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| RNA, 2000 Mar, 6(3), 434 - 48 In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase; Geslain R et al.; Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS) . The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli . The effects were interpreted in the light of the crystal structure of ArgRS . Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme . Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation . Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme . Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. Bioorg Med Chem Lett, 2000 Mar 6, 10(5), 407 - 9 Irreversible inhibition of type I dehydroquinase by substrates for type II dehydroquinase; Bello CG et al.; Mechanistic differences between type I and type II dehydroquinases have been exploited in the design of type specific inhibitors . (2R)-2-Bromo-3-dehydroquinic acid (3), (2R)-2-fluoro-3-dehydroquinic acid (5) and 2-bromo-3-dehydroshikimic acid (4), all excellent substrates for type II dehydroquinase, are shown to be irreversible inhibitors of type I dehydroquinase. Plant J, 2000 Jan, 21(2), 189 - 98 Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism; Wuthrich KL et al.; Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC) . RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR) . The gene was expressed at all stages of leaf development and in roots . By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha . The gene of A . thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies . With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated . The reaction required reduced ferredoxin and was sensitive to oxygen . AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments . Upon transport, it was processed to a mature form of 31 kDa . The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO. Mol Biochem Parasitol, 2000 Feb 25, 106(1), 121 - 9 Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii; Odberg-Ferragut C et al.; A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned . The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively . The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa . Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants . Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs . The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies . Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T . gondii . Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages . Southern blot analysis indicated that SOD occured as a single copy gene in T . gondii genome. Mol Biochem Parasitol, 2000 Feb 25, 106(1), 83 - 91 Comparative study of Leishmania mexicana and Trypanosoma brucei NAD-dependent glycerol-3-phosphate dehydrogenase; Marche S et al.; The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism . Here the physicochemical and kinetic properties of natural G3PDH from T . brucei with the recombinant homologue of L . mexicana which share 63% positional identity are compared . Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction . Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations. Rev Inst Med Trop Sao Paulo, 2000 Jan-Feb, 42(1), 9 - 15 Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives; Quiroga M et al.; Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries . The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E . coli in 44 children since their birth and during the first 20 months of their lives . In all of the children studied, we detected at least one category of diarrheagenic E . coli through the 20 months of the study . 510 diarrheagenic E . coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC) . Neither EHEC nor EIEC were identified . The median age for diarrheagenic E . coli colonization was 7.5 months . The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months . A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants . For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast-feeding were associated with an increase of asymptomatic infections. J Membr Biol, 2000 Mar 15, 174(2), 135 - 40 The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI; Ding PZ et al.; The melibiose carrier from Escherichia coli is a sugar-cation cotransport system . Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices . Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI . The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS) . Studies were carried out on both intact cells and inside out vesicles . Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C) . PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C) . Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS . It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel. Br J Pharmacol, 2000 Apr, 129(7), 1309 - 14 The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice; Yoshino S et al.; 1 . We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved . 2 . CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21 . Varying doses of LPS from E . coli were i.p . injected on day 50 . 3 . Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35 . Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50 . An i.p . injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion . Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells . The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-gamma, IL-1beta, and TNF-alpha . LPS from S . enteritidis, S . typhimurium, and K . neumoniae and its component, lipid A from E . coli also reactivated the disease . Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA . 4 . These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans. Appl Environ Microbiol, 2000 Apr, 66(4), 1734 - 6 Genetic and biochemical characterization of a highly thermostable alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus; Debeche T et al.; The gene encoding an alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated . Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90 degrees C) and pH (stable in the pH range 4 to 12) . On the basis of amino acid sequence similarities, this 56, 071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system . However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides. Appl Environ Microbiol, 2000 Apr, 66(4), 1680 - 4 Engineering desiccation tolerance in Escherichia coli; Billi D et al.; Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp . strain PCC 6803 . Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide . The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration. Appl Environ Microbiol, 2000 Apr, 66(4), 1393 - 9 Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process; McLaggan D et al.; The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems . The fate of the ESG has not previously been investigated . In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E . coli . The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released . N-ethylmaleamic acid is not toxic to E . coli cells even at high concentrations . The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm . The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione . The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems. Appl Environ Microbiol, 2000 Apr, 66(4), 1311 - 20 Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains; Ren Q et al.; To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions . Experiments with different substrates (fatty acids with different chain lengths) or different E . coli hosts failed to produce PHAs with altered physical properties . Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E . coli hosts . By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor . Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point . The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties. Nat Struct Biol, 2000 Apr, 7(4), 298 - 303 Structural insights into substrate binding by the molecular chaperone DnaK; Pellecchia M et al.; How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones . We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro . We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding . Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site . Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures . Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity. Extremophiles, 2000 Feb, 4(1), 43 - 51 MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension; Li WT et al.; All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold . In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly . In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension . The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4 . The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1 . Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E . coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones. J Membr Biol, 2000 Mar 1, 174(1), 31 - 40 Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-{X}-R/K-R/K, of the lactose permease of Escherichia coli; Pazdernik NJ et al.; The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG{X}(R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9 . In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail . Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression . Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability . By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport . A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity . An analysis of site-directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3 . To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function . One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys) . Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes. Kansenshogaku Zasshi, 2000 Feb, 74(2), 134 - 42 {The prevalence of virulence-related genes, eaeA, aggR and astA, of localized and aggregative-adherent Escherichia coli (EPEC and EAggEC) in healthy children and age-matched patients with diarrhea}; Moriya K et al.; The prevalence of virulence-related genes of localized- and aggregated-adherent Escherichia coli (EPEC and EAggEC), such as eaeA, aggR and astA was compared between E . coli isolated from 0 to 5 year old children with and without diarrhea in Saga Prefecture . In the case of eaeA, 233 cases in Aichi Prefecture were included . The subjects were 74 diarrheal patients from which no diarrheagenic bacteria were detected besides E . coli . The control subjects were 304 nursery school children without diarrhea, and E . coli was isolated from 278 children in which 105 strains were of 0-serotype . EaeA-positive E . coli was isolated from nine (12.2%) Saga cases, 19 (8.2%) Aichi cases and 6 (5.7%) control subjects; aggR-positive E . coli was isolated from 10 (13.5%) cases and 6 (5.7%) control subjects and astA-positive E . coli from 10 (13.5%) cases and 14 (13.3%) control subjects . No significant difference (p > 0.05) was observed in the prevalence of eaeA, aggR and astA between healthy and diarrheal children, even in age-matched and 0-serotypable E . coli limited comparisons . The pathogenicity of EPEC and EAggEC should be investigated, considering other known or unidentified factors. Anal Chem, 2000 Mar 15, 72(6), 1134 - 43 Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification; Chen X et al.; Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins . A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides . A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization . Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence . Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification . Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing . Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern . Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra . This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications. J Biochem (Tokyo), 2000 Apr, 127(4), 665 - 71 Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine:pyruvate/alanine:glyoxylate aminotransferase; Oda T et al.; In the rat liver, two mRNAs for serine:pyruvate (or alanine:glyoxylate) aminotransferase are generated from a single gene by alternative transcription initiation . The longer mRNA encodes a precursor of a mitochondrial enzyme that has a mitochondrial targeting signal at the N-terminus and is translocated into mitochondria . The shorter mRNA encodes a peroxisomal enzyme of mature size that is imported into peroxisomes . We have been interested in the mechanism of selective targeting to mitochondria of the precursor protein that also contains a peroxisomal targeting signal in the molecule . In this study, we examined the effect of the mitochondrial targeting signal on the conformation of the protein and on the function of the peroxisomal targeting signal in the precursor molecule . The results suggest that the mitochondrial targeting signal causes the conformation of the protein to become unfolded and that this conformational change in turn causes repression of the putative peroxisomal targeting signal contained in the precursor protein. J Biochem (Tokyo), 2000 Apr, 127(4), 627 - 33 A new approach to gene mutation analysis using "GFP-Display"; Aoki T et al.; The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides . This simple detection method using SDS/urea gels was designated GFP-display . The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide . K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli . Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed . These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature . Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7 . Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed . GFP-display could be coupled with an in vitro translation system . Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours . GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies. J Biochem (Tokyo), 2000 Apr, 127(4), 559 - 67 Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions; Mihara H et al.; We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate . All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine . These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme . The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine . When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine . Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine . Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine . We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS . The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected . This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization. J Biochem (Tokyo), 2000 Apr, 127(4), 537 - 41 The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli; Uga H et al.; We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids . The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e . an iteron conserved position and a mini-F specific position . Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE . This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA . These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding . Thus the binding mode may be common to in the case of mini-F like plasmids. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 512 - 5 Crystallization and preliminary X-ray analysis of shikimate dehydrogenase from Escherichia coli; Maclean J et al.; Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour-diffusion method using ammonium sulfate as a precipitant . Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution . The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 A, beta = 122.2 degrees (at 100 K) . Native crystals diffract to 2.3 A in-house on a rotating-anode X-ray source . The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 A(3) Da(-1). Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 509 - 11 Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia coli L--asparaginase II (Ser58Ala mutant); Kozak M et al.; Periplasmic Escherichia coli L-asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P2(1)2(1)2) . Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0) . The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9 A and diffract to 2.3 A resolution . The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 501 - 3 Crystallization and preliminary X-ray analysis of squid neuronal Sec1; Bracher A et al.; Sec1 protein family members are involved in the regulation of all intracellular SNARE-mediated (SNARE = soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) vesicle-fusion processes in a step preceding membrane fusion and have been shown to interact with t-SNAREs . To better understand the structural basis and the role of Sec1 in the regulation of the SNARE-complex formation, neuronal Sec1 from the squid Loligo pealei has been expressed and crystallized; this invertebrate protein shows a high sequence homology to the human neuronal Sec1, Munc18a . Here, the production of diffraction-quality native crystals, which belong to space group P3(1)21 and diffract to 3.3 A resolution, is described . In addition, selenomethionyl n-Sec1 crystals in space groups P3(1)21 and P2(1) have been generated . Preliminary analysis of the monoclinic space group indicates that these crystals diffract to a resolution higher than 2.5 A. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 489 - 91 The NADP(H)-binding component (dIII) of human heart transhydrogenase: crystallization and preliminary crystallographic analysis; Peake SJ et al.; Transhydrogenase is a membrane protein which uses the energy of the proton motive force to drive the reduction of NADP(+) by NADH . The enzyme has three domains: dII spans the membrane, while dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively . DIII from human heart transhydrogenase has been expressed in Escherichia coli . The purified protein has been crystallized with bound NADP(+) using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant . The crystals belong to the tetragonal space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 58.1, c = 251.0 A . A 2.1 A resolution native data set has been collected with an R(merge) of 6 . 8%. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 472 - 4 Crystallization of native and selenomethionyl yeast orotidine 5'-phosphate decarboxylase; Miller BG et al.; Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant . Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A . Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 469 - 71 Crystallization and X-ray diffraction studies of the fatty-acid responsive transcription factor FadR from Escherichia coli; van Aalten DM et al.; FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized . Crystals of two binary complexes were obtained . The FadR-CoA complex crystallized in space group C222(1), with unit-cell parameters a = 61.3, b = 102.0, c = 91.3 A . The FadR-octanoyl-CoA complex crystallized in space group P6(5)22, with unit-cell parameters a = b = 59.7, c = 296.2 A . Both crystal forms diffracted to 3.5 A on a rotating-anode generator . In both crystal forms, the asymmetric unit contains one subunit . The protein is known to be a homodimer; each subunit consists of two domains of unknown fold . For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported. Exp Cell Res, 2000 Apr 10, 256(1), 282 - 90 Heat-inducible expression of a reporter gene detected by transient assay in zebrafish; Adam A et al.; Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system . Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene . Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes . However, their induction kinetics were different, which might be due to differences in their 5' UTRs . Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level . These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish . Biochemistry (Mosc), 2000 Mar, 65(3), 388 - 92 Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase; Parfenyev AN et al.; Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded . Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity . The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition . These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution . Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues. Biochemistry (Mosc), 2000 Mar, 65(3), 373 - 87 Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase; Avaeva SM et al.; The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated . The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis . The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied . X-Ray data on E . coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed . It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+ . Different properties of these cations account for active site deformation . Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition . This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi . It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site . As a result, Ca2+ is a powerful inhibitor of all known PPases . Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered. Biochemistry (Mosc), 2000 Mar, 65(3), 361 - 72 Active site interactions in oligomeric structures of inorganic pyrophosphatases; Avaeva SM; Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants . However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear . Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem . The abundant experimental data requires generalization and comprehensive analysis . A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme . Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry . In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding . The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers. Biochemistry (Mosc), 2000 Mar, 65(3), 315 - 23 Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications; Shiba T et al.; In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E . coli polyphosphate kinase (PPK) and their applications . With regard to the first subject, it has been shown that E . coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted . Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression . Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression . Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS . The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration . Recently E . coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs . This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method . PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E . coli . The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute. Biochemistry (Mosc), 2000 Mar, 65(3), 309 - 14 Polyphosphates and enzymes of polyphosphate metabolism in Escherichia coli; Nesmeyanova MA; This review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E . coli and their regulation by exogenous orthophosphate and other physiological and genetic factors. Protein Sci, 2000 Jan, 9(1), 64 - 72 Conformational substates in different crystal forms of the photoactive yellow protein--correlation with theoretical and experimental flexibility; van Aalten DM et al.; The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies . Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies . These studies have shown that conformational changes during the photocycle are limited to the chromophore and its immediate environment . However, spectroscopic studies suggest the presence of large conformational changes in the protein . Here, we address this apparent discrepancy in two ways . First, we obtain a description of large concerted motions in the ground state of the yellow protein from NMR data and theoretical calculations . Second, we describe the high-resolution structure of the yellow protein crystallized in a different space group . The structure of the yellow protein differs significantly between the two crystal forms . We show that these differences can be used to obtain a description of the flexibility of the protein that is consistent with the motions observed in solution. Protein Sci, 2000 Jan, 9(1), 53 - 63 The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase; Sakash JB et al.; As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used . These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function . The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides . Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP . PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP . These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response . Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer . The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP . The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated . These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site . 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP . Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect. Protein Sci, 2000 Jan, 9(1), 37 - 48 Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase; Knapp JE et al.; The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A . E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal {His}6 tag . The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method . The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution . The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms . The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface . Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD . Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E . coli E2oCD . The remaining E2 components can be divided into three groups based on active-site sequence similarity . Analysis of the surface properties of both crystal forms of E . coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o. Protein Sci, 2000 Jan, 9(1), 20 - 8 NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization; Yu WF et al.; Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds . However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence . We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer . Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant . Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx . Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31 . A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4) . Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site . In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. Anal Chem, 2000 Mar 1, 72(5), 1006 - 14 A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification; Hoving S et al.; We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage . The mass differences between a series of N-terminally degraded peptides give short sequences of defined length . Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database . The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing . The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples . We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins . We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis. Pharmacogenetics, 2000 Feb, 10(1), 59 - 66 Characterization of human polymorphic DNA repair methyltransferase; Inoue R et al.; The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis . More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts . We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3 . Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation . In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme . Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence . The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples . On the other hand, E . coli and human cells expressing V2 cDNA showed a significantly reduced level of survival . In these cells, V2 protein was hardly detected, even though mRNA was produced normally . An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences . There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo . Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views . From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. Pharmacogenetics, 2000 Feb, 10(1), 49 - 57 Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis; Blackburn AC et al.; Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest . The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic . This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124 . Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively . These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively . The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography . Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate . This report demonstrates the discovery of a functional polymorphism by analysis of the EST database. J Photochem Photobiol B, 2000 Jan, 54(1), 67 - 71 H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background; Asad LM et al.; Pretreatment with 2.5 mM H2O2 protects E . coli cells against UV-C killing, a phenomenon independent of LexA cleavage . In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain {lexA (Ind-)} . Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response. J Hered, 2000 Jan-Feb, 91(1), 24 - 30 Detection of RFLP markers associated with antibody response in meat-type chickens: haplotype/genotype, single-band, and multiband analyses of RFLP in the major histocompatibility complex; Yonash N et al.; Improving disease resistance in poultry by direct selection or by selecting for immune response is hardly feasible due to the quantitative nature of these traits, their low heritability, and the difficulties associated with reliable measurements . In this situation, marker-assisted selection (MAS) is expected to be a more effective breeding approach . The major histocompatibility complex (MHC), known to affect immune response and disease resistance, was examined as a set of candidate genes for association between DNA markers and antibody response . Backcross (BC1) and F2 families were generated from a cross between lines divergently selected for high or low antibody response to Escherichia coli vaccination . Restriction fragment length polymorphism (RFLP) analysis of the highly polymorphic MHC class IV (B-G) region suggested an association with antibody response to several antigens (E . coli, SRBC, NDV) . The multiband data generated with the class IV probe were used to compare the efficacies of three alternative analyses: "single-band" (carriers versus noncarriers of each RFLP band separately), "multiband" (multiple regression on all RFLP bands), and "genotype" (determined from family analysis of RFLP patterns/haplotypes) . Groups of birds identified by the "multiband" analysis were identical to the haplotype-based genotypes, suggesting that the laborious step of haplotype determination can be omitted without unduly sacrificing power of analysis. Microbios, 2000, 101(399), 89 - 103 Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12; Zabel MD et al.; The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase . In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene . Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E . coli entered the stationary phase . However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor . The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen . The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr . Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen . The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase . Tryptophan, indole, and several indole derivatives had no effect. J Gene Med, 1999 Jan-Feb, 1(1), 31 - 42 Selective uptake and sustained expression of AAV vectors following subcutaneous delivery; Donahue BA et al.; BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors are capable of long-term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system . In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins . METHODS: rAAV vectors encoding the human factor IX, human interferon-alpha 2a, murine erythropoietin (epo), and Escherichia coli lacZ genes were used for subcutaneous delivery into mature immunocompetent mice . Expression of factor IX and interferon in mouse serum was measured by ELISA . Expression of Epo was monitored by an increase in hemotocrit and by RIA . The tissue tropism of AAV transduction was determined by histochemistry following administration of the lacZ vector . RESULTS: Long-term protein expression (at least one year) is demonstrated in the serum of immunocompetent mice following subcutaneous delivery of AAV vectors encoding the human factor IX and interferon genes . The murine epo gene delivered via this route resulted in levels of Epo that correlate with increased hematocrits of up to 90% for a duration of nine months . rAAV encoding the lacZ gene revealed that the panniculus carnosus, a skeletal muscle layer of the skin, was transduced upon subcutaneous administration . CONCLUSIONS: This study shows that long-term expression of secreted proteins can be achieved using rAAV vectors injected subcutaneously as a single administration . The observation that the panniculus carnosus is the primary tissue transduced by rAAV illustrates the high tropism of rAAV for skeletal muscle. J Gene Med, 1999 Mar-Apr, 1(2), 84 - 92 Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells; Kilger E et al.; BACKGROUND: EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses . Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro . These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV . In addition, mini-EBVs can be modified in E . coli to alter their genetic composition or adopt new genes . METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1 . Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro . These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors . RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV . These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation . Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established . CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines. Mol Reprod Dev, 2000 May, 56(1), 34 - 44 New approach to cell lineage analysis in mammals using the Cre-loxP system; Sato M et al.; The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals . We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E . coli beta-galactosidase (beta-gal) . When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal . When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity . This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence . We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres . One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage . When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos . These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene . Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals . Arch Insect Biochem Physiol, 2000 Apr, 43(4), 165 - 72 Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification; Uno T et al.; A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins . The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family . To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein . The recombinant protein was purified to homogeneity with glutathione S-Sepharose . The fusion protein bound {(35)S} GTPgammaS and {(3)H} GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively . The binding of {(35)S} GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides . The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho . Bound {(35)S} GTPgammaS and {(3)H} GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2) . These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP . Arch . Arch Insect Biochem Physiol, 2000 Apr, 43(4), 147 - 64 Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda; Gardiner EM et al.; Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs . In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda . For both species, hemocyte densities per microl of blood increased with instar . Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar . Hematopoietic organs were located in the meso- and metathorax of S . Frugiperda and P . Includens . These organs contained large numbers of hemocytes in S . Frugiperda, but contained few hemocytes in P . Includens . The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes . Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA . BrdU labeling indices for both species also fluctuated with the molting cycle . Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S . Frugiperda but not in P . Includens . Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation . Arch . J Cell Physiol, 2000 May, 183(2), 182 - 95 Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration; Zhu D et al.; The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain . In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain {ARD}) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function . Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 {S1, aa1-aa217}, subdomain 2 {S2, aa218-aa381}, subdomain 3 {S3, aa382-aa612}, and subdomain 4 {S4, aa613-aa834}) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44 . Microinjection of antiglutathione S-transferase (GST)-tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections . Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration . Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration . Avian Dis, 2000 Jan-Mar, 44(1), 185 - 91 Iss from a virulent avian Escherichia coli; Foley SL et al.; No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols . Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability . The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E . coli Iss protein . In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E . coli, and expression was induced . The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography . The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing . Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E . coli. Avian Dis, 2000 Jan-Mar, 44(1), 179 - 84 Cloning and sequencing of the iss gene from a virulent avian Escherichia coli; Horne SM et al.; Control of colibacillosis is important to the poultry industry . We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis . Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis . The iss gene was amplified from a virulent avian E . coli isolate and sequenced . The sequences of the gene and the predicted protein product were compared with those of iss from a human E . coli isolate and lambda bor . The iss gene from the avian E . coli isolate has 96.8% identity with the iss gene from the human E . coli isolate and 89.4% identity with lambda bor . The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor . The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences . In sum, iss from this avian E . coli isolate is very similar to iss from a human E . coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E . coli isolate, Iss proteins from avian and human E . coli isolates have only 87% identity . The strong association of iss with E . coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control. Avian Dis, 2000 Jan-Mar, 44(1), 170 - 8 Cloning and expression of the VP2 gene of an infectious bursal disease virus; Yu L et al.; A serotype I infectious bursal disease virus (IBDV) strain HZ96 was isolated in Hangzhou, China, in 1996 and attenuated by adaptation to chicken embryo fibroblast cells . The VP2 gene of strain HZ96 was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced . Compared with the VP2 sequences of other IBDV strains, HZ96 is most related to two attenuated strains, CU-1 and PBG98, and two attenuated Chinese strains, Harbin and CJ801bkf . HZ96 shares nucleotide sequence homology 98.9% with CU-1 and PBG98, 98.5% with Harbin, and 98.6% with CJ801bkf . Most of the sequence variations observed between HZ96 and other strains are located in the middle variable region from nucleotides 637 to 996 . Similar to other attenuated IBDVs, HZ96 has unique substitutions at residues 279 (Asp to Asn) and 284 (Ala to Thr), suggesting that these two substitutions may be directly related to adaptation of the virus to cell culture and attenuation of its virulence . As part of our effort to develop a submit vaccine for IBDV, the VP2 gene of HZ96 was cloned into a heat-inducible expression vector and expressed in Escherichia coli system . A protein band with expected molecular weight of 52 kD was detected by direct protein staining and western blotting. Avian Dis, 2000 Jan-Mar, 44(1), 59 - 65 Alterations in macrophage-produced cytokines and nitrite associated with poult enteritis and mortality syndrome; Heggen CL et al.; Poult enteritis and mortality syndrome (PEMS) is an acute, transmissible, infectious intestinal disease associated with high mortality and morbidity in turkey poults . Earlier studies demonstrated immune dysfunction, involving both humoral and cell-mediated immunity, associated with PEMS . The current study examined cytokines and metabolites produced by macrophages from poults exposed to PEMS agent(s) . Six trials were conducted with six separate hatches of poults . Poults in the PEMS group were exposed to PEMS agent(s) via contact exposure at 7 days of age whereas uninfected poults served as control poults . Abdominal macrophages were harvested from control (uninfected) and PEMS poults at various times postexposure and cultured for 18-24 hr in the presence of Escherichia coli lipopolysaccharide . Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) bioactivities and nitrite levels in macrophage culture supernatants were quantified . Macrophage supernatants from PEMS poults had greater IL-1-mediated stimulation index compared with the macrophage supernatants from uninfected control poults in both trials . However, this increase was significant only in trial 1 . IL-6 activity tested in three separate trials was significantly higher in PEMS macrophage supernatants over the controls . On the contrary, TNF-alpha production by macrophages was decreased in PEMS macrophage culture supernatants . Nitrite levels in PEMS macrophage culture supernatants were significantly higher in two out of three trials . These findings suggest that the enhanced production of proinflammatory cytokine/metabolites by activated macrophages in PEMS poults may be responsible, at least in part, for the physiological intestinal inflammation, gut motility, and anorexia that characterize this disease. Proc R Soc Lond B Biol Sci, 2000 Mar 7, 267(1442), 515 - 22 Pervasive compensatory adaptation in Escherichia coli; Moore FB et al.; To investigate compensatory adaptation (CA), we used genotypes of Escherichia coli which were identical except for one or two deleterious mutations . We compared CA for (i) deleterious mutations with large versus small effects, (ii) genotypes carrying one versus two mutations, and (iii) pairs of deleterious mutations which interact in a multiplicative versus synergistic fashion . In all, we studied 14 different genotypes, plus a control strain which was not mutated . Most genotypes showed CA during 200 generations of experimental evolution, where we define CA as a fitness increase which is disproportionately large relative to that in evolving control lines, coupled with retention of the original deleterious mutation(s) . We observed greater CA for mutations of large effect than for those of small effect, which can be explained by the greater benefit to recovery in severely handicapped genotypes given the dynamics of selection . The rates of CA were similar for double and single mutants whose initial fitnesses were approximately equal . CA was faster for synergistic than for multiplicative pairs, presumably because the marginal gain which results from CA for one of the component mutations is greater in that case . The most surprising result in our view, is that compensation should be so readily achieved in an organism which is haploid and has little genetic redundancy This finding suggests a degree of versatility in the E . coil genome which demands further study from both genetic and physiological perspectives. Br J Cancer, 2000 Mar, 82(5), 1035 - 40 Decreased GTPase activity of K-ras mutants deriving from human functional adrenocortical tumours; Lin SR et al.; Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K-ras gene mutation . In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61) . To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K-Ras oncogene and the alterations of its biocharacteristics, mutant K-Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV . Mutant K-Ras genes were expressed at high levels in Escherichia coli and the resultant K-Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding . The purified K-Ras protein from E . coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase-activating protein for Ras . The results showed that the wild-type cellular K-Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K-Ras gene, which mutated at codon 60 . With regards to the codon 15, 16, 18 and 31 mutant K-Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K-Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K-Ras protein . These results indicated that mutations at these hot spots of K-Ras gene were indeed activated K-Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove. Glycoconj J, 1999 Aug, 16(8), 457 - 63 Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom; Oda Y et al.; Colominic acid is an alpha2,8-linked sialic acid polymer produced by Escherichia coli . We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC . Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition . SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom . SC did not inhibit phospholipase A2 activity in bee venom . This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively . SC with a higher sulfur content and a larger molecular mass showed more potent activity . The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important . For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity . A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity. Biosci Biotechnol Biochem, 2000 Feb, 64(2), 348 - 54 Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells; Kawasaki Y et al.; Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC) . We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo . In the in vitro study, BLF was found to inhibit the adherence of ETEC . This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells . In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC. Int J Biochem Cell Biol, 2000 May, 32(5), 499 - 508 Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs; Zackova M et al.; Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form . This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode . The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles . The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition . These transport activities were further characterized by testing various inhibitors of the two different transport modes . The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes . This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport . Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents . Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only . These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway . This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria . This might be considered as a side function of this carrier. Int J Biochem Cell Biol, 2000 May, 32(5), 481 - 8 Determination of interactions between human thrombopoietin and its receptor MPL by yeast two-hybrid system and affinity biosensor; Hsieh DP et al.; The binding of human thrombopoietin to the extracellular domain of its receptor MPL prompts a cascade transduction of intracellular signals, leading to the development of megakaryocyte precursors and the production of circulating platelets . We have used a yeast two-hybrid system to reveal, via in vivo interactions between different deletion constructs of MPL and thrombopoietin, that the extracellular subunit 1 of MPL is the ligand binding site and the N-terminal domain of thrombopoietin alone is sufficient for the binding . The extracellular portion of MPL was heterologously expressed in E . coli and its specific affinity with thrombopoietin was visualized in vitro by resonance mirror biosensor technique. J Mol Biol, 2000 Apr 7, 297(4), 947 - 59 Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization; Tan S et al.; We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex . We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization . We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal . J Mol Biol, 2000 Apr 7, 297(4), 933 - 45 Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies; Iyer R et al.; By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range . Inhibitors require at least two amine groups to be effective . For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups . Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative . But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups . Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction . From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine . The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore . Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine . Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine . Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction . Biochemistry, 2000 Apr 4, 39(13), 3856 - 60 Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli; Kersten MV et al.; Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme . Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites . The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme . Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation . Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites . In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation. Biochemistry, 2000 Apr 4, 39(13), 3827 - 34 Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum; Nakamura A et al.; Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum . CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half . We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties . Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin . When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6 . A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin . The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+) . CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner . A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation . The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells. Biochemistry, 2000 Apr 4, 39(13), 3817 - 26 Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli; D'souza VM et al.; The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated . Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion . Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively . Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity . Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site . Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry . Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of {CoCo(MetAP)} also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry . EPR studies on {CoCo(MetAP)} also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6 . EPR studies on {Fe(III)_(MetAP)} and {Fe(III)Fe(III)(MetAP)} also suggested no spin-coupling between the two metal ions . (1)H nuclear magnetic resonance (NMR) spectra of {Co(II)_(MetAP)} in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171 . Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E . coli are discussed. Biochemistry, 2000 Apr 4, 39(13), 3751 - 62 A three-step kinetic mechanism for peptide binding to MHC class II proteins; Joshi RV et al.; Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M(-)(1) s(-)(1)) and dissociation (<10(-)(5) s(-)(1)) processes . Various linear and branched pathways have been proposed to account for these data . Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins . Our experiments identified an obligate intermediate in the binding reaction . The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change . The same mechanism is observed for different peptide antigens . In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding . The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed. Biochemistry, 2000 Apr 4, 39(13), 3745 - 50 The glucose transporter of the Escherichia coli phosphotransferase system: linker insertion mutants and split variants; Beutler R et al.; The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate . It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus . Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity . They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains . Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32% . IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex . It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly. Biochemistry, 2000 Apr 4, 39(13), 3690 - 8 Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast; Dove JE et al.; Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover . This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue . In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ . In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli . With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized . Active-site mutants are used to investigate further the nature of these spectral species . Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II) . In the following paper in this issue {Schwartz, B., Dove, J . E., and Klinman, J . P . (2000) Biochemistry 39, 3699-3707}, the initial reaction of precursor protein with oxygen is characterized kinetically . Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate. Biochemistry, 2000 Apr 4, 39(13), 3656 - 65 Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution; Lebbink JH et al.; The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date . To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli . This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature . Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail . Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers . Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme . Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold . In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C . Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site . The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta-D-galactopyra noside hydrolysis . This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates. Biochemistry, 2000 Apr 4, 39(13), 3647 - 55 Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase . 2 . 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II); Lin Y et al.; 13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe . {U-(13)C}ATP and AKe.{U-(13)C}AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP . (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e . , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation-(13)C distances . An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes . Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide . These distances, together with the interproton distances determined previously from TRNOESY experiments {Lin, Y., and Nageswara Rao, B . D . (2000) Biochemistry 39, 3636-3646}, led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes . These conformations differ significantly from the nucleotide conformations in crystals of AKe . AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography. Biochemistry, 2000 Apr 4, 39(13), 3636 - 46 Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase . 1 . Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy; Lin Y et al.; Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe . AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations . The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees . These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding . The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees . These solution-state conformations are significantly different from those in published data from X-ray crystallography . A computation of the ligand NOEs, made by using the program CORCEMA {Moseley, H . N . B., Curto, E . V., and Krishna, N . R . (1995) J . Magn . Reson . B108, 243-261} with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex {Berry, M . B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G . N . , Jr . (1994) Proteins: Struct., Funct., Genet . 19, 183-198}, showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone. Biochemistry, 2000 Apr 4, 39(13), 3525 - 32 Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA; Minasov G et al.; The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present . Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes {Lima, W . F., and Crooke, S . T . (1997) Biochemistry 36, 390} . Therefore, the hybrid substrates may not adopt a canonical A-form geometry . Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes . This is particularly relevant to the RNase H-dependent pathway of antisense action . Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA) . Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H {Damha, M . J., et al . (1998) J . Am . Chem . Soc . 120, 12976} . Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA {Berger, I., et al . (1998) Nucleic Acids Res . 26, 2473} and {3.3.0}bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation . On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand . This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids . Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H . Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications. Scand J Immunol, 2000 Apr, 51(4), 392 - 9 CD14-dependent and independent pathways in lipopolysaccharide-induced activation of a murine B-cell line, CH12 . LX; Kimura S et al.; Using a lipopolysaccharide (LPS)-responsive murine B-cell line, CH12 . LX, we assessed the possible role of CD14 in LPS-induced activation of B cells . Flow cytometric analysis indicated that CH12.LX cells expressed the CD14 molecule with a lower intensity than did the macrophage cell line J774.1 . A reverse transcription-polymerase chain reaction and Northern blot analysis revealed low, but significant, levels of CD14 mRNA in CH12.LX cells, whose cDNA was identical to that of the mouse macrophage CD14 gene . After stimulation with LPS, CH12.LX cells proliferated, accompanied by up-regulations of CD14, transforming growth factor (TGF)-beta and interleukin (IL)-6 mRNA, and increased IgM and IgA secretion . In the absence of serum or with the addition of anti-CD14 monoclonal antibodies, however, LPS-stimulation induced neither the up-regulation of CD14 and TGF-beta mRNA nor an increase in IgA secretion . These findings indicate that CD14 expression is not restricted to myeloid cells, but is involved in some cellular activation events of murine B cells elicited by LPS . Furthermore, a CD14-independent pathway may also exist in the LPS-induced activation of B cells that leads to proliferation, IL-6 production and the enhancement of IgM (but not IgA) secretion. J Bacteriol, 2000 Apr, 182(8), 2285 - 91 Intrinsic polymerase activities of UmuD'(2)C and MucA'(2)B are responsible for their different mutagenic properties during bypass of a T-T cis-syn cyclobutane dimer; O'Grady PI et al.; In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD'(2)C complex (DNA polymerase V {polV}) or its plasmid-encoded homologs, such as MucA'(2)B . Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains . We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (epsilon subunit) or holE (theta subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of epsilon, or the dnaE antimutator allele spq-2 . The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD'C, MucAB, or MucA'B proteins were expressed from a low-copy-number plasmid . With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype . We also demonstrate that the MucA'(2)B complex is more efficient in promoting translesion replication than the UmuD'(2)C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA'(2)B than by UmuD'(2)C . These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication . Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass. J Bacteriol, 2000 Apr, 182(8), 2277 - 84 Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin; Ray WK et al.; Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols . In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli . Well-characterized rhodaneses are comprised of two structurally similar ca . 15-kDa domains . Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses . The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E . coli, encodes the 12-kDa rhodanese . As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine . The K(m)s for SSO(3)(2-) and CN(-) were 78 and 17 mM, respectively . The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer . GlpE exhibited a k(cat) of 230 s(-1) . Thioredoxin 1 from E . coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases . The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%) . This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases . GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins for which sulfurtransferase activity has been confirmed. J Bacteriol, 2000 Apr, 182(8), 2269 - 76 Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis; Liu Z et al.; To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required . The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T . ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed . To knock out the T . ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E . coli to T . ferrooxidans ATCC 33020 by conjugation under the best conditions determined . The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination . These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain . However, the T . ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T . ferrooxidans. J Bacteriol, 2000 Apr, 182(8), 2238 - 44 A second {2Fe-2S} ferredoxin from Sphingomonas sp . Strain RW1 can function as an electron donor for the dioxin dioxygenase; Armengaud J et al.; The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp . strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme . An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J . Armengaud, B . Happe, and K . N . Timmis, J . Bacteriol . 180:3954-3966, 1998) . In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli . This third ferredoxin thus far identified in Sphingomonas sp . strain RW1 contained a putidaredoxin-type {2Fe-2S} cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy . The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp . strain RW1 . In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp . strain RW1 . RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3 . In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase. J Bacteriol, 2000 Apr, 182(8), 2218 - 29 Cellular responses to postsegregational killing by restriction-modification genes; Handa N et al.; Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability . The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex . In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair . A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability . Many cells formed long filaments, some of which were multinucleated and others anucleated . In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated . Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants . The cells induced the SOS response in a RecBC-dependent manner . These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes . These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA. J Bacteriol, 2000 Apr, 182(8), 2207 - 17 A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport; Cosgriff AJ et al.; In vivo recombination has been used to make a series of AroP-PheP chimeric proteins . Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan . Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP . Phenylalanine is the residue at the corresponding position in PheP . The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane . Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P . These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110 . Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins. J Bacteriol, 2000 Apr, 182(8), 2134 - 41 A novel phenanthrene dioxygenase from Nocardioides sp . Strain KP7: expression in Escherichia coli; Saito A et al.; Nocardioides sp . strain KP7 grows on phenanthrene but not on naphthalene . This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate . The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome . A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced . The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified . The gene cluster, phdAB, was located 8 . 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase . The phdCD gene cluster was located 2.9 kb downstream of the phdB gene . PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases . The PhdC sequence showed features of a {3Fe-4S} or {4Fe-4S} type of ferredoxin, not of the {2Fe-2S} type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases . PhdD also showed moderate (less than 40%) sequence identity to known reductases . The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters . The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E . coli . E . coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes . It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity . The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp . strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases. J Gastroenterol Hepatol, 2000 Feb, 15(2), 182 - 91 Amino acid substitutions in codons 9-11 of hepatitis C virus core protein lead to the synthesis of a short core protein product; Yeh CT et al.; BACKGROUND: Previous in vitro experiments have indicated that if the ninth codon of the hepatitis C virus (HCV) core gene is mutated from arginine to lysine, a short 16-kDa (P16) instead of a 21-kDa (P21) core protein will be produced . In this study, we aimed to investigate whether similar mutations existed in patients with chronic HCV infection and whether such mutations led to the expression of P16 . METHODS: The core gene was isolated from patients' sera by reverse transcription-polymerase chain reaction and sequenced . RESULTS: Three of 10 patients with hepatocellular carcinoma were found to have mutant viruses with missense mutations at codons 9-11: arginine-to-glycine mutation at codon 9 (case 1); lysine-to-glutamine mutation at codon 10 (case 5); and lysine-to-asparagine/threonine-to-alanine double mutations at codons 10 and 11 (case 8) . Site-directed mutagenesis and in vitro translation experiments revealed that P16 was expressed by all three mutants . Using gel-purified P21 and P16 proteins obtained from transformed Escherichia coli, the serum titres of anti-P21 and anti-P16 were assayed . Unequal titres of anti-P16 and anti-P21 were found in only cases 1, 5 and 8 . A rabbit antibody directed against P16 but not P21 was thus generated for immunohistochemical analysis . P16 was detected in the nuclei of hepatocytes in the peri-hepatoma tissue of a single case (case 1) . CONCLUSIONS: These data indicate that missense mutations at codons 9-11 can occur during chronic HCV infection, which results in the expression of P16 core protein. Bioseparation, 1999, 8(1-5), 255 - 67 Flow injection analysis of intracellular beta-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification; Tocaj A et al.; A continuous integrated process for on-line quantification of intracellular components has been developed . By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (beta-galactosidase) from the cell interior . The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell . The cell debris does not interfere in the binding assay as it is carried out in an expanded bed . The time for an assay cycle is at present approx . 35 min . This integrated system can be used for quantification of proteins down to at least 10(-7) mol/L. Bioseparation, 1999, 8(1-5), 219 - 28 Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli; Brobjer M; A capture step was developed using the expanded bed adsorption technology to separate a protein of interest on a cation exchanger from a crude Escherichia coli homogenate . This method was developed in bench-top scale using a STREAMLINE 25 column (Amersham Pharmacia Biotech, Sweden) and STREAMLINE SP . The development was based on earlier experiments performed in a packed bed column (SP-Sepharose FF) to investigate the conditions for sample application, wash and elution . The packed bed method was transformed into an expanded bed method by slightly modifying the wash procedure and cleaning in place (CIP) . This method was then scaled-up to pilot scale and used for production of the fusion protein according to cGMP . The yield over the step in pilot scale was 70-85% compared with only 30-50% in small scale . Pressure build-up, attachment of biomass to the adsorbent and collapses of the expanded bed were phenomena seen in small scale but not in pilot scale . The scale-up of the step significantly improved the performance of the step. Bioseparation, 1999, 8(1-5), 53 - 67 On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed; Clemmitt RH et al.; The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described . An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation . The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method . Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock . The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating . The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor . The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed. Bioorg Khim, 1999 Dec, 25(12), 923 - 9 {Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells}; Ptitsyn LR et al.; An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells . The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E . coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter . The regulated low-level biosynthesis of Kil protein increased the permeability of E . coli outer membrane for periplasmic proteins . This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium . As a result, the E . coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium . The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively . The hEGF preparation isolated possessed biological activity both in vivo and in vitro. Bioorg Khim, 1999 Dec, 25(12), 883 - 91 {Structural and functional characteristics of ATP-dependent Lon-proteinase from Escherichia coli}; Rotanova TV; Enzymic and structural peculiarities of the ATP-dependent Lon protease from Escherichia coli and its mutant and modified forms were studied . Amino acid residues important for the function of proteolytic and ATPase sites and for the transmission of the interdomain signals of the activity coupling were found . It was shown that the protein substrates are hydrolyzed only by the full-size enzyme, whereas the isolated proteolytic domain displays a peptide-hydrolyzing activity. J Biol Chem, 2000 Mar 31, 275(13), 9863 - 9 The MutL ATPase is required for mismatch repair; Spampinato C et al.; Members of the MutL family contain a novel nucleotide binding motif near their amino terminus, and the Escherichia coli protein has been found to be a weak ATPase (Ban, C., and Yang, W . (1998) Cell 95, 541-552) . Genetic analysis has indicated that substitution of Lys for Glu-32 within this motif of bacterial MutL results in a strong dominant negative phenotype (Aronshtam, A., and Marinus, M . G . (1996) Nucleic Acids Res . 24, 2498-2504) . By in vitro comparison of MutL-E32K with the wild type protein, we show the mutant protein to be defective in DNA-activated ATP hydrolysis, as well as MutS- and MutL-dependent activation of the MutH d(GATC) endonuclease and the mismatch repair excision system . MutL-E32K also acts in dominant negative manner in the presence of wild type MutL in vitro, inhibiting the overall mismatch repair reaction, as well as MutH activation . As judged by protein affinity chromatography, MutL and MutL-E32K both support formation of ternary complexes that also contain MutS and MutH or MutS and DNA helicase II . These findings imply that the MutL nucleotide binding center is required for mismatch repair and suggest that the dominant negative behavior of the MutL-E32K mutation is due to the formation of dead-end complexes in which the MutL-E32K protein is unable to transduce a signal from MutS that otherwise results in mismatch-dependent activation of the MutH d(GATC) endonuclease or the unwinding activity of helicase II. J Biol Chem, 2000 Mar 31, 275(13), 9673 - 83 Molecular cloning, genomic organization, and biochemical characterization of myristoyl-CoA:protein N-myristoyltransferase from Arabidopsis thaliana; Qi Q et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions . We have isolated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant . This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da . The primary structure is 50% identical to the mammalian NMTs . Analyses of Southern blots, genomic clones, and database sequences suggested that the A . thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations . The recombinant AtNMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase . The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA among the acyl CoA donors and also inhibition by the bovine brain NMT inhibitor NIP(71) . The AtNMT1 expression profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximately 1.7 kb transcript and approximately 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developmentally very active . NMT activity was also evident in all these tissues . Subcellular distribution studies indicated that, in leaf extracts, approximately 60% of AtNMT activity was associated with the ribosomal fractions, whereas approximately 30% of the activity was observed in the cytosolic fractions . The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter . The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants. J Biol Chem, 2000 Mar 31, 275(13), 9510 - 7 HSP25, a small heat shock protein associated with dense bodies and M-lines of body wall muscle in Caenorhabditis elegans; Ding L et al.; HSP25, a previously uncharacterized member of the alpha-crystallin family of small heat shock proteins in Caenorhabditis elegans, has been examined using biochemical and immunological techniques . HSP25 is the second largest of 16 identifiable small heat shock proteins in the nematode and is expressed at all developmental stages under normal growth conditions . Recombinant HSP25 produced in Escherichia coli exists predominantly as small oligomers (dimers to tetramers) and possesses chaperone activity against citrate synthase in vitro . In C . elegans, HSP25 is localized to dense bodies and M-lines in body wall muscle, to the lining of the pharynx, and to the junctions between cells of the spermathecal wall . Affinity chromatography of nematode extracts on a column of immobilized HSP25 resulted in specific binding of vinculin and alpha-actinin but not actin, as revealed by Western blotting . These results suggest a role for HSP25 in the organization or maintenance of the myofilament lattice and adherens junctions in C . elegans. J Biol Chem, 2000 Mar 31, 275(13), 9468 - 75 Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center; Brino L et al.; DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle . Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids . To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues) . Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process . Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover . The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E . coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center . We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule . Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis . A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure. J Biol Chem, 2000 Mar 31, 275(13), 9263 - 9 Ribosome-mediated folding of partially unfolded ricin A-chain; Argent RH et al.; After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates . It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin . Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro . We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state . This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain . By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity . The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum . Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death. Biophys J, 2000 Apr, 78(4), 2093 - 106 Proteins with similar architecture exhibit similar large-scale dynamic behavior; Keskin O et al.; We have investigated the similarities and differences in the computed dynamic fluctuations exhibited by six members of a protein fold family with a coarse-grained Gaussian network model . Specifically, we consider the cofactor binding fragment of CysB; the lysine/arginine/ornithine-binding protein (LAO); the enzyme porphobilinogen deaminase (PBGD); the ribose-binding protein (RBP); the N-terminal lobe of ovotransferrin in apo-form (apo-OVOT); and the leucine/isoleucine/valine-binding protein (LIVBP) . All have domains that resemble a Rossmann fold, but there are also some significant differences . Results indicate that similar global dynamic behavior is preserved for the members of a fold family, and that differences usually occur in regions only where specific function is localized . The present work is a computational demonstration that the scaffold of a protein fold may be utilized for diverse purposes . LAO requires a bound ligand before it conforms to the large-scale fluctuation behavior of the three other members of the family, CysB, PBGD, and RBP, all of which contain a substrate (cofactor) at the active site cleft . The dynamics of the ligand-free enzymes LIVBP and apo-OVOT, on the other hand, concur with that of unliganded LAO . The present results suggest that it is possible to construct structure alignments based on dynamic fluctuation behavior. Biophys J, 2000 Apr, 78(4), 1852 - 61 Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis; Milligan CJ et al.; The positively charged S4 region of voltage-dependent potassium channels moves outward during depolarization, leading to channel opening, but possible movement of the negatively charged S2 region may be more complex . Here we have studied possible movement of the S2 region of the slowly activating human voltage-dependent potassium channel hKv2.1 . For this, cysteine mutants in the S2 region were expressed in Xenopus oocytes by injection of cRNA . Whole-cell currents were measured using the two-electrode voltage-clamp technique, and the effect of the membrane-impermeable cysteine-binding reagent parachloromercuribenzenesulfonate (PCMBS) was studied . For mutant S223C (located just outside the membrane in the S2 region), PCMBS inhibited currents and caused faster deactivation of tail currents . The time course of reactivity of PCMBS on tail current amplitudes was faster at more negative holding potentials . There was no effect of PCMBS on potassium channel currents for mutants D225C, N226C, A230C, and V232C . These data suggest that residue S223 is exposed to the extracellular phase at normal resting potentials, making it accessible to PCMBS, but upon depolarization there is a conformational change, making it less accessible, possibly by a local rather than global movement of S2 residues into the membrane . Voltage-dependent movements of nearby residues could also explain the results. Biophys J, 2000 Apr, 78(4), 1748 - 64 Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress; Cayley DS et al.; To obtain turgor pressure, intracellular osmolalities, and cytoplasmic water activity of Escherichia coli as a function of osmolality of growth, we have quantified and analyzed amounts of cell, cytoplasmic, and periplasmic water as functions of osmolality of growth and osmolality of plasmolysis of nongrowing cells with NaCl . The effects are large; NaCl (plasmolysis) titrations of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and cell water to approximately 20% and approximately 50% of their original values, and increase periplasmic water by approximately 300% . Independent analysis of amounts of cytoplasmic and cell water demonstrate that turgor pressure decreases with increasing osmolality of growth, from approximately 3.1 atm at 0.03 Osm to approximately 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm . Analysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations as a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pressure decreases with increasing osmolality, and verifies that cytoplasmic and periplasmic osmolalities are equal . We propose that MDO play a key role in periplasmic volume regulation at low-to-moderate osmolality . At high growth osmolalities, where only a small amount of cytoplasmic water is observed, the small turgor pressure of E . coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity . From these findings, we deduce that the activity of cytoplasmic water exceeds its mole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolality and exceeds unity at high osmolality, presumably as a consequence of macromolecular crowding . These novel findings are significant for thermodynamic analyses of effects of changes in growth osmolality on biopolymer processes in general and osmoregulatory processes in particular in the E . coli cytoplasm. Biochem Biophys Res Commun, 2000 Apr 2, 270(1), 100 - 7 Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III; Khandekar SS et al.; Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP . In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E . coli KAS III . Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active . Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer . Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2 . Protein Expr Purif, 2000 Apr, 18(3), 394 - 403 Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation; Lajmi AR et al.; GCN4 is a yeast transcriptional regulatory protein; its DNA-binding domain is a basic region/leucine zipper (bZIP) structure that comprises a dimer of alpha-helices capable of high-affinity, sequence-specific recognition of the DNA major groove . We are exploiting what nature has evolved by manipulating the bZIP motif as a molecular recognition scaffold; thus we reduced the elegantly minimal bZIP to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold . These Ala-based mutants are unusual proteins for expression as they are short ( approximately 100 amino acids) and hydrophobic (Ala-mutated basic regions, leucine-zipper dimerization domains) . Hydrophobicity posed a major problem throughout the expression, isolation, and purification stages; inclusion body formation and protein aggregation were significant hurdles throughout protein production . We describe measures that solved these problems, including use of high concentrations of denaturant in all steps of protein isolation and purification and use of temperature-dependent renaturing techniques to obtain folded, functional protein . Despite these difficulties, we ultimately retrieved 5-10 mg/L of broth of active, correctly folded protein after the complete purification procedure . Homogeneity of the proteins was established by chromatography, electrophoresis, and mass spectrometry . Furthermore, characterization by circular dichroism and DNase footprinting analysis demonstrates that these alanine-based mutants retain the structure and function of the native GCN4 DNA-binding domain . Remarkably, the most heavily mutated protein, containing 24 alanines of 27 total amino acids in the DNA-binding basic region, still binds the AP-1 site, the target of native GCN4 . Protein Expr Purif, 2000 Apr, 18(3), 378 - 87 Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme; Koeplinger KA et al.; A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli . This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag . The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB) . The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid . The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT . SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8 . Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme . Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA . SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8 . This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8 . Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage . Protein Expr Purif, 2000 Apr, 18(3), 355 - 60 Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form; Shanklin J; Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein . The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase . The E . coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus . The plasmid was designated pAasH . The activity of Aas in membranes was assessed from several cell lines . Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d-thiogalactopyranoside (IPTG) or lactose, respectively . When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid . Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography . The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body . Protein Expr Purif, 2000 Apr, 18(3), 346 - 54 tRNA-assisted overproduction of eukaryotic ribosomal proteins; Dieci G et al.; Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli . We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery . In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E . coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons . In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts . The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step . Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM) . This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies . Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome . Protein Expr Purif, 2000 Apr, 18(3), 338 - 45 Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer; Amrhein I et al.; Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli . The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK . The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells . Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer . Protein Expr Purif, 2000 Apr, 18(3), 320 - 6 Expression in Escherichia coli and simple purification of human Fhit protein; Pawelczyk T et al.; The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity . We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit . Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells) . The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent . Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide . There were no unspecific cleavage products . The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity . The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol . Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5) . The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical . This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A . Protein Expr Purif, 2000 Apr, 18(3), 277 - 85 Purification and characterization of recombinant forms of murine Tcl1 proteins; Du Bois GC et al.; The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined . This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells . The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1) . The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases . The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes . Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues . The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies . Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins . In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85 . The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column . The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers . The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy . Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination . Protein Expr Purif, 2000 Apr, 18(3), 262 - 8 Overexpression, purification, and refolding of a Porphyromonas gingivalis cysteine protease from Escherichia coli; Margetts MB et al.; This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis . This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease . Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains . The Rgp-1 protease domain was expressed in E . coli, purified, refolded, and assayed for activity . These expression studies demonstrated that prior to the formation of inclusion bodies in the E . coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate . When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity . This is the first report on the expression and purification of active Rgp-1 from E . coli . Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P . gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease . Avian Dis, 2000 Jan-Mar, 44(1), 105 - 13 High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus; Guy JS et al.; Six-day-old turkeys were inoculated with turkey coronavirus (TCV) and an enteropathogenic Escherichia coli (EPEC) (isolate R98/5) that were isolated from poult enteritis and mortality syndrome (PEMS)-affected turkeys . Turkeys inoculated with only R98/5 did not develop clinically apparent disease, and only mild disease and moderate growth depression were observed in turkeys inoculated with only TCV . Turkeys dually inoculated with TCV and R98/5 developed severe enteritis with high mortality (38/48, 79%) and marked growth depression . R98/5 infection resulted in attaching/effacing (AE) intestinal lesions characteristic of EPEC: adherence of bacterial microcolonies to intestinal epithelium with degeneration and necrosis of epithelium at sites of bacterial attachment . AE lesions were more extensive and were detected for a prolonged duration in dually inoculated turkeys compared with turkeys inoculated with only R98/5 . An apparent synergistic effect in dually inoculated turkeys was indicated by increased mortality, enhanced growth depression, and enhanced AE lesion development . The results suggest that TCV promoted intestinal colonization by R98/5; however, R98/5 did not appear to alter TCV infection . The present study provides a possible etiologic explanation for PEMS. Plant Mol Biol, 1999 Dec, 41(6), 837 - 49 Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development; Zou J et al.; Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration . This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development . The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases . The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner . Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants . Immunoblot analyses performed with a monoclonal antibody to the E1alpha mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein . MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants . Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants . Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time . The potential role for AtPDHK gene manipulation in crop improvement is discussed. Nucleic Acids Res . 2000 Apr 15;28(8):E34. Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions; Masuko M et al.; We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions . We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue . A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak . The Forster distance between two dye residues was 22.3 A (the orientation factor of 2/3) . We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid . Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Forster theory . The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 A . Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein-rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays. Nucleic Acids Res, 2000 Apr 15, 28(8), 1778 - 84 The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion; Blyn LB et al.; Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions . Thiostrepton, which binds to the 'GTPase center' of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds . Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg(2+), L11 and thio-strepton in the formation and maintenance of the rRNA fragment tertiary structure . Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg(2+), L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure. Hepatology, 2000 Apr, 31(4), 937 - 47 Functional significance of endothelin B receptors in mediating sinusoidal and extrasinusoidal effects of endothelins in the intact rat liver; Bauer M et al.; Endothelins (ET) are important regulators of the hepatic microcirculation that act through different receptor subtypes . We investigated functional significance of ET(B) receptors in mediating microhemodynamic effects of ETs in normal and endotoxin (lipopolysaccharide {LPS})-primed rat liver . LPS priming (Escherichia coli O26:B6; 1 mg . kg(-1)) selectively increased ET(B) mRNA and led to a shift in available receptors to the ET(B) subtype . IRL 1620 (an ET(B) agonist) increased portal pressure in a dose-dependent manner, and the increase in ET(B) expression was associated with prolonged portal pressor response in isolated livers . However, lactate dehydrogenase (LDH) release was attenuated and sinusoidal blood flow was better maintained upon ET(B) stimulation in vivo . In isolated livers, portal constriction as well as release of LDH, were substantially increased in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) . In vivo microscopic assessment of sinusoidal perfusion during ET(B) stimulation revealed a disruption of the flow pattern including frequent reversal of the flow direction without significant sinusoid constriction . Sinusoidal flow decreased even further after discontinuation of IRL 1620 . Both effects were mediated at extrasinusoidal sites that probably included postsinusoidal sites . However, after pretreatment with L-NAME, IRL 1620 evoked a significant sinusoidal constriction that colocalized with the body of the stellate cell . We propose that ET(B1)-induced NOS activity attenuates ET(B2) (and presumably ET(A))-mediated portal pressor response and stellate cell constriction . Transcriptional activation of the ET(B) gene may have a permissive effect on liver blood flow and protect against hepatocellular damage under pathophysiological conditions associated with endotoxemia. Genes Dev, 2000 Mar 15, 14(6), 740 - 9 A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange; Zaitsev EN et al.; Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA) . In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA . Our results demonstrate that this "inverse" reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner . Finally, we demonstrate that the RecA protein-dsDNA filament can also pair and promote strand exchange with ssRNA . This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication. Bioorg Med Chem, 2000 Mar, 8(3), 675 - 9 Specific inactivation of Escherichia coli tRNA(Phe) by antisense DNA-treatment under Mg2+-deficient conditions; Kanda T et al.; The preparation of an Escherichia coli tRNA mixture lacking several specific species may be useful for applications ranging from cell-free protein preparation to protein engineering . We have already demonstrated that tRNA(Asp) can be inactivated, or 'knocked out', with practical specificity by an antisense strategy . In the present study, we synthesized five tRNA(Phe)-targeted antisense oligonucleotides and tested if this tRNA can also be inactivated specifically . The salt conditions used previously for the tRNA(Asp) inactivation were not applicable to tRNA(Phe) . Instead, Mg2+-deficient conditions were found to be useful for the inactivation of tRNAPhe by the antisense oligonucleotides . These conditions were also applicable to the inactivation of tRNA(Asp) . The susceptibility to the antisense DNAs can change drastically, depending on the concentration of Mg2+. Bioorg Med Chem, 2000 Mar, 8(3), 657 - 64 Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase; Kiefelt MJ et al.; Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase . Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself . 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds . Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme. Biofizika, 2000 Jan-Feb, 45(1), 103 - 11 {How does environment modify the spatial structure of Dictyostelium discoideum population}; Kravchenko VV et al.; The transformation of the spatial structure of a Dictyostelium discoideum population in response to environmental changes induced by this population was investigated . A comparative analysis of the spatial and temporal characteristics of the D . discoideum colony is given for two cases: (a) when the colony is cultivated on a bacterial lawn, i.e . under conditions close to natural, and (b) in the absence of the bacterial lawn when the colony grows on the nutrient substrate enriched with folic acid . It is shown that the environmental changes induced by cell metabolism modify the spatial structure of the D . discoideum population first, the rate of population propagation falls drastically, which correlates with a decrease in the substrate pH; second, the spatial redistribution of the D . discoideum cell density correlates with the redistribution of folic acid in the substrate . The mechanism of the environment impact on the D . discoideum colony transformation is discussed. Wiad Lek, 1998, 51 Suppl 4, 227 - 32 {Coagulation disorders during treatment with l-asparaginase preparations}; Dobaczewski G; Coagulation disturbances are noticed, during ALL treatment with L-asparaginase, carrying risk of clotting complications . We examined 38 children with ALL (20 boys and 18 girls) aged 2-16 y., treated in 1996-1997 y . according to BFM and New York programmes . They received L-asparaginase of 10,000 and 25,000 U/m2 per dose at the beginning of induction therapy . The therapy started with E.coli L-asparaginase; in 16 cases the drug was changed to Erwinase . Decreasing of fibrinogen, antithrombin III concentration and prothrombin time was noticed . Infectious complications were established in 8 and clotting problems in 3 children . Substitution with antithrombin III was introduced in 15, with fibrinogen in 17 children because of low plasma concentration . In 21 patients treatment modifications according to decreasing of clotting factors concentration were done . Clotting problems strongly influence the treatment of children with ALL . Substitution therapy may improve the effectiveness of therapy. J Biochem (Tokyo), 2000 Mar, 127(3), 517 - 23 Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs; Tsuiki H et al.; We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses . Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues . The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB) . Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen . GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs. J Biochem (Tokyo), 2000 Mar, 127(3), 467 - 73 Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates; Watanabe K et al.; Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2) . This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid . We optimized conditions for induction of enzyme activity in M . commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase . Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity . Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit . Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1 . We cloned and sequenced a cDNA encoding the macrophomate synthase . The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs . Transformed E . coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG . The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase. J Biochem (Tokyo), 2000 Feb, 127(2), 329 - 35 Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-Ray structure analysis; Matsumoto T et al.; The single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair . The tertiary structure of EcoSSB was determined at 2.2 A resolution . This is rather higher resolution than previously reported . Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal . The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities . These concavities include the residues important for ssDNA binding . An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids . It seems likely to play a central role in the binding of ssDNA. J Biochem (Tokyo), 2000 Feb, 127(2), 263 - 8 Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T; Harada K et al.; To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique . The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament . This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160 . These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy. J Biochem (Tokyo), 2000 Jan, 127(1), 163 - 9 Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase; Lehnerer M et al.; A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R) . Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence . The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis . Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system . Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein . While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6 . 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments . Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems . Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R . The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit . All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R. J Biochem (Tokyo), 2000 Jan, 127(1), 151 - 61 Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli; Furukawa H et al.; The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli . The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as {3H}L-quinuclidinyl benzilate ({3H}QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C . No {3H}QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C . The MBP-M2 mutant expressed in E . coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of {(3)H}QNB with carbamylcholine and atropine . The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol . The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated {35S}GTP gamma S binding activity in the presence of GDP . The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated {(35)S}GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol . These results indicate that the MBP-M2 mutant expressed in E . coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor. J Biochem (Tokyo), 2000 Jan, 127(1), 143 - 9 Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme; Tsuruta H et al.; Psychrophilic phosphatase I from Shewanella sp . is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3 . 1.3.48) with a histidine as its catalytic residue {Tsuruta and Aizono (1999) J . Biochem . 125, 690-695} . Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques . The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase) . The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography . The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity. J Biochem (Tokyo), 2000 Jan, 127(1), 129 - 35 A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure; Yamamoto K et al.; Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies . Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose . Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells . The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns . Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin . In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin . This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues . Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies. J Biochem (Tokyo), 2000 Jan, 127(1), 37 - 41 An easy cell-free protein synthesis system dependent on the addition of crude Escherichia coli tRNA; Kanda T et al.; The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis . Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin . This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E . coli tRNA and a small amount of human placental RNase inhibitor . The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor . Chloramphenicol acetyltransferase was synthesized with this system and found to be active. FEMS Microbiol Lett, 2000 Apr 1, 185(1), 89 - 93 Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli; Bouzari S et al.; The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E . coli (EPEC) by slide agglutination was evaluated using hybridization and PCR . The adherence property of these strains was assayed using 3h HeLa cells adherence assay . The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes . In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent . Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P</=0.0001) . These results indicated that since over 60% of the strains identified by serogrouping carried at least one of the putative virulence markers, it therefore seems that this simple test is still of value in our setting although the need for a confirmatory test is also indicated. FEMS Microbiol Lett, 2000 Apr 1, 185(1), 71 - 7 Mutation of cytochrome bd quinol oxidase results in reduced stationary phase survival, iron deprivation, metal toxicity and oxidative stress in Azotobacter vinelandii; Edwards SE et al.; Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss . Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor . Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression . Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress . Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity . We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity. J Mol Biol, 2000 Mar 31, 297(3), 809 - 18 Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme; von Ahsen O et al.; Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds . Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR) . The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate . Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins . The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds . Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR . The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR . However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin . We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step . J Mol Biol, 2000 Mar 31, 297(3), 791 - 802 Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster; Ikura T et al.; Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0 . Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction . The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant . Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state . This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues . Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior . J Mol Biol, 2000 Mar 31, 297(3), 585 - 97 Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments; Frank EG et al.; The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis) . When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination . In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments . To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro . As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers . Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove . This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination . At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies . These data revealed preferential binding at filament ends (most likely, at one end) . End-specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis. J Mol Biol, 2000 Mar 31, 297(3), 543 - 51 NMR structure of activated CheY; Cho HS et al.; The CheY protein is the response regulator in bacterial chemotaxis . Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state . The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein . Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d . of 0.58(+/-0.08) A . Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state . The coupled rearrangement may be a more general mechanism for activation of receiver domains . J Mol Biol, 2000 Mar 31, 297(3), 537 - 42 Identification of the RecA protein-loading domain of RecBCD enzyme; Churchill JJ et al.; Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand . The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi . Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading . We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding . This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins . Mol Gen Genet, 2000 Feb, 263(1), 159 - 64 The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid; Ishiwa A et al.; Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64 . The PilV proteins determine the recipient specificity in liquid matings . A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA', -C, and -C' proteins, while E . coli B was recognized only by the PilVA' protein . To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E . coli K-12 waa (rfa) mutants and E . coli B transformants as recipient cells . E . coli K-12 waa mutants lack receptors for specific PilV proteins . E . coli B cells carrying waaJ or waaJKL genes of E . coli K-12 were recognized by donors expressing the PilVC' protein or the PilVC and -C' proteins, respectively, in addition to the PilVA' protein . Addition of E . coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings . We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Mol Gen Genet, 2000 Feb, 263(1), 131 - 6 SecG is an auxiliary component of the protein export apparatus of Escherichia coli; Flower AM et al.; SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus . These three proteins co-purify and can be co-immunoprecipitated, demonstrating that they are closely associated . While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous . It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise . However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds . The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality . We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary--as suggested previously by biochemical data--and is physiologically important only when cells are otherwise compromised. J Biochem (Tokyo), 2000 Mar, 127(3), 355 - 7 Effect of Arg145Gly mutation in human cardiac troponin I on the ATPase activity of cardiac myofibrils; Takahashi-Yanaga F et al.; In order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined . Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C.I-depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I . In the Ca(2)(+)-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca(2)(+)-sensitivity with mutant troponin I was higher than that with wild-type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I . These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca(2)(+)-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C. Redox Rep, 1999, 4(5), 237 - 41 Assessing the effect of reactive oxygen species on Escherichia coli using a metabolome approach; Tweeddale H et al.; A two-dimensional thin-layer chromatographic analysis of {14C}-labelled metabolites in Escherichia coli was employed to follow metabolic shifts in response to superoxide stress . Steady-state challenge with paraquat at concentrations inducing SoxRS-controlled genes resulted in several alterations in metabolite pools, including a striking increase in valine concentration . Elevated valine levels, together with increased glutathione and alkylperoxidase, are proposed to constitute an intracellular protection mechanism against reactive oxygen species . As shown by this example of metabolome analysis, novel cellular responses to environmental challenge can be revealed by following the total complement of metabolites in a cell. Eur J Pharmacol, 2000 Mar 17, 391(3), 317 - 20 A novel dual regulator of tumor necrosis factor-alpha and interleukin-10 protects mice from endotoxin-induced shock; Fukuda T et al.; A pyrimidylpiperazine derivative, N-{1-(4- inverted question mark{4-(pyrimidin-2-yl)piperazin-1-yl}methyl inverted question markphenyl)cycloprop yl} acetamide (Y-39041), is a dual cytokine regulator of tumor necrosis factor (TNF)-alpha and interleukin-10 production . Lipopolysaccharide-induced TNF-alpha release in BALB/c mice was inhibited by the oral treatment with the compound at 10-100 mg/kg (about 80% suppression) while interleukin-10 release was augmented (about 10-fold increase at 30 mg/kg) . In addition, Y-39041 (30 mg/kg, p.o.) completely protected mice from lipopolysaccharide-induced death by the treatment before and after lipopolysaccharide injection . The finding that Y-39041 suppresses TNF-alpha production and stimulates interleukin-10 production at the same time provides new insights for the treatment of septic shock, rheumatoid arthritis and Crohn's diseases. Arch Biochem Biophys, 2000 Apr 1, 376(1), 206 - 16 Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver; Hanna IH et al.; Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol . The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates . The purified recombinant protein was also used to raise polyclonal antibodies in rabbits . Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein . Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus . The identity of the cleavage boundary was verified by protein sequence analysis . Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations . An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site . Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6 . Arch Biochem Biophys, 2000 Apr 1, 376(1), 163 - 70 Redistribution of cyclic GMP in response to sodium butyrate in colon cells; Crane JK; The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides . Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured . Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide . Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C . Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment . Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin . The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal . In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa . Arch Biochem Biophys, 2000 Apr 1, 376(1), 124 - 40 Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues; Isaev-Ivanov VV et al.; The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described . The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA) . RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R . M . Story, I . T . Weber, and T . A . Steitz (1992) Nature (London) 355, 374-376) . Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively . Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA . Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA . The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP . By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine . In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain . Arch Biochem Biophys, 2000 Apr 1, 376(1), 101 - 8 Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis; Karr DB et al.; Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties . Magnesium and glycerol were absolute requirements for maintaining enzyme activity . The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences . There was no immunological cross-reaction of antibodies from B . japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E . coli with the B . japonicum enzyme . B . japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta . The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min . Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM . The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid . The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis . The calcium-dependent hysteretic behavior of B . japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis . Arch Biochem Biophys, 2000 Apr 1, 376(1), 82 - 90 Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp . W80 strain; Takeda T et al.; A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system . The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids . In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane . The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively . The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity . The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants . The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants . The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli . Arch Biochem Biophys, 2000 Apr 1, 376(1), 47 - 58 Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1 . Expression and characterization of recombinant ADP-glucose pyrophosphorylase; Igarashi RY et al.; A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene . The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes . The genomic fragment also contained an apparent truncated sequence corresponding to the C-terminus of the glgB gene (branching enzyme) . The presence of active branching enzyme activity in crude sonicates of Rb . sphaeroides cells indicates that the genome contains a full-length glgB at another location . The structure of this operon in relation to other glg operons is further discussed . The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family . The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level expression in E . coli . The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the native strain . The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step . Arch Biochem Biophys, 2000 Apr 1, 376(1), 26 - 33 Detection of oxidative base DNA damage by a new biochemical assay; Sattler U et al.; Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses . Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years . Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples . 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions . We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells . DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well . Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction . The trapped protein was subsequently recognized by a specific antibody . A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence . This new assay offers various potentialities, specifically in the field of technology of ROS producers . Biochem J, 2000 Mar 1, 346 Pt 2, 441 - 5 Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase; Ho TY et al.; The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein . A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function . To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase . The deleted mutants were expressed in E . coli with the use of pET expression vectors, then purified to homogeneity . Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3'-exonuclease activity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease activities . Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues . Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His(371) with Ala . The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability . In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His(371) is the active-site residue that has an essential role in PRV DNase activity. Biochem J, 2000 Mar 1, 346 Pt 2, 423 - 31 Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies; Chow LP et al.; The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens . On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified . This allergen was also present in A . fumigatus culture filtrates . Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A . fumigatus and showed 42-49% identity of amino acids with two proteases from P . cyclopium and T . album and with the Pen c 1 allergen from P . citrinum . Asp f 13 coding sequences were expressed in Escherichia coli as a {His}(6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography . Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A . fumigatus . To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed . At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule . In contrast, IgE from A . fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein. Biochem J, 2000 Mar 1, 346 Pt 2, 375 - 84 Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form; Busch JL et al.; Desulfovibrio africanus ferredoxin III (Da FdIII) contains one {4Fe-4S}(2+/1+) cluster and one {3Fe-4S}(1+/0) cluster, bound by seven Cys residues, in which the {3Fe-4S} cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu . The {3Fe-4S} core of this ferredoxin is so far unique in showing rapid bi-directional {3Fe-4S}<-->{4Fe-4S} cluster interconversion with a wide range of metal ions . In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS . Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx . 6 mg.l(-1)), but without incorporation of metal clusters . The nucleotide sequence confirms the protein sequence reported previously {Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim . Biophys . Acta 913, 20-26} . Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions . Absorption and EPR spectroscopy show that both {3Fe-4S} and {4Fe-4S} clusters are correctly inserted . Thin-film electrochemistry provides evidence that the {3Fe-4S} cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein . Nevertheless the protein has lower stability than native Da FdIII during chromatography . The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m . The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd . The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag . This may account for the lower stability of the ht compared with the native protein. Biochem J, 2000 Mar 1, 346 Pt 2, 355 - 8 Enzyme activity and dynamics: xylanase activity in the absence of fast anharmonic dynamics; Dunn RV et al.; The activity and dynamics of a simple, single subunit enzyme, the xylanase from Thermotoga maritima strain Fj SS3B.1 have been measured under similar conditions, from -70 to +10 degrees C . The internal motions of the enzyme, as evidenced by neutron scattering, undergo a sharp transition within this temperature range; they show no evidence for picosecond-timescale anharmonic behaviour (e.g . local diffusive motions or jumps between alternative conformations) at temperatures below -50 degrees C, whereas these motions are strongly activated at higher temperatures . The activity follows Arrhenius behaviour over the whole of the temperature range investigated, -70 to +10 degrees C . The results indicate that a temperature range exists over which the enzyme rate-limiting step is independent of fast anharmonic dynamics. Biochem J, 2000 Mar 1, 346 Pt 2, 275 - 80 Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms; Vanlingen S et al.; We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP(3)R3 as recombinant proteins {ligand-binding site 1 (lbs-1), lbs-2, lbs-3} in the soluble fraction of Escherichia coli . These recombinant proteins contain the complete IP(3)-binding domain and bound IP(3) and adenophostin A with high affinity . Ca(2+) and calmodulin were previously found to maximally inhibit IP(3) binding to lbs-1 by 42+/-6 and 43+/-6% respectively, and with an IC(50) of approx . 200 nM and 3 microM respectively {Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J . Biol . Chem . 274, 12157-12562} . We now report that Ca(2+) inhibited IP(3) binding to lbs-3 with an IC(50) of approx . 700 nM (37+/-4% inhibition at 5 microM Ca(2+)), while IP(3) binding to lbs-2 was not affected by increasing {Ca(2+)} from 100 nM to 25 microM . Calmodulin (10 microM) inhibited IP(3) binding to lbs-3 by 37+/-4%, while IP(3) binding to lbs-2 was inhibited by only 11+/-2% . The inhibition of IP(3) binding to lbs-3 by calmodulin was dose-dependent (IC(50) approximately 2 microM) . We conclude that the IP(3)-binding domains of the various IP(3)R isoforms differ in binding characteristics for IP(3) and adenophostin A, and are differentially modulated by Ca(2+) and calmodulin, suggesting that the various IP(3)R isoforms can have different intracellular functions. Biochem J, 2000 Mar 1, 346 Pt 2, 255 - 63 Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli; Griessler R et al.; Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each approximately 90-kDa subunit contains active-site pyridoxal 5'-phosphate . To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133-->Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation . Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at approximately 45 degrees C . They are manifested by slight increases in unordered structure and (1)H/(2)H exchange, and reflect reversible inactivation of MalP . Aggregation of the MalP dimer is triggered by these conformational changes and starts at approximately 45 degrees C without prior release into solution of pyridoxal 5'-phosphate . It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme . Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 degrees C . Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx . 58 and 67 degrees C, respectively . Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a approximately 15 degrees C higher value on the temperature scale . Apart from greater stability, the Asn-133-->Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme . A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP. Acad Radiol, 2000 Feb, 7(2), 83 - 93 Nontraumatic osteonecrosis: MR perfusion imaging evaluation in an experimental model; Kawamoto S et al.; RATIONALE AND OBJECTIVES: Because the nature and time course of changes in early, nontraumatic osteonecrosis at perfusion and magnetic resonance (MR) imaging are unknown, the authors evaluated this technique in the assessment of early osteonecrosis with a nontraumatic model . MATERIALS AND METHODS: Five rabbits underwent intravenous injection of lipopolysaccharide endotoxin followed by intramuscular injection of methylprednisolone . MR imaging of the femora was performed before and at weekly intervals after endotoxin injection . Histologic findings from the areas of osteonecrosis were correlated with the findings of MR imaging and MR perfusion studies . RESULTS: Histologic evaluation showed osteonecrosis in six femora of four animals 2-4 weeks after endotoxin injection . Findings on T1-weighted images of the femur were normal in all animals; T2-weighted images of one femur showed equivocal changes . On MR perfusion images, the baseline mean peak percentage of enhancement was 52.7% +/- 12.6 . In the six areas without osteonecrosis, the mean percentage of enhancement was similar to the baseline percentage of enhancement at 1 week (62.2% +/- 31.2) . In the four areas with diffuse osteonecrosis, there was essentially no contrast enhancement 1-4 weeks after endotoxin injection . CONCLUSION: T1- and T2-weighted MR imaging is insensitive to the presence of early nontraumatic osteonecrosis . MR perfusion imaging might be useful to detect early nontraumatic osteonecrosis. J Virol, 2000 Apr, 74(8), 3888 - 91 Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase; Vazquez AL et al.; The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases . To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine . All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized . The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate. Cancer Res, 2000 Mar 1, 60(5), 1162 - 7 Association of the Bloom syndrome protein with topoisomerase IIIalpha in somatic and meiotic cells; Johnson FB et al.; Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J . German and N . A . Ellis, The Genetic Basis of Human Cancer, pp . 301-316, 1998) . RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S . Gangloff et al., Mol . Cell . Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F . G . Harmon et al., Mol . Cell, 3: 611-620, 1999) . We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha . These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells . Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans. Lett Appl Microbiol, 2000 Jan, 30(1), 38 - 41 Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads; Madiraju MV et al.; A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli . First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E . coli recA mutant cells . Secondly, plasmid DNA was isolated from recombinant E . coli cells and analysed . Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M . bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA . This protocol was also amenable to other mycobacterial species such as M . avium, M . fortuitum and M . tuberculosis H37Ra . Plasmid recovery from the recombinant M . bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method. Eur J Biochem, 2000 Apr, 267(7), 1966 - 74 Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa; Krawetz JE et al.; The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA . RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death . Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1 . ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini . These deletions also result in RIP activation in vitro . However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent . To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species . We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity . The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed . The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate . The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes. Mutat Res, 2000 Mar 23, 466(2), 161 - 71 Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures; Hamer B et al.; This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay . This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples . The post-treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself . On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation . 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth . The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins . Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test. Mech Dev, 2000 Apr, 92(2), 179 - 91 Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification; Lammel U et al.; Salivary glands are simple structured organs which can serve as a model system in the study of organogenesis . Following a large EMS mutagenesis we have identified a number of genes required for normal salivary gland development . Mutations in the locus small salivary glands-1 (ssg-1) lead to a drastic reduction in the size of the salivary glands . The gene ssg-1 was cloned and subsequent sequence and genetic analysis showed identity to the recently published gene brinker . The salivary gland placode in brinker mutants appears reduced along both the anterior-posterior and dorso-ventral axis . Analysis of the brinker cuticle phenotype revealed a similar loss of anterior-posterior as well as lateral cell fates . The abdominal ventral denticle belts show a reduced number of setae in the first denticle row . Furthermore, we observed a preferential loss of lateral neuroblasts in the anterior parasegment . Together, these phenotypes suggest that brinker not only plays a role in dorso-ventral but also in anterior-posterior axis patterning. Vet Microbiol, 2000 Mar 15, 72(3-4), 295 - 310 Experimental models of porcine post-weaning colibacillosis and their relationship to post-weaning diarrhoea and digestive disorders as encountered in the field; Madec F et al.; The aim of this study was to develop a reliable model system of porcine post-weaning colibacillosis, and in doing so to assess the primary relationship of enterotoxigenic Escherichia coli to post-weaning diarrhoea and digestive disorders as encountered in the field . Six sequential experiments were carried out using 168 SPF piglets weaned into an optimal controlled environment at 28 days of age . The piglets were allocated to 23 treatment groups, 17 of which were inoculated either orally or intragastrically with enterotoxigenic strains of E . coli (LT+, STI+, STII+) possessing adhesive factors including K88 (F4) . The piglets were challenged either once (Day 4 post-weaning) or on several days post-weaning, with the challenge load for each inoculation varying from 10(8) to 10(12) CFU.Overall 14.5% of inoculated pigs developed severe illness and died: these had lesions in their digestive tracts typical of colibacillosis . Diarrhoea occurred on at least 1 day in 50% of inoculated pigs, but was transient (1.7 days on average), appeared very soon after challenge (sometimes within half a day), and was accompanied by signs of depression and low weight gain . Generally a prompt recovery then occurred . In the second 2 weeks post-inoculation daily weight gain reached the same level in most inoculated groups of pigs as in the uninoculated controls . Only a small number of pigs developed a chronic enteritis lasting several days, as is typically observed in field cases . Diarrhoea was more common in the piglets that were tested adhesive positive to the K88 fimbriae receptor, but the disorders were no more severe in these animals . The response of all pigs depended primarily on the inoculum used, and especially on the challenge load . Although enterotoxigenic E . coli are clearly important in the aetiology of post-weaning diarrhoea, other factors are also required for the production of the chronic post-weaning digestive disorders and ill-thrift that are commonly encountered in commercial piggeries. Vet Microbiol, 2000 Mar 15, 72(3-4), 269 - 76 Toxigenic Escherichia coli isolated from pigs in Argentina; Parma AE et al.; The presence of porcine toxigenic E . coli (ETEC, VTEC) in 28 piggeries (5% of total) of the central and northeast region of Argentina was studied for a better understanding of the epidemiology of porcine strains . Samples were taken by rectal swabs from healthy piglets and from those with diarrhoea, in addition to their dams . Between 5-10 colonies were isolated from each one of 223 animals sampled from 1992 to 1997 . By using specific primers each strain was screened by PCR for VT1, VT2all, VT2e, STIa, and LTI toxin genes . Only strains positive for any of the toxins mentioned above were screened for STb . Their O serogroups were determined by agglutination . All of the above enterotoxins and verocytotoxins were found in E . coli isolated from the animals . The STIa gene was detected in E . coli isolated from 27/127 piglets with diarrhoea, in comparison with LTI (4/127 pigs) . No toxin gene was amplified from E . coli isolated from either healthy piglets or their dams . When strains isolated from 48 piglets without diarrhoea but showing delayed growth were analysed by PCR, their toxin profile was determined to be VT1 (1/48 piglets), VT2all (5/48), STIa (1/48), LTI (3/48) and VT2e (3/48) . Serogroup O64 prevailed among ETEC; O138 prevailed for ETEC/VTEC strains . This is the first extensive study regarding porcine toxigenic E . coli in Argentina and constitutes an important database for the implementation of prevention measures. Biochem J, 2000 Apr 1, 347 Pt 1, 69 - 75 Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5; Hagner-Mcwhirter A et al.; In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level . The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 {Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Roden (1980) Biochemistry 19, 495-500} . Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ({4GlcAbeta1-4GlcNSO(3)alpha1-}(n)), or of O-desulphated heparin (predominantly {4IdoAalpha1-4GlcNSO(3)alpha1-}(n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx . 1 h of incubation . Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide . Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h . This isotope effect suggests that the second stage of the reaction, i.e . the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process . Analysis of the 5-(3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate . This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues . Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function {Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmstrom (1996) Biochem . J . 313, 589-596}. Pharmacol Toxicol, 2000 Feb, 86(2), 78 - 82 Effect of loperamide on mucosal guanylyl cyclase activity in rat jejunum following Escherichia coli heat-stable toxin-induced fluid accumulation; Farack UM et al.; Loperamide has antidiarrhoeal activities against secretagogues with different mechanisms of action . Besides its opioid-like effect on intestinal motility and secretion it might exhibit additional antisecretory properties which may not be completely elucidated yet . Direct effects of loperamide on mucosal guanylyl cyclase have never been observed . We therefore investigated the effect of loperamide on intestinal fluid transport altered by heat-stable Escherichia coli enterotoxin which acts by stimulating mucosal guanylyl cyclase . Net fluid movement was determined during a 1 hr incubation period in ligated jejunal loops of anaesthetised female Wistar rats . Transport rates of net fluid movement were calculated from the loop contents measured gravimetrically at the beginning and the end of the experiments . Addition of heat-stable Escherichia coli enterotoxin to the luminal solution resulted in a net secretion of water which was significantly reversed into net absorption by loperamide . The specific activity of the particulate guanylyl cyclase was determined in mucosal scrapings of the jejunum without and with the addition of heat-stable Escherichia coli enterotoxin and/or loperamide . Additions of loperamide of up to 10 micromol/l did not change guanylyl cyclase activity . We conclude that the effect of loperamide counteracting heat-stable Escherichia coli enterotoxin induced changes of intestinal fluid transport does not involve a direct effect on guanylyl cyclase. Biochemistry, 2000 Mar 28, 39(12), 3486 - 90 Disulfide linkage of the b and delta subunits does not affect the function of the Escherichia coli ATP synthase; McLachlin DT et al.; The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits . We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit . The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta . The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain . These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function. Biochemistry, 2000 Mar 28, 39(12), 3266 - 75 Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase); Mowat CG et al.; Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction . The crystal structure of the native yeast enzyme has been determined {Xia, Z.-X., and Mathews, F . S . (1990) J . Mol . Biol . 212, 837-863} as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli {Tegoni, M., and Cambillau, C . (1994) Protein Sci . 3, 303-313}; several key active site residues were identified . In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations . In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site . This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically . Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2) . Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction . Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate . Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state . In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2) . Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution . The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex {Tegoni, M., and Cambillau, C . (1994) Protein Sci . 3, 303-313} . Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context. Biochemistry, 2000 Mar 28, 39(12), 3240 - 7 An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia; Huang X et al.; The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP . The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit . These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E . coli . It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit . To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis . These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit . Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel . With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another . However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P . These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source. Parasitol Res, 2000 Mar, 86(3), 200 - 6 Removal of hydrogen peroxide by a 1-cysteine peroxiredoxin enzyme of the filarial parasite Dirofilaria immitis; Chandrashekar R et al.; Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells . However, the mechanisms of H2O2 removal by the filarial parasites are unclear . Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis . A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D . immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1) . rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol . The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1) . Consistent with the enzyme activity, D . immitis adult worms could detoxify exogenously added H2O2 in vitro . Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products . The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry . In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms . Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system. Electrophoresis, 2000 Feb, 21(3), 645 - 53 Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms; Perrot F et al.; The two-dimensional electrophoretic patterns of cellular proteins from gel-entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms . The amounts of several proteins in immobilized cells were significantly different from those in free bacteria . Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase . Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein . The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed. Electrophoresis, 2000 Feb, 21(3), 497 - 508 Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; Steinberg TH et al.; SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization . Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining . Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm . The fluorescence emission maximum of the dye is approximately 640 nm . Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins . This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues . The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band . This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures . Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest . This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide . The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting . Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry. Pathobiology, 1999, 67(5-6), 253 - 6 Comparison of monocyte functions after LPS- or IL-10-induced reorientation: importance in clinical immunoparalysis; Wolk K et al.; Immunoparalysis is an acquired immunodeficiency which may occur in patients after major surgery, burns, polytrauma and sepsis . It is associated with a modified state of monocytes marked by their altered capacity to induce antigen-specific T cell stimulation and to release various cytokines . However, the pathogenesis of immunoparalysis may differ in various patient groups . It can develop in patients after systemic hyperinflammation induced by gastrointestinal translocation of endotoxin (lipopolysaccharide, LPS) or sepsis, as well as in patients without preceding systemic inflammation but primary anti-inflammation, for instance induced by sympathetic activation . To further elucidate the syndrome, we compared endotoxin tolerance as a model of immunoparalysis after systemic hyperinflammation versus interleukin-10 (IL-10) treatment as a model of primarily anti-inflammation-induced immunoparalysis . In vitro priming of peripheral blood mononuclear cells with either LPS or IL-10 for 24 h led to a strongly or moderately diminished LPS-induced tumor necrosis factor-alpha (TNF-alpha) production, compared to unprimed controls, respectively . Furthermore, LPS-induced reduction of TNF-alpha production capacity persisted over the following days whereas IL-10-primed monocytes rapidly recovered . Similarly, in contrast to persistently diminished MHC class II expression in LPS-treated monocytes, IL-10 only transiently downregulated these molecules . Consequently, in contrast to IL-10-primed monocytes, LPS-primed monocytes were greatly impaired in their capacity to induce antigen-specific T cell proliferation and IFN-gamma production . These data indicate that LPS priming provokes a more profound modulation of monocyte function than IL-10 priming, raising the question of possible variations in the clinical course of immunoparalysis, dependent on its pathogenesis . Mutat Res, 2000 Mar 20, 459(2), 109 - 14 Deoxyxanthosine in DNA is repaired by Escherichia coli endonuclease V; He B et al.; Deoxycytidine, deoxyadenosine and deoxyguanosine undergo spontaneous deamination to form deoxyuridine, deoxyinosine and deoxyxanthosine, respectively . In this manuscript, we show that in addition to its known ability to recognize deoxyuridine and deoxyinosine in DNA, Escherichia coli endonuclease V cleaves DNA containing deoxyxanthosine . However, Alk A protein and human methylpurine glycosylase are unable to recognize deoxyxanthosine . Endonuclease V cleaves DNA containing deoxyxanthosine at the second phosphodiester bond 3' to deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the nick site . This endonucleolytic activity requires Mg(2+) or Mn(2+), and is highly specific for double stranded DNA . Endonuclease V-catalyzed cleavage of DNA containing deoxyxanthosine is a result of its ability to recognize the altered base and not due to its mismatch-specific endonuclease activity . The ability of endonuclease V to recognize both deoxyinosine and deoxyxanthosine suggests that endonuclease V is important for preventing mutations that might arise as a result of deamination of purines. Virus Res, 2000 Feb, 66(2), 197 - 207 RNA-binding activities of cocksfoot mottle sobemovirus proteins; Tamm T et al.; Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs) . ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases . P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase . P3, the coat protein, is encoded by ORF3 . CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed . The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner . The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3 . The RNA-binding activities of His-tagged P3 and native coat protein were similar . P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations . In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl . We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins. J Biotechnol, 2000 Mar 10, 78(2), 115 - 22 Modification with a phosphorylation tag of PKA in the TraT-based display vector of Escherichia coli; Chang H et al.; We have previously developed the TraT display system to express the preS1 peptide of human hepatitis B virus (HBV) and the snake venom rhodostomin (RHO) on the surface of Escherichia coli . In this study, we modified the pT2 vector by adding a thrombin cutting site and a phosphorylation tag of protein kinase A before the multiple restriction enzyme sites . The modified vector allowed us to label the TraT fusion protein (TraT-RHO) with {32P} and to increase the detection sensitivity of TraT-RHO expression bacteria binding to and being internalized into BHK-21 cells . After the thrombin cleavage, the isotope labeled RHO could be detected in a free form . We therefore suggest that the new version of pT2 vector, pT2-KL, will facilitate to identify the counterpart of displayed peptide. J Immunol Methods, 2000 Apr 3, 237(1-2), 175 - 86 A two-phagemid system for the creation of non-phage displayed antibody libraries approaching one trillion members; Ostermeier M et al.; We have designed a two-phagemid system for the construction of very large non-phage displayed Fab antibody libraries in E . coli approaching 10(12) members . The system can accommodate both periplasmic and cytoplasmic Fab expression and should prove useful for the direct selection of functional antibodies by genetic techniques . We successfully alleviate problems of Fab vector instability and report a set of improved 5' primers for the amplification of mouse Ig V(H)95% of mouse Ig V(H) genes and minimize the amount of N-terminal amino acid changes while maintaining the flexibility of periplasmic or cytoplasmic antibody expression in E . coli. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3747 - 52 Arabidopsis RelA/SpoT homologs implicate (p)ppGpp in plant signaling; van der Biezen EA et al.; Arabidopsis RPP5 is a member of a large class of pathogen resistance genes encoding nucleotide-binding sites and leucine-rich repeat domains . Yeast two-hybrid analysis showed that RPP5 specifically interacts with At-RSH1, an Arabidopsis RelA/SpoT homolog . In Escherichia coli, RelA and SpoT determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotides of the bacterial stringent response . Functional analysis in E . coli and in Streptomyces coelicolor A3 (2) showed that At-RSH1 confers phenotypes associated with (p)ppGpp synthesis . We characterized two additional Arabidopsis RelA/SpoT homologs, At-RSH2 and At-RSH3 . At-RSH genes may regulate a rapid plant (p)ppGpp-mediated response to pathogens and other stresses. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3872 - 7 Enhanced visibility of hydrogen atoms by neutron crystallography on fully deuterated myoglobin; Shu F et al.; Although hydrogens comprise half of the atoms in a protein molecule and are of great importance chemically and structurally, direct visualization of them by using crystallography is difficult . Neutron crystallography is capable of directly revealing the position of hydrogens, but its use on unlabeled samples faces certain technical difficulties: the large incoherent scattering of hydrogen results in background scattering that greatly reduces the signal to noise of the experiment . Moreover, whereas the scattering lengths of C, N, and O are positive, that of hydrogen is negative and about half the magnitude . This results in density for hydrogens being half as strong and close to the threshold of detection at 2.0-A resolution . Also, because of its opposite sign, there is a partial cancellation of the hydrogen density with that from neighboring atoms, which can lead to ambiguities in interpretation at medium resolution . These difficulties can be overcome by the use of deuterated protein, and we present here a neutron structure of fully deuterated myoglobin . The structure reveals a wealth of chemical information about the molecule, including the geometry of hydrogen bonding, states of protonation of histidines, and the location and geometry of water molecules at the surface of the protein . The structure also should be of broader interest because it will serve as a benchmark for molecular dynamics and energy minimization calculations and for comparison with NMR studies. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3079 - 83 Isoleucine-15 of rainbow trout carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding; Guan G et al.; Carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) is an enzyme that converts 17alpha-hydroxyprogesterone to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish) . We have previously isolated two types of CR/20beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss) . Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20beta-HSD activity but that the other (type B) did not . Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site . To investigate the structure/function relationships of CR/20beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K . Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T) . Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I) . MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested . Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH. Drug Metab Dispos, 2000 Apr, 28(4), 398 - 402 Thioesterification of 2-arylpropionic acids by recombinant acyl-coenzyme A synthetases (ACS1 and ACS2); Sevoz C et al.; 2-Arylpropionic acids are a class of frequently used nonsteroidal anti-inflammatory drugs exhibiting a potent inhibition of cyclooxygenase isoforms supported by the (+)S-enantiomer alone . Nevertheless, some of these compounds in the (-)R configuration may undergo extensive inversion of configuration to their antipode . The key molecular basis for this mechanism invokes the stereoselective formation of the coenzyme A (CoA) thioester of the 2-arylpropionic acid by long-chain acyl-CoA synthetases (ACSs) . In this report, rat recombinant ACS1 and ACS2 enzymes, constitutively highly expressed in adult rat liver and brain, respectively, have been overproduced in Escherichia coli strains and purified to homogeneity to investigate the involvement of these enzymes in the thioesterification of fenoprofen and ibuprofen . Recombinant ACS1 efficiently catalyzed both nonsteroidal anti-inflammatory drugs with Michaelis-Menten parameters of K(M) = 1686 +/- 93 microM, V(max) = 353 +/- 45 nmol/min/mg protein for (-)R-ibuprofen and K(M) = 103 +/- 12 microM, V(max) = 267 +/- 10 nmol/min/mg protein for (-)R-fenoprofen, and exhibited a marked stereoselectivity in favor of the (-)R-enantiomer . Recombinant ACS2, a closely related sequence with ACS1, exhibited a lower enzymatic efficacy from 7- to 130-fold for (-)R-ibuprofen and (-)R-fenoprofen, respectively . On the basis of these findings and considering the level of tissue expression of the different long-chain ACSs, ACS1 appears to be the major enzyme involved in the first step of the chiral inversion of 2-arylpropionic acids . Nevertheless, the participation of other ACS isoforms of minor quantitative importance could not be excluded in the thioesterification of xenobiotics. J Cell Sci, 2000 Apr, 113 ( Pt 8), 1471 - 80 Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-alpha export; Hood JK et al.; Proteins bearing canonical nuclear localization sequences are imported into the nucleus by the importin/karyopherin-alpha/beta heterodimer . Recycling of the importin-alpha subunit to the cytoplasm requires the action of Cas, a member of the importin-beta superfamily . In the yeast Saccharomyces ceresivisiae, the essential gene CSE1 encodes a Cas homologue that exports the yeast importin-alpha protein Srp1p/Kap60p from the nucleus . In this report, we describe a role for the FXFG nucleoporin Nup2p, and possibly the related Nup1p, in the Cse1p-mediated nuclear export pathway . Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 accumulate Srp1p in the nucleus . Similarly, Cse1p is displaced from the nuclear rim to the nuclear interior in deltanup2 cells . We do not observe any biochemical interaction between Cse1p and Nup2p . Instead, we find that Nup2p binds directly to Srp1p . We have localized Nup2p to the interior face of the nuclear pore complex, and have shown that its N terminus is sufficient for targeting Nup2p to the pore, as well as for binding to Srp1p . Taken together, these data suggest that Nup2p is an important NPC docking site in the Srp1p export pathway. Virology, 2000 Mar 30, 269(1), 225 - 37 Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence; Campbell MS et al.; Tick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, is naturally attenuated for humans but retains demonstrable neurovirulence and peripheral virulence ("neuroinvasiveness") for mice . Previously a mutant, strain E5, less virulent for mice was derived from LGT TP21 . Multiple attempts to prepare a full-length infectious TP21 cDNA from cDNA fragments cloned in E . coli were uniformly unsuccessful . A more informative sequence than that obtained from these cloned cDNA fragments and similar E5 cDNA fragments was derived from RT-PCR fragments that had not been cloned in E . coli . Comparison of the RT-PCR consensus sequence of TP21 and E5 identified only seven amino acid differences that might be responsible for the observed difference in virulence of these strains for mice . Eleven independent infectious cDNA clones of TP21 were recovered using two overlapping long RT-PCR fragments . Importantly, low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of deletion mutants that accumulated late in infection . The four analyzed rescued clones exhibited clone-specific minimal divergence from the consensus sequence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice . Manipulation of these clones should facilitate elucidation of LGT virulence . Bioconjug Chem, 2000 Mar-Apr, 11(2), 258 - 66 Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy; Cheng TL et al.; The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol) . The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity . Conjugate levels in tumors decreased to 36% of peak levels at this time . Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72 . 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors . Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72 . 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy. Allergy, 2000 Feb, 55(2), 141 - 7 Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11; Tsai LC et al.; BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned . This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11 . METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages . The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E . coli . These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays . RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus . The sequence identity of Der f 11 with other known paramyosins is 34-60% . The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2 . These peptides are more reactive than whole rDf642 . CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins . The human IgE and IgG epitopes are scattered throughout the entire molecule . Data also indicate the presence of unique IgE and IgG epitopes in Der f 11. Plant Sci, 2000 May 15, 154(1), 53 - 60 Characterisation of acyl-ACP desaturases from Macadamia integrifolia Maiden & Betche and Nerium oleander L; Gummeson PO et al.; The seed oil in Macadamia integrifolia contains about 30% palmitoleic acid (16:1(Delta9)) and Nerium oleander about 12% isoricinoleic acid (Delta9-hydroxy-18:1(Delta12)) . It has been shown that palmitoleic acid can be produced by acyl-acyl carrier protein (ACP) desaturases and it has also been shown that fatty acid hydroxylation can occur via direct substitution of a hydrogen atom . Therefore it seemed possible that the enzymes responsible for the making of these unusual fatty acids in M . integrifolia and N . oleander were of acyl-ACP desaturase type . Extracts from developing M . integrifolia developing seeds showed a relative ratio of 16:0-ACP to 18:0-ACP desaturation that was about 13 times higher than in sunflower seeds . N . oleander seed extracts catalysed conversion of 18:0-ACP to 18:1(Delta9) but only trace amounts of Delta9-hydroxy fatty acids were formed . A total of four cDNAs were isolated from developing seeds, of both species, using a fragment isolated with PCR amplification . The M . integrifolia acyl-ACP desaturase cDNA was expressed in Escherichia coli . A partly purified fraction of the enzyme showed a 16:0-ACP to 18:0-ACP desaturation ratio about 90-fold less than that in the Macadamia extracts . Expressed N . oleander acyl-ACP desaturase cDNAs showed predominantly 18:0-ACP desaturase activity and no hydroxylase activity . Thus it is not likely that any of the four acyl-ACP desaturases cloned from M . integrifolia or N . oleander is involved in the production of unusual fatty acids. J Extra Corpor Technol, 1999 Jun, 31(2), 67 - 75 Matrix metalloproteinase inhibitor: differential effects on pulmonary neutrophil and monocyte sequestration following cardiopulmonary bypass; McCann UG 2nd et al.; Acute respiratory distress syndrome (ARDS) following cardiopulmonary bypass (CPB), also known as "post-pump" or "post-perfusion syndrome" (PPS), results from sequential priming and activation of neutrophils . We hypothesized that chemically modified tetracycline (CMT-3) an inhibitor of neutrophil matrix metalloproteinase (MMP) and elastase, would prevent PPS . We performed histometric analysis of lung tissue from our porcine PPS model to correlate cellular sequestration and histologic injury with CMT-3 treatment . METHODS: Yorkshire pigs were randomized into five groups: Control (n = 3); CPB (n = 5); femoral-femoral bypass 1 hour; LPS (n = 7), Escherichia coli lipopolysaccharide (1 microgram/kg); CPB + LPS (n = 6); and CPB + LPS + CMT (n = 5), sequential insults and CMT-3 . Protocol histometric analysis defined cellular and tissue components of lung injury . RESULTS: CMT-3 decreased neutrophil sequestration in the CPB + LPS + CMT-3 group (p < 0.0001 vs . CPB + LPS) . There were no differences in monocytes between CPB + LPS and CPB + LPS + CMT treatment groups . CONCLUSIONS: CMT-3 attenuates neutrophil sequestration but has no effect on mononuclear sequestration in our PPS model . This finding supports current research on leukocyte chemokines and has important implications regarding mechanisms of CMT-3 . Despite lack of monocyte response to CMT-3, PPS was prevented by inhibiting neutrophils alone; confirming the primary role of neutrophils in PPS. Carbohydr Res, 2000 Feb 25, 324(3), 161 - 9 Enzymatic synthesis of blood group A and B trisaccharide analogues; Seto NO et al.; Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates . These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations . The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal . Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates . We now report a unique donor specificity for each enzyme type . Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-{alpha-L-Fucp-(1-->2)}-beta-D-Galp-O-(CH2 )7CH3 (4) . Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-{alpha-L-Fucp-(1-->2)}-beta-D-Galp-O-(CH2) 7CH3 (5) . In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc . Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5. Mol Cell Biochem, 2000 Jan, 203(1-2), 95 - 101 Synthesis and phosphorylation of androgen receptor of the mouse brain cortex and their regulation by sex steroids during aging; Thakur MK et al.; To examine the synthesis and phosphorylation of androgen receptor (AR) and their regulation by sex steroids, adult (24 weeks) and old (65 weeks) male and female mice were gonadectomized and administered with testosterone and estradiol . AR amount, synthesis and phosphorylation were measured in the brain cortex by immunoblotting and immunoprecipitation using antibody raised against rat AR transactivation domain (TAD) which was expressed in E . coli as a fusion protein . We found that the amount of AR was high in adult and declined in old mice of both sexes . Administration of testosterone and estradiol significantly down-regulated the level of AR in old male and adult female . Similarly, the rate of AR synthesis also declined with age . Exogenous treatment of gonadectomized mice with testosterone and estradiol reduced the extent of synthesis significantly in all groups except in old female . No sex-dependent variation was noticed either in the level or synthesis of AR . In contrast, the extent of phosphorylation was higher in old mice of both sexes as compared to their adult counterparts . Testosterone and estradiol supplementation resulted in remarkable increase in AR phosphorylation in all groups . Thus it is evident from our findings that the amount and synthesis of AR decrease but phosphorylation of AR increases in the brain cortex with advancing age of mice and they are regulated by testosterone and estradiol in age- and sex-specific manner. Nature, 2000 Mar 9, 404(6774), 205 - 8 Ligand binding and conformational motions in myoglobin; Ostermann A et al.; Myoglobin, a small globular haem protein that binds gaseous ligands such as O2, CO and NO reversibly at the haem iron, serves as a model for studying structural and dynamic aspects of protein reactions . Time-resolved spectroscopic measurements after photodissociation of the ligand revealed a complex ligand-binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein . To observe the structural changes induced by ligand dissociation, we have carried out X-ray crystallographic investigations of carbon monoxy-myoglobin (MbCO mutant L29W) crystals illuminated below and above 180 K, complemented by time-resolved infrared spectroscopy of CO rebinding . Here we show that below 180 K photodissociated ligands migrate to specific sites within an internal cavity--the distal haem pocket--of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing . Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels . We recover most of the ligands in a cavity on the opposite side of the haem group. Int Rev Immunol, 2000, 19(1), 39 - 50 Colitis in HLA-B27/beta 2 microglobulin transgenic rats; Sartor RB; Rats of susceptible genetic backgrounds expressing high copy numbers of the transgene encoding HLA-B27 and human beta 2 mu develop chronic colitis complicated in the advanced stage by adenomatous polyps progressing to adenocarcinoma . Unique features of this model include a spectrum of extraintestinal manifestations resembling to some extent human spondyloarthropathy, with peripheral and axial joint, dermatologic and male genital inflammation . Inflammation is T lymphocyte mediated, although surprisingly CD4+ cells are more active in transferring disease than CD8+ cells, which would be expected to be preferentially activated by Class I MHC peptides . Inflammation is dependent on a nonlymphoid bone marrow-derived cell, expressing high copy numbers of B27, probably APCs . In vitro function of transgenic dendritic cells is deficient, and in vivo competition for peptide binding in the antigen binding site of B27 attenuates arthritis . Normal bacteria are required for disease expression, with B . vulgatus preferentially able to induce colitis, whereas other bacteria such as E . coli stimulate no inflammatory response . Inflammation and resulted complications are modulated by non-MHC genes and are amenable to treatment by bone marrow transplant from normal donors . These results support the hypothesis that gastrointestinal and systemic inflammation in B27 transgenic rats is the result of loss of tolerance to enteric bacteria, as a consequence of defective APC (? dendritic cells) function . Whether disease is the result of selective MHC binding of enteric antigens uniquely capable of inducing disease, lack of appropriate induction of a CD8+ suppressor cell population, or skewed cytokine (IL-12, IL-18) secretion by APCs remains to be determined. Biotechniques . 2000 Mar;28(3):510, 512, 514, 516 passim. Polycationic lipids translocate lipopolysaccharide into HeLa cells; Eldstrom JR et al.; We have investigated the ability of LIPOFECTAMINE, a polycationic lipid reagent used in DNA transfection, to translocate E . coli lipopolysaccharide (LPS) into HeLa cells . Although HeLa cells did not spontaneously take up fluorescein isothiocyanate-labelled LPS (FITC-LPS) from the culture medium, the cells that were co-incubated with greater than 1 g/mL FITC-LPS and LIPOFECTAMINE showed punctate fluorescence . Virtually all cells were loaded on incubation with 100 micrograms/mL FITC-LPS . Confocal scanning laser microscopy showed extensive FITC-LPS loading in the cytoplasm of HeLa cells, but no label was evident in the nuclear regions of these cells . Loading with LPS for up to six hours had no effect on the viability of HeLa cells, beyond the 30% reduction in live cells that is attributable to the toxic effect of LIPOFECTAMINE itself . In contrast to cells treated with etoposide for six hours, LPS-loaded cells did not display apoptotic bodies . Exposure of cells to 4 beta-phorbol 12-myristate 13-acetate led to the induction of the immediate early gene c-fos and resulted in an enhanced c-Fos signal, detected by Western blot analysis . In contrast, LPS loading did not alter the c-fos expression in HeLa cells . The loading of LPS into HeLa cells by means of polycationic lipids results in relatively low acute toxicity, as judged from cell viability, morphology and c-fos expression . Therefore, our method appears well suited to the study of acute actions of LPS in the intracellular compartment of mammalian cells. Biotechniques, 2000 Mar, 28(3), 498 - 500, 504-5 Restriction site-free insertion of PCR products directionally into vectors; Chen GJ et al.; A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency . The method uses a pair of DNA integration primers with two portions . The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector . For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions . This method has been used to clone the E . coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein . It was also applied to convert a construct of the E . coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag. Biotechniques, 2000 Mar, 28(3), 456 - 60, 462 Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags; Kaldalu N et al.; We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags . We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells . The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays . The tags were shown not to interfere with the function of these proteins in vivo and in vitro . Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions . We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M . This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins . We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins . The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins. Biophys Chem, 2000 Feb 14, 84(1), 45 - 64 Thermodynamics of reactions catalyzed by anthranilate synthase; Byrnes WM et al.; Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan . One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l) . This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq) . The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively . Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79 . Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08 . The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products . These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms . For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1 . For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1 . Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway . The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength . Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given. J Biol Chem, 2000 Mar 24, 275(12), 8863 - 71 Purification, cloning, and characterization of an acidic ectoprotein phosphatase differentially expressed in the infectious bloodstream form of Trypanosoma brucei; Bakalara N et al.; We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei . The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites . There was no significant sequence homology with other phosphatases . Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F . Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly . The recombinant N-terminal domains, expressed in E . coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP . The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole . Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy. J Biol Chem, 2000 Mar 24, 275(12), 8844 - 53 Cloning and characterization of a new member of the Nudix hydrolases from human and mouse; Yang H et al.; Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives . Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars . The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence . The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity . At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose . The murine NUDT5 homolog was also cloned and characterized . mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9 . Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver . The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively . The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed. J Biol Chem, 2000 Mar 24, 275(12), 8726 - 32 The CafA protein required for the 5'-maturation of 16 S rRNA is a 5'-end-dependent ribonuclease that has context-dependent broad sequence specificity; Tock MR et al.; The CafA protein, which was initially described as having a role in either Escherichia coli cell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5'-end of 16 S rRNA . The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E . coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids . We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting . We detected CafA cleavage of RNAI and a structured region from the 5'-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site . The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA . Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs . Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro . We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5'-end of the RNA . This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5'-end of an RNA . Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity . Together with the knowledge that CafA can cut RNAI and ompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E . coli. J Biol Chem, 2000 Mar 24, 275(12), 8531 - 9 Recruitment of a foreign quinone into the A(1) site of photosystem I . II . Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy; Zybailov B et al.; Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described . Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone . In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type . Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e . the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type . Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings . They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site . These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9. J Biol Chem, 2000 Mar 24, 275(12), 8523 - 30 Recruitment of a foreign quinone into the A(1) site of photosystem I . I . Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp . pcc 6803; Johnson TW et al.; Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp . PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli . Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway . The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)) . The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity . The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates . PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin . HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type . We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters. J Biol Chem, 2000 Mar 24, 275(12), 8480 - 6 Kinetic studies of cAMP-induced allosteric changes in cyclic AMP receptor protein from Escherichia coli; Malecki J et al.; Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli . The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP . Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites . Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP(2) complex, i.e . after saturation of the high affinity sites . The observed conformational change occurs according to sequential model of allostery and is described by rate constants: k(c) = 9.7 +/- 0.1 s(-1) and k(-c) = 0.31 +/- 0.05 s(-1), for the forward and backward reaction, respectively . Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP(4) complex and results from displacement of equilibrium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affinity sites of one of these forms only . The observed conformational change occurs according to concerted model of allostery and is described by rate constants: k(on) = 28 +/- 1.5 s(-1) and k(off) = 75.5 +/- 3 s(-1) . Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP. J Biol Chem, 2000 Mar 24, 275(12), 8469 - 74 Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation; Gan XQ et al.; The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes . The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region . Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate . On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin . In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate . Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro . These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation. J Biol Chem, 2000 Mar 24, 275(12), 8448 - 55 The C-terminal domain of MutY glycosylase determines the 7,8-dihydro-8-oxo-guanine specificity and is crucial for mutation avoidance; Li X et al.; Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, or A/C mismatches and also has a weak guanine glycosylase activity on G/8-oxoG-containing DNA . The N-terminal domain of MutY, residues 1-226, has been shown to retain catalytic activity . Substrate binding, glycosylase, and Schiff base intermediate formation activities of the truncated and intact MutY were compared . MutY has high binding affinity with 8-oxoG when mispaired with A, G, T, C, or inosine . The truncated protein has more than 18-fold lower affinities for binding various 8-oxoG-containing mismatches when compared with intact MutY . MutY catalytic activity toward A/8-oxoG-containing DNA is much faster than that on A/G-containing DNA whereas deletion of the C-terminal domain reduces its catalytic preference for A/8-oxoG-DNA over A/G-DNA . MutY exerts more inhibition on the catalytic activity of MutM (Fpg) protein than does truncated MutY . The tight binding of MutY with GO mispaired with T, G, and apurinic/apyrimidinic sites may be involved in the regulation of MutM activity . An E . coli mutY strain that produces an N-terminal 249-residue truncated MutY confers a mutator phenotype . These findings strongly suggest that the C-terminal domain of MutY determines the 8-oxoG specificity and is crucial for mutation avoidance by oxidative damage. J Biol Chem, 2000 Mar 24, 275(12), 8283 - 6 Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate; Palenchar PM et al.; ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA . Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese . Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis . We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide . Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo . We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis . To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S(2-) rather than S(0). Infect Immun, 2000 Apr, 68(4), 2369 - 73 Molecular and immunological analyses of the Borrelia turicatae Bdr protein family; Carlyon JA et al.; Here, we describe the molecular and immunological characterization of the bdr gene family of Borrelia turicatae, a relapsing-fever spirochete . Nine bdr alleles belonging to two different subfamilies were sequenced and localized to linear plasmids . Anti-Bdr antiserum was generated and used to analyze Bdr expression in pre- and postinfection isogenic populations . The analyses presented here provide a detailed characterization of the Bdr proteins in a relapsing-fever spirochete species, enhancing our understanding of these proteins at the genus-wide level. Infect Immun, 2000 Apr, 68(4), 2323 - 7 Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach; Gomez M et al.; Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host . Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology . The 3,924 proteins deduced from the M . tuberculosis genome were analyzed with several computer programs . We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties . Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins . Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization . These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M . tuberculosis with high efficiency and 90% accuracy. Infect Immun, 2000 Apr, 68(4), 2254 - 8 Effects of Shiga toxin 2 on lethality, fetuses, delivery, and puerperal behavior in pregnant mice; Yoshimura K et al.; Shiga toxin 2 (Stx2) is produced by enterohemorrhagic Escherichia coli (EHEC) and is known as the major virulence factor of EHEC . The aim of this study was to evaluate the effects of Stx2 on (i) maternal lethality, (ii) fetuses, (iii) delivery period, and (iv) maternal behavior after delivery . Timed pregnant ICR mice were injected intravenously with Stx2 on day 5 of pregnancy (early stage) or on day 15 (late stage) . In early-stage experiments, the number of normal fetuses of mice injected with Stx2 was significantly lower than that of control mice . In late-stage experiments, mothers injected with Stx2 delivered normal numbers of neonates, but could not take care of them . The lethal doses of Stx2 were not different for pregnant and nonpregnant female mice at either stage . We conclude that Stx2 is toxic to the fetus in early pregnancy and affects maternal puerperal behavior in late pregnancy. Infect Immun, 2000 Apr, 68(4), 2171 - 82 Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2; Marches O et al.; Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits . Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions . In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers . To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant . When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant . When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative . In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions . In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli . The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits . Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits . Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant . In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity. Infect Immun, 2000 Apr, 68(4), 2096 - 101 Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains; Bonacorsi SP et al.; Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified . However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM . To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique . The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E . coli strain C5, isolated from a case of neonatal meningitis . Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM . We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains . Of these clones, 44 were clustered in six distinct regions on the chromosome . The sfa and ibe10 genes were located in regions 2 and 6, respectively . A group of genes (cnf1, hra, hly, and prs) known to be present in a pathogenicity island of the uropathogenic strain E . coli J96 colocalized with region 6 . The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group . Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains . Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E . coli to invade the meninges of neonates. Infect Immun, 2000 Apr, 68(4), 1884 - 92 Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment; Fenno JC et al.; Proteins secreted or exported by Treponema denticola have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases . However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment . A prominent 70-kDa protein was isolated from surface extracts of T . denticola ATCC 35405 . A clone expressing a portion of the protein was identified in an Escherichia coli expression library of T . denticola DNA . DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria . Genes encoding OppB, -C, -D, and -F were identified directly downstream of oppA in T . denticola . OppA was present in representative strains of T . denticola and in Treponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii . Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete . Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells . A T . denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog epsilon-aminocaproic acid . Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients. Antimicrob Agents Chemother, 2000 Apr, 44(4), 873 - 8 A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame; Sahli R et al.; Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV) . To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples . After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector . The ligation products are then used to transform tdk mutant bacteria . After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression . An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR) . Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR . In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates . We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated . This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample. Gene, 2000 Jan 25, 242(1-2), 381 - 91 PCR-based gene targeting in the filamentous fungus Ashbya gossypii; Wendland J et al.; We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya gossypii . Short guide sequences with 40-46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination . The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3 . This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants . Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed . This approach was used for the complete deletion of the open reading frame of the A . gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers . We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers . A gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established . Since short target guide sequences are sufficient to direct homologous integration into the A . gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi . Thus functional analysis of A . gossypii genes is already possible, based on single-pass sequence information. Adv Exp Med Biol, 1999, 467, 833 - 40 Enhancement of the specificity of an enzyme-based biosensor for L-tryptophan; Simonian AL et al.; A new selective amperometric biosensor for reagentless L-tryptophan determination has been developed using immobilized tryptophan-2-monooxygenase (TMO, EC 1.13.12.3) . This enzyme-based biosensor provides a rapid-response detection system for concentrations of L-tryptophan between 25 and 1,000 microM in a batch mode system and between 100 and 50,000 microM in a flow-injection mode . The response time was 30 seconds, and the total analysis time was less than 3 minutes . The biosensor retained catalytic activity and fidelity of phenylalanine and tryptophan response for greater than 4 months with repeated usage . The biosensor selectivity to L-tryptophan was dramatically increased relative to phenylalanine when a competitive inhibitor of TMO, indole acetamide (IA), was included . The biosensor was successfully used for L-tryptophan determination in nutrition broth, giving values identical to those determined by HPLC analysis. Adv Exp Med Biol, 1999, 467, 789 - 99 Tryptophans in membrane proteins . X-ray crystallographic analyses; Wallace BA et al.; While tryptophans are generally found in low abundance in soluble proteins, in many integral membrane proteins they comprise a significantly higher proportion of the amino acid composition . Now that crystal structures are available for a number of membrane proteins, it has been possible to examine the distribution and disposition of the tryptophans within these structures . The tryptophan locations with respect to the lipid bilayer (along the direction normal to the membrane surface) are strikingly non-uniform in nearly all of the membrane proteins examined . They tend to cluster at the interface between the polar head group region and the hydrophobic interior, in a relatively uniform layer just below the surface . In many cases, their distributions with respect to the extra- and intra-cellular surfaces tend to be asymmetric . These observations provide evidence for possible structural roles for tryptophans in transmembrane sheets and helices, where they may play a part in the stabilization of the transmembrane segments and perhaps in the orientation and bilayer insertion processes. Adv Exp Med Biol, 1999, 467, 611 - 4 Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase; Fukuoka S et al.; To elucidate the molecular basis underlying quinolinate metabolism, the cDNA encoding human brain QPRTase was cloned. Adv Exp Med Biol, 1999, 467, 443 - 52 From sugar beet molasses to Lyphan . Integrated quality management from the raw material to the drug; Faurie R et al.; L-tryptophan is produced at the AMINO GmbH (Frellstedt, FRG) via biocatalytical condensation of the amino acid L-serine with indole . As a biocatalyst, tryptophan synthetase is used which is produced in high activities by a natural mutant Escherichia coli strain . The enzyme mechanism and specificity and the individual process-parameters for the biotransformation procedure are explained as well as the purification process of educts and products . This includes a detailed description of the quality control of educts, intermediates and final product . The active ingredient L-tryptophan is subsequently used by AMINO's subsidiary company esparma GmbH to produce and distribute the pharmaceutical Lyphan . The quality management system and the production procedure for Lyphan are described and discussed. Eur Surg Res, 2000, 32(1), 18 - 22 In situ perfusion of the liver enhances the efficiency of retrovirus-mediated gene transfer to hepatocytes; Tomori H et al.; To increase the efficiency of retrovirus-mediated gene transfer targeting an individual's liver in vivo, the liver was perfused in situ with the retrovirus vector during hepatic cold ischemia . Four weeks prior to gene transfer, the spleen was transpositioned to the left subcutaneous position to develop a portosplenic shunt, which was performed in order to prevent intestinal congestion during hepatic ischemia . Traditional retrovirus vectors (1 x 10(5) CFU/ml) which encode genes for the Escherichia coli beta-galactosidase (LacZ) were used in this study . Twenty-four hours after partial hepatectomy (70%), the remnant liver was surgically isolated, perfused with 1 ml of vector solution through the portal vein, and kept in contact with the vector for 30 min under cold ischemia (group 1) . Hepatic ischemia could thus be performed without any intestinal congestion, due to the preestablished portosystemic shunt . In group 2, the liver was perfused with 1 ml of vector solution through the portal vein without in situ perfusion of the liver . Animals were sacrificed 1, 3, 7 and 28 days after gene transfer . In X-gal staining, the transferred LacZ was detected positive in 10-15% of the hepatocytes only in group 1, 3 days after gene transfer . Graft histology and a liver function test showed no difference between both groups 24 h after gene transfer . In conclusion, in situ perfusion of the liver greatly enhanced the efficacy of retrovirus-mediated gene transfer, targeting an individual's liver in vivo . J Infect Dis, 2000 Mar, 181(3), 1129 - 32 Safety and immunogenicity of intranasally administered inactivated trivalent virosome-formulated influenza vaccine containing Escherichia coli heat-labile toxin as a mucosal adjuvant; Gluck R et al.; A trivalent influenza virosome vaccine containing hemagglutinin and Escherichia coli heat-labile toxin (HLT) was administered intranasally to young adults and elderly subjects . Symptoms that followed immunization were mild and transient . A significant increase in serum hemagglutination inhibition (HI) antibody was noted for the 3 vaccine strains . There was no significant difference in postimmunization geometric mean titers or seroconversion rates between age groups . The percentage of subjects attaining protective HI titers (>/=40%) was comparable in both groups for the A/Bayern (P=.5) and B/Beijing (P=.3) strains but was higher among young adults (92.2%) versus elderly subjects (76.5%; P=.057) for the A/Wuhan strain . The proportion of subjects with nonprotective baseline titers who attained protective levels after immunization was similar in both age groups for the A/Bayern and B/Beijing components . For the A/Wuhan component, significantly (P=.017) more young adults achieved protective titers versus elderly subjects (85 . 7% and 53.8%, respectively) . Vaccination evoked a significant (P< . 005) increase in anti-HLT antibody titers. Biochem Biophys Res Commun, 2000 Mar 24, 269(3), 737 - 42 Substrate binding to 15beta-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration; Simgen B et al.; CYP106A2 has been expressed in E . coli with a high yield of up to 130 mg per litre of culture, purified to electrophoretic homogenity and found to be active in 15beta-hydroxylation of deoxycorticosterone using the adrenal redox proteins adrenodoxin and adrenodoxin reductase . Inspite of catalytic activity no substrate binding was detectable by UV-Vis spectroscopy . In contrast, an effect of substrate binding has been detected using the CO stretch mode infrared spectrum indicating that deoxycorticosterone binds in the heme pocket near the iron ligand . Biochem Biophys Res Commun, 2000 Mar 24, 269(3), 732 - 6 Equistatin, a protease inhibitor from the sea anemone actinia equina, is composed of three structural and functional domains; Strukelj B et al.; A cDNA encoding a precursor of equistatin, a potent cysteine and aspartic proteinase inhibitor, was isolated from the sea anemone Actinia equina . The deduced amino acid sequence of a 199-amino-acid residue mature protein with 20 cysteine residues, forming three structurally similar thyroglobulin type-1 domains, is preceded by a typical eukaryotic signal peptide . The mature protein region and those coding for each of the domains were expressed in the periplasmic space of Escherichia coli, isolated, and characterized . The whole recombinant equistatin and its first domain, but not the second and third domains, inhibited the cysteine proteinase papain (K(i) 0.60 nM) comparably to natural equistatin . Preliminary results on inhibition of cathepsin D, supported by structural comparison, show that the second domain is likely to be involved in activity against aspartic proteinases . J Surg Res, 2000 Mar, 89(1), 60 - 5 A recombinant rat regenerating protein is mitogenic to pancreatic derived cells; Levine JL et al.; Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to beta- and ductal cells . Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult . The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells . cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR . The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter . After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-beta-d-thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate . This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively . This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis . The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and beta-cell lines, respectively . Antibodies raised to the protein reacted against rat reg I in pancreas . The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells . It is more potent than reg I isolated from pancreatic tissue . This protein can be isolated rapidly, easily, and with a high amount of purity . J Surg Res, 2000 Mar, 89(1), 26 - 30 Effect of endotoxemia on hepatic portal and sinusoidal blood flow in rats; Secchi A et al.; A decrease in liver blood flow leads to dysfunction of hepatocytes and Kupffer cells, with subsequent local and systemic liberation of proinflammatory mediators that may maintain systemic inflammatory response syndrome (SIRS) and may lead to multiple organ dysfunction syndrome (MODS) . There is only limited knowledge on the hepatic micro- and macrocirculation during sepsis or endotoxemia . Therefore, the aim of our study was to investigate alterations in hepatic portal blood flow (PBF) and sinusoidal blood flow (SBF) during endotoxemia . In male Wistar rats endotoxemia was induced by continuous infusion of 2 mg/kg/h lipopolysaccharides from Escherichia coli 026:B6 immediately after baseline measurements (n = 8) . The control group (n = 8) received an equivalent volume of Ringer's solution . Mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), PBF, and SBF were measured at baseline and 60 and 120 min after induction of endotoxemia . PBF was measured using an ultrasonic flow probe that was positioned around the portal vein . SBF was detected by in vivo videomicroscopy of the left liver lobe . In the LPS group MAP decreased, but CO remained at baseline values . During endotoxemia PBF decreased significantly from 23 +/- 3 to 15 +/- 4 mL/min (60 min) and 16 +/- 3 mL/min (120 min) . SBF also significantly decreased to 68.5% (60 min) and 57.1% (120 min) of baseline value . Our results demonstrate that during early endotoxemia hepatic macro- and microcirculatory perfusion is significantly decreased despite unchanged CO . This early reduction of hepatic perfusion might be caused by an increased hepatic vessel resistance as a consequence of liberation of vasoconstrictive mediators or/and by a decrease in intestinal perfusion . Mol Genet Metab, 2000 Feb, 69(2), 101 - 10 Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype; Waters PJ et al.; Missense mutations account for 48% of all reported human disease-causing alleles . Since few are predicted to ablate directly an enzyme's catalytic site or other functionally important amino acid residues, how do most missense mutations cause loss of function and lead to disease? The classic monogenic phenotype hyperphenylalaninemia (HPA), manifesting notably as phenylketonuria (PKU), where missense mutations in the PAH gene compose 60% of the alleles impairing phenylalanine hydroxylase (PAH) function, allows us to examine this question . Here we characterize four PKU-associated PAH mutations (F39L, K42I, L48S, I65T), each changing an amino acid distant from the enzyme active site . Using three complementary in vitro protein expression systems, and 3D-structural localization, we demonstrate a common mechanism . PAH protein folding is affected, causing altered oligomerization and accelerated proteolytic degradation, leading to reduced cellular levels of this cytosolic protein . Enzyme specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein . The F39L, L48S, and I65T PAH mutations were selected because each is associated with a spectrum of in vivo HPA among patients . Our in vitro data suggest that interindividual differences in cellular handling of the mutant, but active, PAH proteins will contribute to the observed variability of phenotypic severity . PKU thus supports a newly emerging paradigm both for mechanism whereby missense mutations cause genetic disease and for potential modulation of a disease phenotype . J Biol Chem, 2000 Mar 24, 275(12), 8794 - 805 Substrate specificity and inhibition studies of human serotonin N-acetyltransferase; Ferry G et al.; Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates . In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized . The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated . Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors . We report the findings of several peptides with low micromolar inhibitory potency . For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed . The assay was based on the separation and detection of N-{(3)H}acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection . The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study . We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate . The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives . An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal. Biol Chem, 2000 Jan, 381(1), 85 - 8 Cloning and expression of functional equistatin; Galesa K et al.; Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains . It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D . In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened . A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence . The mature protein region was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli . Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column . The protein displays physical and inhibitory properties closely similar to the native inhibitor. Biol Chem, 2000 Jan, 381(1), 39 - 47 Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1; Flicker S et al.; Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens . We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli . The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end . The modified Bip1 heavy chain cDNA was co-expressed in E . coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H . His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1 . rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut) . We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution . Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy. Biol Chem, 2000 Jan, 381(1), 1 - 5 Enzymatic halogenation catalyzed via a catalytic triad and by oxidoreductases; van Pee KH et al.; During the search for haloperoxidases in bacteria we detected a type of enzymes that catalyzed the peroxide-dependent halogenation of organic substrates . However, in contrast to already known haloperoxidases, these enzymes do not contain a prosthetic group or metal ions nor any other cofactor . Biochemical and molecular genetic studies revealed that they contain a catalytic triad consisting of a serine, a histidine, and an aspartate . The reaction they catalyze is actually the perhydrolysis of an acetic acid serine ester leading to the formation of peracetic acid . As a strong oxidizing agent the enzymatically formed peracetic acid can oxidize halide ions, resulting in the formation of hypohalous acid which then acts as the actual halogenating agent . Since hypohalous acid is also formed by the heme- and vanadium-containing haloperoxidases, enzymatic halogenation catalyzed by haloperoxidases and perhydrolases in general lacks substrate specificity and regioselectivity . However, detailed studies on the biosynthesis of several halometabolites led to the detection of a novel type of halogenases . These enzymes consist of a two-component system and require NADH and FAD for activity . Whereas the gene for one of the components is part of the biosynthetic cluster of the halometabolite, the second component is an enzyme which is also present in bacteria from which no halometabolites have ever been isolated, like Escherichia coli . In contrast to haloperoxidases and perhydrolases the newly detected NADH/FAD-dependent halogenases are substrate-specific and regioselective and might provide ideal tools for specific halogenation reactions. Cell, 2000 Mar 3, 100(5), 561 - 73 Multivalent binding of nonnative substrate proteins by the chaperonin GroEL; Farr GW et al.; The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES . Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown . We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides . A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains . Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement . As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment. Gene, 2000 Jan 25, 242(1-2), 437 - 48 Sequence, expression and functional analysis of the Coccidioides immitis ODC (ornithine decarboxylase) gene; Guevara-Olvera L et al.; The ornithine decarboxylase (ODC) gene of the human respiratory fungal pathogen, Coccidioides immitis (Ci) was cloned, sequenced, chromosome-mapped, and expressed in Escherichia coli (Ec) . The genomic, cDNA and translated sequences are presented . Transformation of an ODC null mutant strain of Ec (EWH 319) with the Ci ODC gene was conducted to confirm function of the protein encoded by the fungal gene . Activity of the enzyme by the bacterial transformant was inhibited by 1, 4-diamino-2-butanone (DAB), a known inhibitor of eukaryotic ODC . Temporal expression of the Ci ODC gene during the parasitic cell cycle is constitutive, based on results of RT PCR . However, results of enzyme activity assays of cell homogenates obtained at different stages of parasitic cell development in vitro showed that the functional protein is present only during periods of isotropic growth and segmentation, and these morphogenetic events can be arrested by the addition of DAB . The observed absence of a difference in steady-state mRNA transcript amounts, and the developmentally correlated variation in levels of enzyme activity, suggest a translational or post-translational mechanism of ODC regulation . Since no PEST sequence was detected in the Ci ODC, enzyme regulation by programmed protein degradation as reported for many other eukaryotic ODCs may not occur in this case . ODC activity appears to play a key role in the morphogenesis of Ci, and the enzyme could be a rational target for therapy of disseminated coccidioidomycosis. Ann Clin Lab Sci, 2000 Jan, 30(1), 92 - 8 Expression of stress proteins HSP 72 & HSP 32 in response to endotoxemia; Ofenstein JP et al.; Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP) . However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70 . We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90 . Fifteen rats received intravenously either 3 mg/kg of endotoxin (E . coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6) . Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot . In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C . In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C . In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C . HSP 90 & HSP 70c (constitutive) were present in all groups . Consistent with observations by others, HSP 72 was not induced in Group 1 . HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2) . However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3) . Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells . In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated. Circ Res, 2000 Mar 17, 86(5), 514 - 9 NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells; Lynn S et al.; Arsenic is atherogenic, carcinogenic, and genotoxic . Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells . By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 micromol/L . DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol . Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production . The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1, 2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase . Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase . Treatment with arsenite also increased the mRNA level of p22phox . These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage . The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis. Alcohol, 2000 Feb, 20(2), 193 - 203 Acute alcohol intoxication and gadolinium chloride attenuate endotoxin-induced release of CC chemokines in the rat; Bukara M et al.; This work tests the hypotheses that Kupffer cells are a major source of CC-chemokines (MIP-1alpha, MCP-1, RANTES) during acute endotoxemia and that acute ethanol intoxication modulates Escherichia coli lipopolysaccharide (LPS, 1 mg/Kg, i.v.)-induced chemokine release in the rat . LPS stimulated the release of CC-chemokines into the circulation, hepatic sequestration of leukocytes and liver injury . LPS-induced serum chemokines peaked at 1-3 h and could not be detected at 24-h posttreatment . Splenectomy significantly suppressed LPS-induced RANTES release, but not MIP-1alpha and MCP-1 . Kupffer cell depletion by gadolinium chloride or acute ethanol intoxication significantly attenuated LPS-induced CC-chemokine release and hepatic injury . Hepatic sequestration of leukocytes during endotoxemia was also suppressed by acute ethanol . LPS downregulated the expression of MIP-1alpha and MCP-1 mRNAs and upregulated RANTES mRNA in Kupffer cells at 3-h post endotoxin . The expression of mRNAs was further suppressed in ethanol plus the LPS-treated group . Ethanol also suppressed the LPS-mediated priming of Kupffer cells for enhanced CC-chemokine release in vitro . Ethanol alone significantly upregulated the expression of CC-chemokine mRNA, and primed the Kupffer cells for enhanced RANTES release . CC-chemokine release and mRNA expression in hepatic sinusoidal endothelial cells were not significantly altered by ethanol, except for MCP-1 release . These data show that acute ethanol may be beneficial in tissue injury during acute endotoxemia. Chin J Biotechnol, 1999, 15(2), 91 - 7 Cloning and expression of truncated PTHrP receptor in Escherichia coli; Dou L et al.; To evaluate the effects of recombinant soluble parathyroid hormone related protein receptor(sPTHrPR), the RNA prepared from renal cell carcinoma was reverse-transcribed and amplified by a polymerase chain reaction(PCR) . A 543 base pair fragment corresponding to extracellular domain of PTHrPR was obtained and cloned . Sequencing was performed by the dideoxy chain-termination method . Comparison of the nucleic acid sequence with the published data revealed that only one nucleotide had changed . A high level expression vector referred to as pET-3a/sPTHrPR was constructed . The binding specificity of the expression products was proved by indirect ELISA . Our preliminary experiment also showed that the expression products can block the biological activity of PTHrP. Arzneimittelforschung, 2000 Feb, 50(2), 140 - 7 Synthesis and pharmacological screening of novel non-acidic gastroprotective antipyretic anti-inflammatory agents with anti-platelet properties . 5-Alkyl/cycloalkylamino substituted 2-amino-5H-{1}benzopyrano{4,3-d} pyrimidines; Bruno O et al.; The preparation and screening of antipyretic, anti-inflammatory, analgesic, gastroprotective and antiplatelet activities of original non-acidic aminobenzo-pyranopyrimidine derivatives are described . Major and dose-dependent analgesic and antipyretic properties were detected in all the compounds after oral administration (6.25-100 mg kg-1) in the mouse writhing test and in the E . coli lipopolysaccharide-induced fever in the rat, respectively . Unlike the reference drug indometacin (ED50 ulcer = 9.1 mg kg-1), no gastrolesivity was displayed by all the new compounds up to 150 mg kg-1 so that therapeutical indices equal or higher than that of indometacin were calculated . Furthermore, at 100 mg kg-1 they were able to prevent ethanol-induced gastric mucosal injury in rats . At a 1 mmol/l concentration the compounds under study were as effective as acetylsalicylic acid in inhibiting in vitro platelet aggregation induced by adenosine diphosphate. J Allergy Clin Immunol, 2000 Mar, 105(3), 475 - 81 Allergen provocation augments endotoxin-induced nasal inflammation in subjects with atopic asthma; Eldridge MW et al.; BACKGROUND: Recent epidemiologic and in vivo studies have suggested that inhaled endotoxin plays an important role in asthma pathogenesis . OBJECTIVE: The present study examines the effect of nasal allergen provocation on subsequent endotoxin challenge in subjects with atopic asthma . METHODS: By using a split-nose randomized crossover design, individual nares of 12 asthmatic subjects underwent challenge and lavage as follows . Immediately after a baseline nasal lavage, one nares received normal saline, and the other received dust mite antigen . Four hours later, both nares were exposed to either saline or endotoxin . Dust mite antigen (Dermatophagoides farinae) and endotoxin (Escherichia coli 026:B6) doses were 100 AU and 1000 ng, respectively . Postchallenge lavages were done at 8 and 24 hours after the initial challenge . The subjects then returned a minimum of 3 weeks later for crossover to the study arm . Nasal lavage fluid was analyzed for total and differential cell counts, IL-8, IL-6, intercellular adhesion molecule 1, GM-CSF, eosinophil cationic protein, myeloperoxidase, and soluble CD14 . RESULTS: A significant increase in the total inflammatory cell count was seen at 8 hours for the dust mite/endotoxin exposure compared with the saline/saline and saline/endotoxin exposures . Differential cell counts revealed a similar neutrophilic and eosinophilic inflammation for the dust mite/endotoxin exposure at 8 hours . CONCLUSIONS: These data demonstrate an interaction between allergen and endotoxin exposure in asthmatic subjects, suggesting that a prior allergen challenge significantly augments the endotoxin-induced inflammation . Moreover, these data provide further evidence that concomitant exposure to allergen and endotoxin may be an important factor in asthma pathogenesis. Biochim Biophys Acta, 2000 Mar 16, 1478(1), 69 - 77 Destabilase from the medicinal leech is a representative of a novel family of lysozymes; Zavalova LL et al.; Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase . Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity . The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear . Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes . New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates . It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein. Biochim Biophys Acta, 2000 Mar 16, 1478(1), 9 - 18 A new cytolysin from the sea anemone, Heteractis magnifica: isolation, cDNA cloning and functional expression; Wang Y et al.; We purified a new cytolysin (HMgIII) from the sea anemone, Heteractis magnifica . HMgIII, which has a molecular mass of approximately 19 kDa, functions as both a cytolysin and a hemolysin . The full-length HMg III cDNA was obtained by reverse transcriptase-polymerase chain reaction, using primers designed from its N-terminal amino acid sequence and an internal conserved region of two other sea anemone cytolysins: equinatoxin II (EqT II) and cytolysin III . The cDNA contained an open reading frame of 633 bp, which encodes a protein of 211 amino acids . The nascent HMg III protein contained a prepropeptide of 34 amino acids, which includes a signal peptide of 19 amino acids . The mature HMg III has a predicted molecular mass of 19 kDa and a pI of 9.1, and shares 91%, 89%, 65% and 63% amino acid sequence similarity with cytolysin III, cytolysin ST I, tenebrosin-C and equinatoxin (EqT II), respectively . The predicted secondary structure of the mature HMg III comprises 16% alpha-helix, 23% extended strand and 60% random coils . The characteristic amphiphilic alpha-helix of cytolysins is located at the N-terminus of the processed HMg III . Recombinant HMg III (rHMg III) was expressed in Escherichia coli as a fusion protein containing a 6xHisTag at the N-terminus . The hemolytic and cytotoxic activities of the purified rHMg III were comparable to those of the native HMg III . The hemolytic activities of both proteins were similarly potentiated with 8-anilino-1-naphthalenesulfonate (ANS) . Increasing the length of the peptide tag on the N-terminal of rHMg III correlated with decreasing hemