in Escherichia coli; Ueguchi C et al.; The Escherichia coli bgl operon is of interest, since its expression is silent (phenotypically Bgl-), at least under standard laboratory conditions . Here we attempted to identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl by a random insertion mutagenesis with mini-Tn10 . These collected mutations, conferring the phenotype of Bgl+, were localized in three loci on the genetic map, two of which appeared to be hns and bglJ, which were previously implicated as the factors affecting the Bgl phenotype . The other locus at 1 to 2 min on the genetic map appeared to be a new one . In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein . Genetic analyses revealed that overproduction of LeuO in the wild-type cells causes the phenotype of Bgl+ . A leuO deletion mutant was also characterized in terms of expression of bgl . From these results, the possible function of LeuO in bgl expression will be discussed from an evolutionary and/or ecological point of view.

J Bacteriol, 1998 Jan, 180(1), 175 - 7
Stability of the Escherichia coli division inhibitor protein MinC requires determinants in the carboxy-terminal region of the protein; Sen M et al.; Certain mutations in the C-terminal region of the Escherichia coli division inhibitor protein MinC cause loss of function of the division inhibitor by making MinC more sensitive to degradation by Lon protease, implying a possible role for the C-terminal region in regulating the stability and cellular concentration of MinC.

J Bacteriol, 1998 Jan, 180(1), 171 - 4
Differential expression of the OmpF and OmpC porin proteins in Escherichia coli K-12 depends upon the level of active OmpR; Lan CY et al.; We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase . Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell . This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.

J Bacteriol, 1998 Jan, 180(1), 90 - 5
Role of the cold-box region in the 5' untranslated region of the cspA mRNA in its transient expression at low temperature in Escherichia coli; Fang L et al.; Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced in Escherichia coli . However, when the 5' untranslated region (5' UTR) of cspA mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed . It has been proposed that this effect is due to highly conserved 11-base sequences designated the "cold box" existing in the 5' UTRs of cspA, cspB, and csdA . Here, we demonstrate that the overproduction of the 5' UTR of not only cspA but also cspB and csdA mRNAs causes derepression of all three genes at the same time . Conversely, when the cold-box region was deleted from the cspA 5' UTR its derepression function was abolished . The amount of mRNA from the chromosomal cspA gene was much higher in cells overproducing the wild-type 5' UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5' UTR . The stability of the chromosomal cspA mRNA in cells overproducing the wild-type 5' UTR was almost identical to that in cells overproducing the cold-box-deleted 5' UTR . Therefore, the derepression of cspA caused by overproduction of 5' UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in cspA transcription in cold shock-adapted cells . The role of the cold-box region was further confirmed with a cspA mutant strain containing a cold-box-deleted cspA gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.

J Bacteriol, 1998 Jan, 180(1), 73 - 82
RimM and RbfA are essential for efficient processing of 16S rRNA in Escherichia coli; Bylund GO et al.; The trmD operon is located at 56.7 min on the genetic map of the Escherichia coli chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m1G37)methyltransferase (TrmD), and r-protein L19, in that order . Previously, we have shown that strains from which the rimM gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency . The slow growth and translational deficiency were found to be partly suppressed by mutations in rpsM, which encodes r-protein S13 . Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes . Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the nusA operon at 68.9 min on the chromosome . For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a deltarimM mutant . Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA.

J Bacteriol, 1998 Jan, 180(1), 1 - 9
Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes; MacGregor BJ et al.; These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA) . In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters . The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E . coli Esigma32 . Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7 . A point mutation at position -34 that is towards the E . coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements . In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides . When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E . coli heat shock promoters (Esigma38) . (R . K . Karls, J . Brooks, P . Rossmeissl, J . Luedke, and T . J . Donohue, J . Bacteriol . 180:10-19, 1998).

Planta, 1997 Dec, 203(4), 422 - 9
New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L . and bougainvillea spectabilis Willd; Bolognesi A et al.; New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L . (two proteins) and from the leaves of Bougainvillea spectabilis Willd . (one protein) . These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range . All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases . The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1 . The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis . When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested . An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

Microbiology, 1997 Dec, 143 ( Pt 12), 3889 - 98
The ParB protein encoded by the RP4 par region is a Ca(2+)-dependent nuclease linearizing circular DNA substrates; Grohmann E et al.; The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases . The ParB protein was overexpressed by means of an inducible tac-promoter system . Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity . The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region . The nuclease activity in vitro is stimulated by the presence of Ca2+ ions . EDTA (5 mM) completely inhibits nuclease activity . By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated . Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

Microbiology, 1997 Dec, 143 ( Pt 12), 3819 - 25
An unusual illegitimate recombination occurs in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii; Wang J et al.; In this study, we describe an unusual illegitimate recombination in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii . A 96 bp DNA segment was deleted from the ospA structural gene of B . afzelii strain R9 . The nature of the rearrangement suggested that it arose by a strand slippage mechanism, which was stimulated by a 18-mer palindromic sequence and 5-mer short direct repeats at both termini of the deleted DNA . The deleted sequence could form a complex hairpin structure suggesting that it may have played important roles in pausing of replication and slippaging of the nascent strand across the replication fork . In addition, the mutant strain was isolated from a chronic Lyme disease patient, implying that the variation mechanism may have been used by the borrelial strain to avoid host immune elimination.

Microbiology, 1997 Dec, 143 ( Pt 12), 3795 - 805
Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli; Cunningham L et al.; Escherichia coli contains two differentially regulated aconitase genes, acnA and acnB . Two acnA promoters transcribing from start points located 407 bp (P1acnA) and 50 bp (P2acnA) upstream of the acnA coding region, and one acnB promoter (PacnB) with a start point 95 bp upstream of the acnB coding region, were identified by primer extension analysis . A 2.8 kb acnA monocistronic transcript was detected by Northern blot hybridization, but only in redox-stressed (methyl-viologen-treated) cultures, and a 2.5 kb acnB monocistronic transcript was detected in exponential- but not stationary-phase cultures . These findings are consistent with previous observations that acnA is specifically subject to SoxRS-mediated activation, whereas acnB encodes the major aconitase that is synthesized earlier in the growth cycle than AcnA . Further studies with acn-lacZ gene fusions and a wider range of transcription regulators indicated that acnA expression is initiated by sigma 38 from P1acnA, and from P2acnA it is activated directly or indirectly by CRP, FruR, Fur and SoxRS, and repressed by ArcA and FNR . In contrast, acnB expression is activated by CRP and repressed by ArcA, FruR and Fis from PacnB . Comparable studies with fum-lacZ fusions indicated that transcription of fumC, but not of fumA or fumB, is initiated by RNA polymerase containing sigma 38 . It is concluded that AcnB is the major citric acid cycle enzyme, whereas AcnA is an aerobic stationary-phase enzyme that is specifically induced by iron and redox-stress.

Microbiology, 1997 Dec, 143 ( Pt 12), 3785 - 93
The molecular basis for the differential regulation of the hlyE-encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved activating region 1 contact of HlyX; Green J et al.; The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation . The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant . However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E . coli K-12 strains produce an otherwise latent haemolysin . The gene encoding the 'latent' haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site . Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells . Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes . One major transcript (tsp1) initiating at 78.5 bp downstream of the FNR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected . From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter . Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing beta to beta 11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved . The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.

Int J Parasitol, 1997 Nov, 27(11), 1419 - 28
Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle; Estuningsih SE et al.; Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F . gigantica antigen, as vaccines against tropical fasciolosis . The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E . coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's) . Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant . Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant . There was no correlation between total antibody titres to FABP and protection . The protection observed in cattle vaccinated with native FABP of F . gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke . This is the first report of vaccination of cattle against F . gigantica with a purified protein.

Biotechniques, 1997 Dec, 23(6), 1087 - 92
Inexpensive handheld device for the construction of high-density nucleic acid arrays; Schummer M et al.; We report an easy-to-use, 384-pin handheld arraying and replicating device (ARD) for constructing high-density replicas of nucleic acids and E . coli transformants . We have modified an existing 384-pin tool to include a novel guide system to ensure vertical pin movement and enhance reproducibility . An asymmetric rectangular multiplexing frame is designed to increase the array density to 1536 dots on a standard microplate-size nylon membrane and to reduce the time and effort involved in producing array replicas . Our initial studies used the ARD to construct 1536-dot arrays of ovarian cDNA clones . We have hybridized these arrays with 32P-labeled probes, which resulted in distinctive signals for either visual interpretation or semiautomatic spot detection and signal integration.

Biotechniques, 1997 Dec, 23(6), 1070 - 2, 1074-5
Long PCR facilitates concise cloning and sequencing with a minimal tiling set of templates; Burland V et al.; We demonstrate a rapid cloning and sequencing strategy for kilobase-size DNA segments using DNase I and long PCR . In a single-tube protocol, deletions were formed in a plasmid insert by two enzymatic cuts, one at a fixed site and one at random . The doubly cut molecules were recircularized to generate a library of plasmids carrying deletions of various sizes and transformed into E . coli . The plasmid inserts were directly amplified from transformant colonies by long PCR and sized on a high-resolution agarose gel . A minimal tiling set, selected from the amplified material, was used directly as templates for long-read sequencing . The system is useful for inserts up to about 3.5 kb for de novo sequencing (both strands) or 6 kb for confirmatory sequencing (one strand).

Biotechniques, 1997 Dec, 23(6), 1062 - 8
Lac repressor inducible gene expression in human breast cancer cells in vitro and in a xenograft tumor; Lee AV et al.; We have studied the lac repressor (lacR) system in two breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro and in vivo . Breast cancer cell lines were stably transfected with lacR and tested for inducibility by transient transfection with a lac operator/luciferase reporter plasmid . The level of expression of lacR did not appear to correlate with the basal or maximal activation of induction by isopropyl beta-D-thiogalactoside (IPTG) . Stable transfection with the same reporter gene resulted in up to 40-fold (MDA-MB-231) and 50-fold (MCF7) induction . In the absence of IPTG, a low level of basal reporter gene expression was seen in all clones . Detailed analysis showed that induction was rapid (maximal at 24 h), reversible (a return to basal expression by 24 h) and dose-dependent . To test if this system was also inducible in vivo, cells were grown as a xenograft tumor in nude mice . Mice were given IPTG (0.53 mmol) by intraperitoneal injection, and the tumors were biopsied at several time points following administration . IPTG caused a 10-fold increase in luciferase activity after 8 h, which persisted for 24 h . Thus, this system allows tightly controlled inducible in vivo and in vitro gene expression with low basal expression, and it may provide an important tool for the study of lethal genes in human breast cancer cells.

Biochim Biophys Acta, 1997 Nov 8, 1349(1), 13 - 24
A novel high-molecular mass mammalian ethanolamine kinase; Uchida T; The present report describes, for the first time, a mammalian kinase highly specific for ethanolamine (Km = 41 microM) . Ethanolamine kinase catalyzes the initial step in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis . Although plant and protozoan kinases are known to exhibit remarkable specificity for ethanolamine, no equivalent mammalian kinase has been characterized previously . The easily shocked (eas) mutant of Drosophila has been characterized and found to lack ethanolamine kinase activity while still possessing normal choline kinase activity (Pavlidis, P., Ramaswami, M., and Tanouye, M.A . (1994) Cell 79, 23-33) . The gene compensating this mutation encodes a sequence resembling that of choline kinase . In the present report, the eas gene product, expressed in Escherichia coli as fusion protein, is found to have highly specific ethanolamine kinase activity . Anti-eas antibody revealed a protein with a molecular mass of approximately 86 kDa in rat tissues and human HeLa cells by Western blotting, but did not bind rat choline kinase isozymes . Rat liver kinase activity specific for ethanolamine was separated chromatographically and by an immunological procedure using anti-choline kinase antibody, associated with the 86 kDa protein, and immunoprecipitated by anti-eas antibody . This 86 kDa protein is characterized as ethanolamine kinase . Relations with previously reported kinases are discussed.

Scand J Immunol, 1997 Dec, 46(6), 587 - 96
Enrichment and characterization of dendritic cells from rat renal mesangium; Gieseler R et al.; Dendritic cells (DC) initiate primary immune reactions and are distributed throughout most tissues . The most potent DC population of the kidney has long been suggested to reside within the glomerular mesangium . Using LEW.1A rats, we enriched and characterized such low-density cells . Mesangial DC generally exhibited round to oval cell bodies and cytoplasmic veils . Phenotypically, these cells were 100% OX-6++, 45% OX-42++, 35% ED1low, 10% OX-62low, and negative for ED2 and alpha-naphtylbutyrate esterase . Introducing a new monoclonal antibody, R3, which stains a subset of splenic DC, we showed strong antigen expression on 60% of mesangial DC . Correlating cell populations were detected immunohistochemically . Functionally, mesangial DC potently stimulated allogeneic mixed leucocyte reactions, but did not phagocytose opsonized Escherichia coli . In addition to their striking phenotypic similarity with autologous splenic DC, mesangial DC exhibited 88% of the allostimulatory activity of splenic DC . Calculation indicated approximately two mesangial DC per glomerulum . We suggest that these cells comprise different maturation-dependent subsets . The OX-62 integrin especially appears to be expressed only on mature mesangial DC, which may correlate to lymphoid veiled cells or interdigitating DC . An employment of mesangial DC in experimental models of acute allograft rejection or glomerulonephritis is discussed.

Protein Sci, 1997 Dec, 6(12), 2677 - 80
Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum; Flick KE et al.; The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene . This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron . The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E . coli . Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex . The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A . The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton . These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.

J Virol, 1998 Jan, 72(1), 104 - 12
Repression of the A8L gene, encoding the early transcription factor 82-kilodalton subunit, inhibits morphogenesis of vaccinia virions; Hu X et al.; The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively . A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles . The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle . To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the Escherichia coli lac operator system . The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels . Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer . Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication . Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect . Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.

Electrophoresis, 1997 Oct, 18(11), 2071 - 7
Difference gel electrophoresis: a single gel method for detecting changes in protein extracts; Unlu M et al.; We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples . This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images . The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them . Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels . DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels . Moreover, an inducible protein from E . coli was detected after 15 min of induction and identified using DIGE preparatively.

Clin Exp Allergy, 1997 Nov, 27(11), 1307 - 13
Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen; Asturias JA et al.; BACKGROUND: Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of the crossreactivity found among pollen and food allergic patients . This allergen is usually under-represented in natural extracts used for allergy diagnosis . OBJECTIVES: To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy . METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers . Expression in Escherichia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose . RESULTS: Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced . Full-length C . dactylon profilin gene was expressed in Escherichia coli as non fusion protein . Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained . The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen . Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower profilin . CONCLUSIONS: Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.

Mol Cell Biol, 1998 Jan, 18(1), 608 - 15
Human IAP-like protein regulates programmed cell death downstream of Bcl-xL and cytochrome c; Duckett CS et al.; The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes . Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors . hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis . hILP and Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway . Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-xL . In contrast, hILP, but not Bcl-xL, inhibited apoptosis induced by microinjection of cytochrome c . These data suggest that while Bcl-xL may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.

Mol Mar Biol Biotechnol, 1997 Dec, 6(4), 357 - 63
A functional study of the salmon GnRH promoter; Husebye H et al.; The Pa and Pb promoters of the Atlantic salmon (Salmo salar) GnRH gene were fused together or individually to the Escherichia coli lacZ gene, and their transcriptional activities were measured in transient expression assays in zebrafish (Danio rerio) . In 48-hour embryos, both promoters were preferentially expressed in the brain, whereas a cytomegalovirus (CMV) promoter-lacZ fusion gene displayed high levels of activity in nonbrain tissues . Pa and Pb exhibited different cell specificity in the forebrain . Pb was active in large neuron-like cells exclusive in the olfactory placode region, whereas Pa appeared active in nonneuron-like cells in the forebrain . In Atlantic salmon forebrain tissue, both Pa and Pb exhibited endogenous activity, as assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis . However, only the Pb transcript contained the prepro-GnRH exon II-IV sequences, suggesting that Pa activity may not be related to GnRH production in this species.

Mol Mar Biol Biotechnol, 1997 Dec, 6(4), 345 - 50
Expressed sequence tags of medaka (Oryzias latipes) liver mRNA; Hirono I et al.; A medaka liver cDNA library was constructed in lambda ZAPII . The number of clones in this library is approximately 5 x 10(6) . Three hundred sixty-one clones were randomly selected and, of these, 33 clones with over 1 kb were sequenced . These sequences were compared with GenBank and the dbEST (Re.96.0) . Twenty-five clones of these 33 clones encoded 18 different genes and 2 different expressed sequence tags (ESTs) . Sequences of 10 of the 18 clones had not previously been reported in fish . The codon usage of medaka genes is similar to that of Xenopus, mouse, and human genes, but not similar to that of yeast and Escherichia coli genes.

J Microsc, 1997 Nov, 188 ( Pt 2), 96 - 105
X-ray microscopy and imaging of Escherichia coli, LPS and DNA; Rajyaguru JM et al.; Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs . X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources . We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material . To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy . This approach eliminates the specimen preparation steps . Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist) . This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc . The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV . The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image . By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules . This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 67 - 72
Regulation of fumarase (fumB) gene expression in Escherichia coli in response to oxygen, iron and heme availability: role of the arcA, fur, and hemA gene products; Tseng CP; Three distinct fumarases, FumA, FumB and FumC, have been reported in Escherichia coli . While the fumA and fumC gene products are expressed under aerobic cell growth conditions, the FumB fumarase appears to be more abundant during anaerobic growth . To study the transcriptional regulation of the fumB gene, a fumB-lacZ operon fusion was constructed and analyzed in a single copy under a variety of cell culture conditions . Expression of fumB-lacZ was fourfold higher under anaerobic than aerobic growth conditions . This anaerobic response is modulated by the ArcA and Fnr proteins, which function independently as anaerobic activators of fumB gene expression . Cellular iron limitation in a fur mutant caused fumB-lacZ expression to decrease sevenfold while cellular heme limitation decreased fumB gene expression twofold . In addition, fumB-lacZ expression was shown to vary depending on the DNA superhelicity . This study further delineates the regulation of the fumB gene in cell growth.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 19 - 25
Identification of gene products from the Azotobacter vinelandii nifBfdxNnifOQ operon; Bosch R et al.; The Azotobacter vinelandii nifBfdxNnifOQ operon is required for synthesis of the nitrogenase iron-molybdenum cofactor . To further characterize the roles of its gene products, specific antibodies against NifB and NifO were generated, and the NifB, NifO and NifQ gene products were visualized and identified in nitrogen-fixing A . vinelandii cell extracts by a combination of two-dimensional gel electrophoresis of radiolabelled extracts and immunological detection methods . The three proteins showed apparent pI and M(r) values similar to those expected from sequence data, except for NifO, which showed an apparent M(r) of ca . 23 kDa (vs . 16 kDa expected).

Lett Appl Microbiol, 1997 Nov, 25(5), 325 - 6
Improved detection of enteroaggregative Escherichia coli using formalin-fixed HEp-2 cells; Spencer J et al.; Certain strains of enteroaggregative Escherichia coli cause detachment of HEp-2 cells during adhesion tests, preventing observation of the aggregative adherent phenotype . The use of formalin-fixed HEp-2 cells prevents cell detachment and facilitates the detection of enteroaggregative E . coli.

Comp Biochem Physiol B Biochem Mol Biol, 1997 Sep, 118(1), 13 - 5
Developmental changes in UDP-N-acetylglucosamine 2-epimerase activity of rat and guinea-pig liver; Gal B et al.; The activity of UDP-N-acetylglucosamine 2-epimerase was determined in the liver of rats and guinea-pigs of different ages . The activity of this enzyme in rats was low at birth, increased to a maximum value on day 15, and fell gradually until day 30 . Thereafter, it increased up to the 60th day . The activity profile of the enzyme from guinea-pig liver was very similar . However, guinea-pig activity was 2-5 times lower than in rats . Both rats and guinea-pigs displayed similar liver sialic acid contents which increased from birth to 2 months of age . Rats also showed a N-glycolylneuraminic acid content that decreased from birth to 2 months . From these results we can inferred that postnatal UDP-N-acetylglucosamine 2-epimerase activity seems to be correlated with age and the developmental states of rats and guinea-pigs.

J Mol Biol, 1997 Dec 12, 274(4), 608 - 21
Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure; Blickling S et al.; DHDPS is the first enzyme unique to the lysine biosynthetic pathway in plants and bacteria and catalyses the formation of (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid . It is feedback-regulated in plants by L-lysine . The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved to a resolution of 2.8 A . The molecule is a homotetramer composed of a dimer of dimers . Comparison with the structure of Escherichia coli DHDPS showed a novel quaternary structure by a profound rearrangement of the dimers forming the tetramer . The crystal structure of the enzyme in the presence of L-lysine revealed substantial changes . These changes together with the novel quaternary structure provide a structural basis for the strong inhibition of plant DHDPS enzymes by L-lysine.

J Mol Biol, 1997 Dec 12, 274(4), 546 - 61
The Escherichia coli dnaA gene: four functional domains; Sutton MD et al.; The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101 . Twenty-eight novel missense mutations of the E . coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector . Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here . Results suggest that E . coli DnaA protein contains four functional domains . Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain . The second domain maps to a region near the C terminus and is involved in DNA binding . The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize . Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.

J Mol Biol, 1997 Dec 12, 274(4), 446 - 53
Crystal structure of herbicide-detoxifying maize glutathione S-transferase-I in complex with lactoylglutathione: evidence for an induced-fit mechanism; Neuefeind T et al.; Glutathione S-transferases (GSTs) -I and -III are involved in herbicide metabolism in maize and have been intensively studied . Starting with plant tissue from Zea mays var . mutin recombinant GST-I was prepared by heterologous expression in Escherichia coli . The enzyme was crystallized in the presence of lactoylglutathione, a ligand formerly never observed in a GST structure and known as an intermediate of the pharmacologically relevant glyoxalase system . The crystal structure of GST-I has been determined at 2.5 A resolution and exhibits the GST-typical dimer of two identical subunits, each consisting of 214 residues . Compared with other plant GSTs the three-dimensional structure of GST-I primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region . Furthermore, a comparison of the ligand-bound GST-I structure with the apo structure of GST-III indicates the movement of a ten-residue loop upon binding of the ligand to the active site . This is the first structure-based evidence for an induced fit mechanism of glutathione S-transferases, which has previously been postulated for class pi enzymes . Together with GST-III, GST-I may explain herbicide resistance and selectivity in maize as well as in other agronomic relevant crops.

Anal Biochem, 1997 Dec 15, 254(2), 187 - 91
An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay; Kadono-Okuda K et al.; We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with {alpha-32P}GTP . The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E . coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting . Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina . The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules . This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity . Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

Protein Sci, 1997 Dec, 6(12), 2681 - 3
HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies; Franken P et al.; Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction . Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase . One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure . The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A . The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A . Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

Protein Sci, 1997 Dec, 6(12), 2650 - 4
Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant; van den Akker F et al.; Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general . For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present . The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution . Its structure appears to be essentially the same as the wild-type LT-I structure . The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis . In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I . The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E . coli infections . The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.

Protein Sci, 1997 Dec, 6(12), 2636 - 8
Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli; Zhou M et al.; Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time . Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5 . These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector . They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A . A complete native data set has been collected at 3.1 A resolution for these crystals.

Protein Sci, 1997 Dec, 6(12), 2578 - 88
Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain; Gast K et al.; The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding . Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2 . It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly . Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2 . Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting . These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure . This finding will be discussed in terms of early events in protein folding.

Protein Sci, 1997 Dec, 6(12), 2504 - 11
Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu; Strop P et al.; Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features . The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation . Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone . Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold . The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution) . The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate . The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group . Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.

Comput Chem, 1997, 21(4), 237 - 56
Artificial neural networks for molecular sequence analysis; Wu CH; Artificial neural networks provide a unique computing architecture whose potential has attracted interest from researchers across different disciplines . As a technique for computational analysis, neural network technology is very well suited for the analysis of molecular sequence data . It has been applied successfully to a variety of problems, ranging from gene identification, to protein structure prediction and sequence classification . This article provides an overview of major neural network paradigms, discusses design issues, and reviews current applications in DNA/RNA and protein sequence analysis.

J Radiat Res (Tokyo), 1997 Sep, 38(3), 165 - 71
Mutational specificity of the ferrous ion in a supF gene of endonuclease III/VIII deficient Escherichia coli; Shimamura H et al.; When 125 microM Fe2+/EDTA treated plasmid pUB3 was used to transfect an Escherichia coli NKJ2004 (nth nei) host, which is totally defective in glycosylases for thymine glycol and 5-hydroxycytosine, a 3.7 fold increase in mutation frequency was observed . Among 46 supF mutants sequenced, 28 had base substitutions, with G:C-->C:G transversion predominant (14 cases), followed by G:C-->T:A transversion (6 cases) and G:C-->A:T transition (6 cases) . The results are consistent with our previous Fe2+ mutagenesis results where, in the wild type host, 78% were base substitutions, with G:C-->C:G transversion (59%) predominant, followed by G:C-->T:A transversion (28%) and G:C-->A:T transition (11%) . Treatment of pUB3 DNA with Fe2+/EDTA did not yield formation of Endonuclease III sensitive sites . The possibility of 5-hydroxycytosine as the causative lesion for Fe2+ induced G:C-->C:G transversion is discussed.

Protein Expr Purif, 1997 Nov, 11(2), 201 - 8
High-level expression of ovine growth hormone in Escherichia coli: single-step purification and characterization; Appa Rao KB et al.; The gene for ovine growth hormone (oGH) was expressed without signal sequences in Escherichia coli . A recombinant plasmid expression vector has been constructed which directs the synthesis of a fusion protein containing a stretch of six histidine residues (His6) at the amino-terminus under the control of a T5 promoter . Upon induction with isopropyl-beta-D-thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular protein . The recombinant ovine growth hormone containing His tag was recovered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity Ni2+.NTA resin that has selectivity for proteins with neighboring histidine residues . Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and amino terminal analysis demonstrated the authenticity of the fusion protein . The purified RoGH after refolding was found to be functionally active in terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio immuno assay . Yields of the purified expressed protein were found to be 32 microg/ml at a shake-flask level . Thus, results indicate that a combination of E . coli expression and affinity purification by Ni2+.NTA chromatography promises to be a rapid method to produce oGH for use in structure-function studies .

Protein Expr Purif, 1997 Nov, 11(2), 185 - 94
Expression, purification, and characterization of rat aromatic L-amino acid decarboxylase in Escherichia coli; Jebai F et al.; A cDNA encoding rat aromatic L-amino acid decarboxylase (AADC) was successfully expressed in Escherichia coli using a T7 RNA polymerase expression system . Two types of expression vectors were tested and revealed to be equivalent to produce AADC . The enzyme was purified in both cases . The ratio of recovery of the pure active recombinant protein was better when the purification of the protein was made easier by addition of a short His-Tag at the C-terminal moiety of AADC, as achieved in the case of pET-20b+ vector expression . Spectral characteristics of the bound pyridoxal-5'-phosphate were essentially identical to the spectral properties of rat AADC . Kinetic constants Km and Vmax of recombinant AADC for the natural substrates L-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mM and 8444 U/mg, and 0.066 mM and 1813 U/mg, respectively . These values were in good agreement with previously reported values for AADC of the rat and other mammalian species .

Protein Expr Purif, 1997 Nov, 11(2), 179 - 84
Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus; Lu C et al.; Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification . Previously the protein has been purified from P . furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system . We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No . 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction . However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside . The protein was purified by heating (to denature E . coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns . The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions . This bacterial expression system appears to be the method of choice for production of Pfu .

Protein Expr Purif, 1997 Nov, 11(2), 169 - 78
Refolding of a recombinant collagen-targeted TGF-beta2 fusion protein expressed in Escherichia coli; Han B et al.; In this study, a tripartite transforming growth factor-beta (TGF-beta2) fusion protein bearing an N-terminal purification tag and an auxiliary collagen binding decapeptide has been constructed and expressed at high levels in Escherichia coli . The resulting recombinant protein accumulates in an insoluble and biologically inactive inclusion-body complex . The insoluble protein was solubilized in guanidine hydrochloride and a Ni-chelating affinity column was utilized to isolate the 13.5-kDa TGF-beta2 fusion protein, which was then refolded into its native conformation under controlled redox conditions . The formation of native homodimers was monitored by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels and the bioactivity determined by a quantitative TGF-beta assay system using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter/luciferase reporter plasmid . To optimize yields, renaturation conditions including denaturants, limiting protein concentrations, redox ratios, dialysis conditions, and refolding kinetics were studied and monitored by bioactivity . These studies demonstrate that recombinant TGF-beta2 fusion proteins can be produced in E . coli and renatured into biologically active homodimers . Furthermore, they confirm that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen . Taken together, these studies advance the technology necessary to generate large quantities of targeted TGF-beta fusion proteins for specific biomedical applications .

Protein Expr Purif, 1997 Nov, 11(2), 162 - 8
Purification of ribonucleases Sa, Sa2, and Sa3 after expression in Escherichia coli; Hebert EJ et al.; The genes for three small ribonucleases from different strains of Streptomyces aureofaciens have been cloned and expressed in Escherichia coli . The purification of these ribonucleases from the periplasmic space is described . The yields range from 10 to 50 mg of protein per liter of culture medium . The molar absorption coefficients, isoelectric pH values, and pH of optimum activity are reported .

J Mol Biol, 1997 Nov 14, 273(5), 1020 - 31
Affinities and selectivities of divalent cation binding sites within an RNA tertiary structure; Bukhman YV et al.; A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA, nucleotides 1051 to 1108, adopts a specific tertiary structure normally requiring both monovalent (NH4+ or K+) and divalent (Mg2+) ions to fold; this ion-dependent structure is a prerequisite for recognition by ribosomal protein L11 . Melting experiments have been used to show that a sequence variant of this fragment, GACG RNA, is able to adopt a stable tertiary structure in the presence of 1.6 M NH4Cl and absence of divalent ions . The similarity of this high-salt structure to the tertiary structure formed under more typical salt conditions (0.1 M NH4Cl and several mM MgCl2) was shown by its following properties: (i) an unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii) selectivity for NH4+ over K+ or Na+, (iii) stabilization by L11 protein, and (iv) further stabilization by added Mg2+ . Delocalized electrostatic interactions of divalent ions with nucleic acids should be very weak in the presence of >1 M monovalent salt; thus stabilization of the tertiary structure by low (<1 mM) Mg2+ concentrations in these high-salt conditions suggests that Mg2+ binds at specific site(s) . GACG RNA tertiary structure unfolding in 1.6 M NH4Cl (Tm approximately 39 degrees C) is distinct from melting of the secondary structure (centered at approximately 72 degrees C), and it has been possible to calculate the free energy of tertiary structure stabilization upon addition of various divalent cations . From these binding free energies, ion-RNA binding isotherms for Mn2+, Mg2+, Ca2+, Sr2+ and Ba2+ have been obtained . All of these ions bind at two sites: one site favors Mg2+ and Ba2+ and discriminates against Ca2+, while the other site favors binding of smaller ions over larger ones (Mg2+ >Ca2+ >Sr2+ >Ba2+) . Weak cooperative or anticooperative interactions between the sites, also dependent on ion radius, may also be taking place .

Biochim Biophys Acta, 1997 Dec 4, 1330(2), 113 - 20
Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits; Ghaim JB et al.; Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E . coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides . The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.

Reprod Toxicol, 1997 Nov-Dec, 11(6), 771 - 9
In vivo and in vitro developmental toxicity in LPS-induced zinc-deficient rabbits; Pitt JA et al.; Lipopolysaccharide (LPS) was used to induce maternal hypozincemia in order to test the hypothesis that altered zinc homeostasis is developmentally toxic in the rabbit . Treatment of dams on Gestation Day (GD) 8 with LPS (0.67 microgram/kg i.v.) caused total resorption of 78% (7 of 9) of the litters whereas GD 10 treatment increased the percentage of resorbed implantations (18-fold), but resulted in only 14% (1 of 7) totally resorbed litters . Cotreatment with zinc oxide (ZnO) on GD 10 decreased the resorption rate by 44%, indicating that hypozincemia was partially responsible for the resorptions . However, ZnO had no effect on resorption rate in GD 8 LPS-treated dams . No malformations were observed with LPS dosing on either gestation day . To determine whether LPS-induced Zn deficiency had any direct effects on rabbit embryos, normal GD 9 embryos were cultured for 48 h in serum from LPS-treated dams (0.53 +/- 0.01 microgram/mL Zn) or from controls (1.74 +/- 0.07 micrograms/mL Zn) . Embryo growth and development were normal in both groups, indicating a lack of any direct embryo effects of Zn deficiency . Finally, maternal plasma progesterone and the Zn content of conceptus tissues were measured 24 h after LPS injection on GD 10 . Despite a marked decrease in maternal serum Zn, no significant changes in embryo, visceral yolk sac, yolk sac cavity-exoceolomic fluid, or placental Zn were found . However, maternal progesterone levels were decreased 33 and 28% in the LPS and LPS + ZnO groups, respectively . Taken together, these results indicate that rabbits may differ from rodent species in their lesser susceptibility to the teratogenic potential of transient maternal Zn deficiency, as well as in their resistance to conceptus Zn changes . Nonetheless, Zn deficiency may be responsible for an increase in resorption rate in the rabbit.

Yakugaku Zasshi, 1997 Nov, 117(10-11), 910 - 21
{Lethal drug interactions of the new antiviral, sorivudine, with anticancer prodrugs of 5-fluorouracil}; Watabe T et al.; In 1993 eighteen Japanese patients with cancer and herpes zoster, a viral disease, died from interactions of the new oral antiviral drug, sorivudine (SRV: 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil), with oral anticancer prodrugs of 5-fluorouracil (5-FU) within 40 d after SRV was approved by the Japanese government and began to be used clinically . Before the death, most of these patients had severe symptoms of toxicity, including diarrhea with bloody flux and marked decreases in white blood cell and platelet counts . All of these patients received SRV daily for several days while being administered long-term anticancer chemotherapy with one of the oral 5-FU prodrugs . There was no acute toxic symptom in patients who received SRV alone or SRV and the other types of anticancer drugs . A toxicokinetic study was carried out using rats to investigate the mechanism of the acute death in the patients caused by drug interactions between SRV and 5-FU prodrugs . Rats were orally coadministered SRV with tegafur (FT: 1-(2-tetrahydrofuryl)-5-fluorouracil), a 5-FU prodrug that most of the patients were considered to receive before the death . All the rats receiving SRV and FT once daily showed extremely elevated levels of 5-FU in the plasma and tissues, including bone marrow and small intestines, and died within 10 d, while the animals given the same repeated dose of SRV or FT alone were still alive over 20 d without any appreciable toxic symptom . Before their death, there were a marked damage of bone marrow, a marked atrophy of intestinal membrane mucosa, marked decreases in white blood cells and platelets, diarrhea with bloody flux, and severe anorexia as reported for the patients . Data obtained by in vivo and in vitro studies indicated that (E)-5-(2-bromovinyl)uracil (BVU), generated from SRV by the gut flora and absorbed through the intestinal membrane, was reduced in the presence of NADPH to a reactive form by hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the tissue 5-FU levels from FT, bound covalently to DPD as a suicide inhibitor, and markedly retarded the catabolism of 5-FU . An irreversible inactivation by BVU of rat and human DPDs, expressed in E . coli for the latter, was observed in the presence of NADPH with their purified preparations in a manner reciprocal to radiolabelling of the enzyme proteins with {14C}BVU . SRV showed no inhibitory effect on the rat and human DPDs in the presence of NADPH.

Yakugaku Zasshi, 1997 Nov, 117(10-11), 749 - 63
{Mechanism of decoding mRNA in protein biosynthesis}; Nagano K et al.; Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA and have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of T psi C-loop in P-site tRNA base pair exist along with the conventional base pairs of adjacent codon-anticodon interactions . This A-P tRNA pair model can be translocated to the P-E tRNA model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain . On the other hand, it is noteworthy that C1378 of E . coli 16S rRNA cross-links to the 32 position on the anticodon loop of A-site tRNA in the pre-translocational state, and also to the same position of E-site tRNA in the post-translocational state, instead of the corresponding position of P-site tRNA . It resulted in a relationship between the A-P and P-E tRNA docking pair models in the pre- and post-translocational states, respectively, caused by a rotation with the angle of 25 degrees around the axis of rotation symmetry . Furthermore, nucleotide sequence analysis showed that CGAGC1107 of 16S rRNA is complementary with the conserved GT psi CG57 of tRNA . When it is combined with the P-E tRNA pair model, the crystallographically obtained L-shaped tRNA model fits both A-site codon with base pairings and the free T psi C-loop of P-site tRNA without base pairings . The base pairings between the GT psi CG57 of tRNA and the CGAGC1107 of 16S rRNA destabilize the bound aminoacyl-tRNA and result in a flow of discarding noncognate and near-cognate ternary complexes until cognate one arrives at the A-site codon . Recognition of a cognate ternary complex could occur, starting from breaking a hydrogen bond between N3 atom of U33 and O5' atom of A36 in the aminoacyl-tRNA, with base pairings of the codon-anticodon interactions, the conserved A 1394 in the 16S rRNA to the conserved U33 in the anticodon loop of the tRNA . The exposed U33 of the aminoacyl-tRNA is paired with A1394 in the recognition mode of A site, and finally passed to A1398 of A-site tRNA in the A-P tRNA pair model of the pre-translocational state . The exposure of U33 base at the A site is a key event in the mechanism of codon recognition.

Plant Physiol, 1997 Dec, 115(4), 1721 - 7
Oxidative stress causes ferredoxin-NADP+ reductase solubilization from the thylakoid membranes in methyl viologen-treated plants; Palatnik JF et al.; The flavoenzyme ferredoxin-NADP+ reductase (FNR) is a member of the cellular defense barrier against oxidative damage in Escherichia coli . We evaluated the responses of chloroplast FNR to methyl viologen, a superoxide radical propagator, in wheat (Triticum aestivum L.) plants and chloroplasts . Treatments with the herbicide showed little effect on the levels of FNR protein or transcripts, indicating that expression of this reductase is not upregulated by oxidants in plants . Viologens and peroxides caused solubilization of active FNR from the thylakoids into the stroma, converting the enzyme from a membrane-bound NADPH producer to a soluble NADPH consumer . This response appeared specific for FNR, since other thylakoid proteins were unaffected by the treatments . The reductase-binding protein was released together with FNR, suggesting that it might be the target of oxidative modification . Stromal accumulation of a functional NADPH reductase in response to oxidative stress is formally analogous to the induction of FNR synthesis observed in E . coli under similar conditions . FNR solubilization may be playing a crucial role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid . The unchecked accumulation of NADPH might otherwise increase the risks of oxidative damage through a rise in the Mehler reaction rates and/or the production of hydroxyl radicals.

Plant Physiol, 1997 Dec, 115(4), 1431 - 42
Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules; Dahiya P et al.; The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue . A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein . Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B . The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B . In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C . Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles . Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule . These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.

Plant Physiol, 1997 Dec, 115(4), 1371 - 83
Biochemical and molecular biological characterization of CAC2, the Arabidopsis thaliana gene coding for the biotin carboxylase subunit of the plastidic acetyl-coenzyme A carboxylase; Sun J et al.; The biotin carboxylase subunit of the heteromeric chloroplastic acetyl-coenzyme A carboxylase (ACCase) of Arabidopsis thaliana is coded by a single gene (CAC2), which is interrupted by 15 introns . The cDNA encodes a deduced protein of 537 amino acids with an apparent N-terminal chloroplast-targeting transit peptide . Antibodies generated to a glutathione S-transferase-CAC2 fusion protein react solely with a 51-kD polypeptide of Arabidopsis; these antibodies also inhibit ACCase activity in extracts of Arabidopsis . The entire CAC2 cDNA sequence was expressed in Escherichia coli and the resulting recombinant biotin carboxylase was enzymatically active in carboxylating free biotin . The catalytic properties of the recombinant biotin carboxylase indicate that the activity of the heteromeric ACCase may be regulated by light-/dark-induced changes in stromal pH . The CAC2 gene is maximally expressed in organs and tissues that are actively synthesizing fatty acids for membrane lipids or oil deposition . The observed expression pattern of CAC2 mirrors that previously reported for the CAC1 gene (J.-K . Choi, F . Yu, E.S . Wurtele, B.J . Nikolau {1995} Plant Physiol 109: 619-625; J . Ke, J.-K . Choi, M . Smith, H.T . Horner, B.J . Nikolau, E.S . Wurtele {1997} Plant Physiol 113: 357-365), which codes for the biotin carboxyl carrier subunit of the heteromeric ACCase . This coordination is probably partially established by coordinate transcription of the two genes . This hypothesis is consistent with the finding that the CAC2 and CAC1 gene promoters share a common set of sequence motifs that may be important in guiding the transcription of these genes.

Genetics, 1997 Dec, 147(4), 1653 - 64
Genetic analysis of the Chlamydomonas reinhardtii I-CreI mobile intron homing system in Escherichia coli; Seligman LM et al.; We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease . The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii . I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release . I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance . Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity . These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage . Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not . The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.

Hum Gene Ther, 1997 Nov 20, 8(17), 2043 - 55
Regression of experimental brain tumors with 6-thioxanthine and Escherichia coli gpt gene therapy; Ono Y et al.; The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer . Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 microM, whereas parental C6 cells were not affected at a concentration of 500 microM . In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure . In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities . This "modified" bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19 . In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX) . In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment . This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005) . These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer.

Biophys J, 1997 Dec, 73(6), 3078 - 88
Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system; Morein S et al.; The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed . The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli . Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles . The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations . 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H{II}) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25 . 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable . Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w) . Higher peptide content results in coexistence of L alpha and isotropic phases . Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C . Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system . The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.

Nature, 1997 Dec 18-25, 390(6661), 691 - 4
Neuregulin-beta induces expression of an NMDA-receptor subunit; Ozaki M et al.; Neuregulins (also known as ARIA, NDF, heregulin, GGF) are a family of widely expressed growth and differentiation factors . Neuregulins secreted from motor neurons accumulate at maturing neuromuscular junctions, where they stimulate transcription of genes encoding specific acetylcholine receptors . How these factors function at central synapses, however, is unknown . In the maturing cerebellum, neuregulins are concentrated in glutamatergic mossy fibres that innervate granule cells in the internal granule-cell layer . We have analysed the effects of neuregulins on the expression of genes encoding NMDA (N-methyl-D-aspartate) receptors in the cerebellum, because receptor composition changes dramatically as expression of the receptor NR2C subunit is specifically induced in neurons in the internal granule-cell layer during synaptogenesis . Here we report that addition of a neuregulin-beta isoform to cultured cerebellar slices specifically increases the expression of NR2C messenger RNAs by at least 100-fold; effects are only minor with a neuregulin-alpha isoform . This stimulation of NR2C expression requires synaptic activity by NMDA receptors, as well as neuregulin-beta . Addition of the NMDA-receptor-channel blocker AP-5 prevents upregulation of the NR2C subunit by neuregulin, whereas an AMPA/kainate-receptor antagonist does not . Consistent with these effects of neuregulin, we find that granule cells express its receptors ErbB2 and ErbB4 before the NR2C subunit of the NMDA receptor . Our results indicate that neuregulins regulate the composition of neurotransmitter receptors in maturing synapses in the brain, in a manner analogous to the neuromuscular junction.

J Neuroimmunol, 1997 Dec, 80(1-2), 165 - 71
Spread of HSV-1 to the suprachiasmatic nuclei and retina in T cell depleted BALB/c mice; Matsubara S et al.; Following uniocular anterior chamber inoculation of the KOS strain of HSV-1 in euthymic BALB/c mice, virus spreads from the injected eye to the brain, and from the brain to the optic nerve and retina of the uninjected eye by day 7 post inoculation (p.i.), but the optic nerve and retina of the injected eye are not infected with virus . Infection of the optic nerve and retina of the injected eye is observed only in athymic mice or in mice depleted of both CD4+ and CD8+ T cells . To determine the role of T cells in virus spread, adult female BALB/c mice were thymectomized and T cell depleted . Mice were co-injected with the KOS strain of HSV-1 and RH116, a thymidine kinase-negative mutant of KOS containing the Escherichia coli lac Z gene . Animals were sacrificed on days 3-7 p.i., and the eyes and brains were examined for blue-stained, virus-infected cells . A difference in the timing of virus infection was observed in the area of the suprachiasmatic nuclei only in mice depleted of both CD4+ and CD8+ T cells, and in this group, the contralateral suprachiasmatic nucleus was infected two days earlier . Since one route by which virus could infect the retina of the injected eye is via connections of the contralateral suprachiasmatic nucleus to the ipsilateral optic nerve, these findings suggest that (a) retinitis observed in the injected eyes of mice depleted of both CD4+ and CD8+ T cells results from virus infection of the contralateral suprachiasmatic nucleus followed by spread of virus to the ipsilateral optic nerve and retina and (b) early HSV-1 infection of the contralateral suprachiasmatic nucleus is prevented by a T cell dependent mechanism.

J Neuroimmunol, 1997 Dec, 80(1-2), 131 - 6
Highly purified oligo-His tagged human recombinant alpha(1)-AChR is immunogenic in vivo and suitable for T cell stimulation in vitro in experimental and human myasthenia gravis; Voltz R et al.; Using recombinantly expressed proteins for selection of antigen-specific T cell lines carries a high risk of selecting T cells specific for contaminating proteins . This risk is especially high for very hydrophobic proteins which are notoriously difficult to purify, such as the integral membrane protein acetylcholine receptor (AChR) . We prepared a highly purified recombinant AChR by adding an oligo-histidine affinity-tag to the human alpha(1)-AChR and expressing it in E . coli . This allowed purification by Ni-NTA chromatography and subsequent electroelution from preparative SDS gel as purification steps, resulting in complete purity as assessed by silver stain on SDS-PAGE . This protein preparation induced fatal experimental allergic myasthenia gravis in Lewis rats . Furthermore, the protein could be used to select T cell lines from immunized Lewis rats and patients with myasthenia gravis . However, even with this highly purified protein, one of 8 Lewis rat T cell lines and 3 of 7 human T cell lines cross-reacted to E . coli control proteins . The results show that oligo-histidine tagged, highly purified human alpha(1)-AChR is highly immunogenic in vivo and in vitro.

Biophys J, 1997 Dec, 73(6), 3257 - 64
Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR; Poon J et al.; Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon . It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes . This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.

FEBS Lett, 1997 Nov 24, 418(1-2), 53 - 7
The in vitro DNA binding properties of NDP kinase are related to its oligomeric state; Mesnildrey S et al.; Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function . While the human NDPK-B binds specifically single-strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not . We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild-type does not . These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.

FEBS Lett, 1997 Nov 24, 418(1-2), 35 - 8
The 'assembly-promoting sequence region' of microtubule-associated protein 4 failed to promote microtubule assembly; Katsuki M et al.; In order to study the function of the bovine MAP4 microtubule-binding domain (the assembly-promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system . When the fragment was mixed with purified tubulin at 37 degrees C, the fragment caused a time- and dose-dependent turbidity increase, and the fragment bound to tubulin . However, the products were cold-stable, and amorphous aggregates were observed by electron microscopy . Using axonemes as the seeds for microtubule assembly, the microtubule-elongating activity of the fragment was examined . A dose-dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose-dependently elongated from the axonemes . Consequently, the AP sequence region does not nucleate microtubules, but elongates them.

Genes Cells, 1997 Sep, 2(9), 547 - 57
Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3; Sekine Y et al.; BACKGROUND: An insertion element IS3 is flanked by terminal inverted repeat (IR) sequences . IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB, from which three proteins are produced . OrfAB is a transframe protein produced by -1 translational frameshifting between orfA and orfB, and it is known to be IS3 transposase . OrfA and OrfB are the proteins produced without frameshifting, but their functions have not been elucidated . RESULTS: A plasmid carrying an IS3 mutant that produces only transposase generates miniplasmids--which are the IS3-mediated intramolecular transposition products--as well as characteristic IS3 circles and linear IS3 molecules . OrfA inhibited the generation of these small molecules to a lesser degree, but OrfB did not . OrfB, together with OrfA, however, inhibited the generation more strongly than OrfA alone . OrfA also inhibited the intermolecular transposition of mini-IS3 with the chloramphenicol-resistance gene flanked by IRs to a reduced frequency, and OrfB together with OrfA inhibited it almost completely . OrfA and/or OrfB did not, however, repress transcription from the promoter in the left-terminal region preceding orfA . CONCLUSIONS: The results obtained above show that OrfA and OrfB are not repressors but are inhibitors of transpositional recombination promoted by transposase . OrfA with an alpha helix-turn-alpha helix DNA-binding motif may compete with transposase to bind to terminal IRs . OrfA, together with OrfB that has a DDE motif conserved in retroviral integrases, may inhibit the formation of an active transpososome consisting oftransposase, two terminal IRs and target DNA for the strand transfer reaction . IS3 with a limited size, 1258 bp in length, uses strategies of translational frameshifting and coupling to produce transposase as well as negative regulators to make its copies at a low level, which minimizes a deleterious effect of transposition on bacterial hosts.

Arch Virol, 1997, 142(10), 2059 - 64
Analysis of the non-sense mutants of varicella-zoster virus thymidine kinase; Suzutani T et al.; Three non-sense mutants of varicella-zoster virus (VZV) thymidine kinase (TK) gene, VZTK325, VZTK278 and VZTK224, were isolated . The mutants had a single nucleotide substitution at codons 326, 279 and 225, which changed the codon of TGG (tryptophan) to the stop codon TGA . The wild type (WT) and mutant TKs were expressed in E . coli cells and their characteristics were evaluated . VZTK224 lost TK activity, but VZTK325 and VZTK278 maintained 74.8% and 21.2% of the WT TK activity . On the other hand, all mutants lost the thymidylate kinase activity . Moreover, VZTK325 and VZTK278 polypeptides were heat-labile . These data suggest that the carboxy-terminal portion of herpesvirus TK plays an important role in the stable folding of TK and thymidylate kinase activity.

Arch Virol, 1997, 142(10), 2021 - 33
The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle; De Diego M et al.; A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed . The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E . coli . Experimental and field sera from naive, vaccinated and infected cattle were examined . Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection . In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates) . A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results . Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection . The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys . The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.

Biotechnol Prog, 1997 Nov-Dec, 13(6), 864 - 8
Insertion of stabilizing loci in vectors of T7 RNA polymerase-mediated Escherichia coli expression systems: a case study on the plasmids involving foreign phospholipase D gene; Mishima N et al.; Plasmids carrying stabilizing loci were used in the expression of phospholipase D (PLD) gene fused with pelB signal sequence by a recombinant strain of Escherichia coli BL21 (DE3) using T7 RNA polymerase mediated expression system . By checking the living cell number and the percentage of the plasmid-bearing cells, it was found that the plasmids involving PLD gene were not stable under noninduced conditions and that, after the induction, the number of plasmid-bearing cells were rapidly decreased to almost zero . Then, a biologically stabilizing locus such as par B, ccd, or par was inserted into the plasmids . The newly constructed plasmids were maintained very stably in the recombinant cells until the cells were induced . However, after the induction, almost all the recombinant cells were rapidly killed due to highly toxic PLD . Using the best one of the stabilized PLD-expressing plasmids, PLD production was improved 2-fold.

Vaccine, 1997 Dec, 15(17-18), 1851 - 7
Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response; Kurar E et al.; Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system . As a test of this new methodology, the immune response to the in vivo-expressed Brucella abortus ribosomal L7/12 gene in the muscle cells of mice was examined . To accomplish this goal the eukaryotic expression systems pcDNA3 and p6 were used . Single intramuscular injection of the L7/L12 gene driven by the human cytomegalovirus (CMV) promoter (pcDNA3) or bovine MHC 1 promoter (p6) resulted in intracellular expression of the B . abortus L7/L12 immunodominant protein encoded by this gene . This application facilitated directed antigen presentation to the immune system and established specific antibody and T-cell responses compared with vector only (pcDNA3) negative controls and B . abortus S19 injected positive controls . Although pcDNA3-encoded L7/L12 gene-inoculated mice possessed significant protection, p6-L7/L12 did not engender significant protection against B . abortus S2308 infection compared to positive control mice . These data suggest a promising antigen-specific response, and L7/L12 nucleic acid vaccination may be an initial step in the development of genetically engineered candidate vaccines against brucellosis . This study for the first time focuses on DNA immunization of a gene from B . abortus.

Vaccine, 1997 Dec, 15(17-18), 1827 - 33
Protective immunity against heterologous challenge with encephalomyocarditis virus by VP1 DNA vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene; Sin JI et al.; For DNA vaccination studies, recombinant VP1 protein of encephalomyocarditis virus (EMCV) was produced from Escherichia coli, and eukaryotic VP1 expression vector, pCT-Gs-VP1, was generated and used as a DNA vaccine . Mice were immunized intramuscularly (i.m.) with pCT-Gs-VP1 in the presence or absence of plasmid DNA expressing granulocyte-macrophage colony stimulating factor (GM-CSF), and were subsequently analyzed for their anti-VP1 immune responses with recombinant VP1 in ELISA . Immunization of mice with pCT-Gs-VP1 resulted in VP1-specific immune response and 43% protection from subsequent lethal heterologous challenge of EMCV . Coinjection of mice with pCT-Gs-VP1 and plasmid DNA encoding GM-CSF was shown to increase the seroconversion rate of the immunized mice with a single DNA injection, and enhanced to a higher degree VP1-specific immunity, which appeared to result in better protection (about 80%) from lethal virus challenge . Thus, our results provide evidence for the potential use of GM-CSF to induce better immune response and resistance against viral infection in DNA vaccination.

Am J Respir Crit Care Med, 1997 Dec, 156(6), 1846 - 54
Endotoxin impairs agonist-induced calcium mobilization in rat mesangial cells; Murray PT et al.; We hypothesized that endotoxin would impair agonist-induced calcium (Ca2+) mobilization in rat mesangial cells, owing to the induction of nitric oxide synthase (NOS) and augmented nitric oxide (NO) synthesis . We measured basal and bradykinin-induced peak free cytosolic Ca2+ concentrations through microspectrofluorimetry with fura-2 in confluent mesangial cells, and assayed conditioned medium for nitrite accumulation . Prior to measurement, cells were incubated overnight in serum-supplemented medium, with or without endotoxin, 1-arginine, indomethacin, meclofenamate, or N omega-nitro-L-arginine methyl ester (L-NAME) . Endotoxin (1 mg/ml) decreased bradykinin-induced peak Ca2+ responses by 35 to 60% (p < 0.0001) and increased nitrite accumulation > 6-fold (p < 0.01) . Arginine supplementation further (> 9-fold, p < 0.0001) increased nitrite accumulation without changing the effect on Ca2+ . Inhibition of NOS abolished increments in nitrite concentration but had no effect on impaired Ca2+ responses . Cyclooxygenase (COX) inhibitors, present during incubation with endotoxin, but not afterward, normalized bradykinin-stimulated calcium responses . Thrombin-stimulated Ca2+ responses were similarly affected . We conclude that neither NO nor prostaglandins act directly to impair agonist-induced Ca2+ mobilization following endotoxin exposure; however, this effect may be an indirect effect of COX products, including reactive oxygen intermediates.

Am J Respir Crit Care Med, 1997 Dec, 156(6), 1825 - 33
Effects of inter-alpha-inhibitor in experimental endotoxic shock and disseminated intravascular coagulation; Jourdain M et al.; We investigated the effects of human inter-alpha-inhibitor (I alpha I) on hemodynamics, oxygenation, and coagulation parameters in a porcine model of endotoxic shock . Four groups of six animals were studied: (1) control, (2) I alpha I group receiving 30 mg/kg I alpha I over 30 min, (3) LPS group receiving 5 micrograms.kg/min Escherichia coli endotoxin over 30 min, and (4) LPS + I alpha I group receiving 30 min after endotoxin 30 mg/kg/30 min I alpha I . We measured hemodynamic and oxygenation parameters, usual coagulation markers and plasma levels of thrombin-antithrombin complexes, antithrombin III activity, plasminogen activator tissue type, plasminogen activator inhibitor type 1, von Willebrand factor, tumor necrosis factor-alpha, and I alpha I at baseline and at 30, 60, 90, 120, 180, 240, and 300 min . In the I alpha I group, plasma I alpha I levels reached 447 +/- 23 mg/L just after injection and 287 +/- 39 mg/L at 300 min . I alpha I half-life was 7.3 +/- 1.9 h . In the IPS + I alpha I group, I alpha I plasma levels decreased more rapidly, reaching 260 mg/L at 300 min . Compared with the LPS group, administration of I alpha I normalized the mean arterial pressure and cardiac index, improved the LPS-induced pulmonary hypertension, and resulted in the blunted increase in blood lactate and oxygen extraction ratio . A significant decrease in thrombin-antithrombin complexes and plasminogen activator inhibitor type 1 levels were observed . There was no significant difference in plasma tumor necrosis factor-alpha levels . We concluded that in this hypodynamic model of endotoxin shock, I alpha I administration resulted in a marked improvement in the hemodynamic, oxygenation, and coagulation parameters.

Mol Gen Mikrobiol Virusol, 1997, (4), 29 - 31
{Isolation and analysis of two cDNA-clones expressing the human lymphocyte expression library, inducing DNA-binding activity in vitro}; Boiko VP et al.; Two cDNA clones are sequenced which were isolated from human lymphocyte expression library using Southwestern (DNA-binding) screening with 32P-labeled Alu DNA in the presence of 100-fold excess of unbalanced poly (dI-dC) . In one of the sequenced clones (vb22) an open reading frame (ORF) is detected, encoding protein with a new potential DNA-binding (zink-finger) domain, and DNA-binding activity of the protein is directly confirmed after its expression (as GST-fusion protein) in Escherichia coli . The other sequenced clone (wa12) is partially homologous to 15EST sequences present in GENBANK (April, 1996) and cloned from very different human tissues . Connection of these 15 overlapping GENBANK sequences resulted in a longer sequence covering wa12 and having ORF potentially encoding a new 10 kDa polypeptide without any apparent DNA-binding domains . This connected sequence as well as wa12 sequence having only 65 amino acids ORF are unrecognizable by computer software as the protein-coding regions, and we suppose that wa12 transcripts possess DNA-binding activity . Homopyrimidine blocks in RNA longer than 12 nucleotides are known to bind mirror duplex DNA sequences to form triplexes whose stability is comparable to that of protein-DNA complexes, and human promoters contain many such blocks.

Mund Kiefer Gesichtschir, 1997 Mar, 1(2), 111 - 4
{Cytokines and cytokine receptors in mouth mucosa of immune suppressed patients}; Schliephake H et al.; In vitro studies on epithelial cells suggest that cyclosporin (CsA) inhibits a pathway of production of IL-6, which is mediated by TNF alpha and E . coli . The aim of the present immunohistochemical study was therefore to evaluate the content and location of cytokines and cytokine receptors in the oral mucosa of 10 patients with CsA medication . Ten patients without immunosuppression served as controls . Specimens were procured during surgical treatment and processed by paraffin embedding and as cryosections . Expression of TNF alpha, IL-1 beta and IL-6-receptor was present both in the epithelial layer and the submucosal tissues, but did not differ very greatly between patients with CsA therapy and the controls . IL-6, however, displayed a positive reaction only in the control group, while it was invisible in the group of patients with CsA medication . The immunohistochemical findings of the present study suggest that there may be a lack of mucosal expression of IL-6 under CsA medication . With regard to the broad spectrum of possible effects of proinflammatory cytokines and their multiple interactions, the functional relevance of these findings merits further investigation.

Gene, 1997 Nov 12, 201(1-2), 203 - 9
Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B; Coia G et al.; A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene . Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv . Following production of the scFv protein using an E . coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA) . This gene construct was introduced into E . coli mutator cells and the transformed cells were used as an inoculum for liquid cultures . After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered . Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels . Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv . The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.

Gene, 1997 Nov 12, 201(1-2), 151 - 8
Hepatitis C virus NS5A protein is phosphorylated in vitro by a stably bound protein kinase from HeLa cells and by cAMP-dependent protein kinase A-alpha catalytic subunit; Ide Y et al.; Hepatitis C virus (HCV) has a positive-strand RNA genome that codes for a polyprotein precursor, which is processed co- and post-translationally by cellular and viral proteinases into three structural and at least six non-structural (NS) proteins . The NS5A protein, expressed in mammalian cells, exists in two phosphorylated forms of 56-kDa and 58-kDa . In this study, we provide evidence for a stable association between NS5A and a protein kinase from HeLa cells and hepatocellular carcinoma (HepG2) cells by co-immunoprecipitation and by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein produced in E . coli . This protein kinase could phosphorylate in vitro the native NS5A on serine residues, (GST)-NS5A, histone H1, and casein as substrates . In addition, the GST-NS5A was also phosphorylated in vitro by the cAMP-dependent protein kinase A-alpha catalytic subunit.

FEBS Lett, 1997 Nov 17, 417(3), 409 - 13
The membrane-located osmosensory kinase, EnvZ, that contains a leucine zipper-like motif functions as a dimer in Escherichia coli; Yaku H et al.; The Escherichia coli EnvZ protein is a membrane-located osmosensor, which is a typical member of histidine kinases involved in His-Asp phosphotransfer signaling . We found that EnvZ has a leucine zipper-like motif in its presumed periplasmic domain . The functional importance of this leucine zipper-like sequence was assessed by introducing a number of appropriate amino acid substitutions . The results collectively suggest that certain leucine residues in the leucine zipper-like structure play an important role in the osmotic signal transduction mediated by EnvZ . When cysteine was substituted for the crucial leucine residues, the EnvZ dimer with disulfide bridge was detected in the cytoplasmic membrane . It was thus demonstrated that the EnvZ osmosensor exists and exerts its signaling ability as a dimer.

FEBS Lett, 1997 Nov 17, 417(3), 400 - 4
Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding; Zahn R et al.; An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof . As an illustration we describe the production of polypeptides comprising residues 23-231, 81-231, 90-231 and 121-231 of the human prion protein (hPrP) . Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin . This 'high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation . After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain . The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins . The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies . Preliminary biophysical characterization of hPrP(23-231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.

FEBS Lett, 1997 Nov 17, 417(3), 329 - 32
Free luciferase may acquire a more favorable conformation than ribosome-associated luciferase for its activity expression; Yang F et al.; A variant of firefly luciferase in which the C-terminal end was extended with 44 amino acid residues served as a model protein in this study . After transcription and translation in vitro, the enzyme activity was measured when still attached to the ribosome and when released from the ribosome by incubation with RNase A or puromycin . It was found that the C-terminally extended luciferase already had activity when linked to the ribosome, but its activity was greatly increased when released from the ribosome . These results indicate that the luciferase is folded during synthesis on the ribosome; however, some conformational adjustments occur after its release from the ribosome which are required for the full expression of its enzymatic activity.

Brain Res, 1997 Oct 31, 773(1-2), 149 - 61
Different receptor mechanisms mediate the effects of endotoxin and interleukin-1 on female sexual behavior; Avitsur R et al.; Activation of the immune system by lipopolysaccharide (LPS) produces physiological, neuroendocrine and behavioral effects, some of which are mediated by cytokine production . We have previously shown that the cytokine interleukin-1 (IL-1) inhibits sexual behavior in female, but not male rats, while producing a comparable suppression of locomotion in both sexes . The present study examined the effects of LPS on sexual behavior and locomotion of male and female rats, and the involvement of IL-1 receptors in mediating the effects of IL-1 and LPS on females' behavior . Peripheral (i.p.) administration of LPS (50 or 250 microg/kg) significantly decreased sexual behavior in females, up to 6 h after administration, while it had no effect on male sexual behavior . However, locomotor activity, measured in the open-field test, was similarly reduced by LPS in both males and females . Pretreatment with the IL-1 receptor antagonist (IL-1ra) either i.p . (10 mg/kg) or intracerebroventricularly (i.c.v.) (50 microg/rat) did not prevent the inhibition of female sexual behavior and locomotion induced by either i.p . (50 microg/kg) or i.c.v . (200 or 400 ng/rat) administration of LPS, respectively . However, identical doses of IL-1ra significantly reversed the effects of IL-1beta, administered either i.p . (5 microg/kg) or i.c.v . (50 ng/rat), respectively . These results demonstrate that both LPS and IL-1beta produce marked inhibition of sexual behavior in female, but not in male rats . However, IL-1 receptors are not required for the effects of LPS on sexual behavior in female rats.

RNA, 1997 Nov, 3(11), 1327 - 36
tRNA recognition for modification: solution probing of tRNA complexed with Escherichia coli tRNA (guanosine-1) methyltransferase; Gabryszuk J et al.; The interaction of Escherichia coli tRNA (guanosine-1) methyltransferase and tRNA(1Leu) transcripts has been probed using cleavage with iodine of phosphorothioate-substituted transcripts, lead acetate, and enzymes specific for single- and double-stranded RNA . All lytic agents protect the anticodon stem-loop and variable loop regions against cleavage, and some protection is also seen in core structures of the tRNA . Residues from both strands of the anticodon stem are protected against cleavage with iodine and lead by enzyme, yet positions G37 and G36, which are crucial for catalysis and binding, are not . This suggests that these residues may undergo structural perturbation in the presence of S-adenosyl methionine . Occupancy of the AdoMet site by the product S-adenosyl-homocysteine, a potent inhibitor of the enzyme, has little or no effect on tRNA binding or protection . Enhanced reactivity with lead is seen at residues located in the anticodon stem-loop, extra-loop, and core (C34, U47c, and G49), which suggests some perturbations in RNA structure might accompany binding.

RNA, 1997 Nov, 3(11), 1220 - 32
Intragenic suppression in tRNA: evidence for crosstalk between the D and the T stems; Ramesh V et al.; We showed previously that introduction of two of the three unique features of Escherichia coli initiator tRNA onto an elongator methionine tRNA conferred significant activity in initiation . Surprisingly, introduction also of the third unique feature, the A11:U24 base pair in the D stem, resulted in total lack of accumulation of the mutant Mi:3 tRNA . We show here that the Mi:3 tRNA gene is transcribed efficiently in vitro . Processing of the Mi:3 precursor transcript shows, however, that both the precursor and the mature Mi:3 tRNA are unstable in E . coli extracts . To understand the basis of instability caused by the A11:U24 base pair in the elongator methionine tRNA background, we have isolated and characterized intragenic suppressor mutations in the tRNA that restore its function in translation initiation . Sequence changes in the T stem that convert the existing A51 x C63 mismatch to a base pair in the Mi:3 tRNA result in accumulation of the tRNAs in vivo . The initiation activity and in vivo levels of accumulation of these suppressors are in the order Mi:3/G51:C63 > Mi:3/A51:U63 >> Mi:3/G51.U63 . These results show that the in vivo accumulation of a tRNA with A11:U24 base pair in the D stem depends upon a base pair between positions 51 and 63 in the T stem . Structural analysis in vitro of the Mi:3 and Mi:3/G51:C63 transcripts suggests that the Mi:3 tRNA is unable to adopt a stable tRNA-like conformation . Various considerations suggest that this is most likely due to a high entropic barrier to tertiary interactions, between the D and the T loops necessary for the formation of a stable tRNA structure.

Am J Respir Cell Mol Biol, 1997 Dec, 17(6), 713 - 26
Elevation of manganese superoxide dismutase gene expression by thioredoxin; Das KC et al.; Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates potentially toxic superoxide radical into hydrogen peroxide and dioxygen . This enzyme is critical for protection against cellular injury due to elevated partial pressures of oxygen . Thioredoxin (TRX) is a potent protein disulfide reductase found in most organisms that participates in many thiol-dependent cellular reductive processes and plays an important role in antioxidant defense, signal transduction, and regulation of cell growth and proliferation . Here we describe induction of manganese superoxide dismutase by thioredoxin . MnSOD mRNA and activity were increased dramatically by low concentrations of TRX (28 microM) . Elevation of MnSOD mRNA by TRX was inhibited by actinomycin D, but not cycloheximide, occurring both in cell lines and primary human lung microvascular endothelial cells . mRNAs for other antioxidant enzymes including copper-zinc superoxide dismutase and catalase were not elevated, demonstrating specificity of induction of MnSOD by TRX . Thiol oxidation by diamide or alkylation by chlorodinitrobenzene inhibited MnSOD induction, further indicating a requirement for reduced TRX . Because both oxidized and reduced thioredoxin (28 microM) induced MnSOD mRNA, the intracellular redox status of externally added Escherichia coli oxidized TRX was determined . About 45% of internalized E . coli TRX was reduced, with 8% in fully reduced form and about 37% in partially reduced form . However, when TRX reductase and nicotinamide adenine dinucleotide (NADPH) were added to the extracellular medium with TRX, more than 80% of E . coli TRX was found to be in a fully reduced state in human adenocarcinoma (A549) cells . Although lower concentrations of oxidized TRX (7 microM) did not induce MnSOD mRNA, this concentration of TRX, when reduced by NADPH and TRX reductase, increased MnSOD mRNA six-fold . In additional studies, MCF-7 cells stably transfected with the human TRX gene had elevated expression of MnSOD mRNA relative to vector-transfected controls . Thus, both endogenously produced and exogenously added TRX elevate MnSOD gene expression . These findings suggest a novel mechanism involving reduced TRX in regulation of MnSOD.

J Vet Med Sci, 1997 Nov, 59(11), 1075 - 7
Inhibitory effect on LPS-induced tumor necrosis factor in calves treated with chlorpromazine or pentoxifylline; Ohtsuka H et al.; The inhibitory effect of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) was investigated in a model of endotoxin shock in Holstein calves following an intravenous administration of Esherichia coli endotoxin (LPS) . Initial correlations with its effects on the levels of tumor necrosis factor (TNF), a pivotal mediator of endotoxin shock, and clinical signs were obtained . The pretreatment of CPZ or DEX significantly decreased the serum levels of TNF, and reduced endotoxic shock . But the pretreatment of PTX hardly reduced the increase of serum TNF levels and endotoxin shock . The levels of serum endotoxin were not significantly different a minute of postinjection of LPS in calves . The results of this study indicate that pretreatment of CPZ or DEX inhibit various biological effects on endotoxin in calves.

J Vet Med Sci, 1997 Nov, 59(11), 1023 - 5
Enhancement of passive immunity with maternal vaccine against newborn calf diarrhea; Kohara J et al.; The effects of a maternal vaccine against newborn calf diarrhea associated with group A bovine rotavirus (BRV), bovine coronavirus (BCV), bovine parvovirus and K99 Escherichia coli (E . coli) were examined on a beef cow-calf herd . After vaccination, serum or colostrum antibody titers to BRV, BCV and E . coli K99 in the vaccinated cows were significantly higher than those in unvaccinated control cows . Serum antibody titers to BRV, BCV and E . coli K99 in calves from the vaccinated cows were also significantly higher than those in calves from the control cows for 3-4 weeks after birth . These results suggested that the immunization of cows with the maternal vaccine enhanced the passive immunity levels in calves against BRV, BCV and K99 E . coli.

J Clin Psychopharmacol, 1997 Dec, 17(6), 467 - 71
The CYP2D6 genotype and plasma concentrations of mianserin enantiomers in relation to therapeutic response to mianserin in depressed Japanese patients; Mihara K et al.; The relationship between therapeutic response to racemic mianserin and steady-state plasma concentrations of S(+)- and R(-)-mianserin was studied in 26 Japanese patients with major depression . The daily dose of mianserin was 30 mg, and the duration of treatment was 3 weeks . Regarding S-mianserin, the proportion of responders (final Montgomery-Asberg Depression Rating Scale score of 10 or less) was significantly higher in the plasma concentration range of 10 to 23 ng/mL than outside (10 of 11 vs . 3 of 15, p = 0.0005) . Such a plasma concentration difference between responders and nonresponders was not found for R-mianserin . In 15 patients, the relationships between the CYP2D6 genotype, determined by allele-specific polymerase chain reaction analysis and Escherichia coli RI restriction fragment length polymorphism, plasma concentrations of the enantiomers, and the therapeutic response were studied . Five patients were homozygous for the wild type (wt) allele (wt/wt), nine were heterozygous for the CYP2D6Ch (Ch) allele causing decreased CYP2D6 activity (Ch/wt), and one patient was heterozygous for the Ch allele and the defect allele CYP2D6D (D) (Ch/D) . The Ch/wt group showed significantly higher plasma concentrations of S-mianserin (mean +/- SD: 15 +/- 6 vs . 8 +/- 1 ng/mL, p = 0.007) and proportion of responders (8 of 9 vs . 1 of 5, p = 0.023) than the wt/wt group . The patient with the Ch/D genotype had the highest plasma concentration of S-mianserin (37 ng/mL) and a poor response . No significant relationship was found between the CYP2D6 genotype and plasma concentration of R-mianserin . The study presented here thus suggests that the CYP2D6 genotype plays a major role in controlling plasma concentration of the S-enantiomer of mianserin, which contributes to a major extent to the antidepressant effect during mianserin treatment.

Acta Chir Hung, 1997, 36(1-4), 393 - 4
Effects of nitric oxide synthase inhibition on the hemodynamic changes in hyperdynamic endotoxemia; Wolfard A et al.; In this study we compared the circulatory effects of the arginine analogue non-specific nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine (NNA), and the specific inducible NOS (iNOS) inhibitor S-methylisothiourea (SMT) and S-(2-aminoethyl)-isothiourea (AEST) in a hyperdynamic endotoxemic dog model . Mean arterial pressure (MAP), cardiac output (CO), and myocardial contractility (MC) were measured . A hyperdynamic circulatory response was elicited with a 2-h infusion of a total dose of 5.3 micrograms/kg E . coli endotoxin (ETX) . NOS inhibitory treatment (2 mg/kg) was administrated from the 45th min of endotoxemia . ETX induced a hyperdynamic circulatory response, and a significant myocardial depression . NNA induced a prolonged, SMT a transient increase in MC, both drugs elevated MAP, but decreased CO . AEST significantly prolonged the elevation in CO, but did not affect MAP . Selective inhibition of the iNOS may be a beneficial in sepsis.

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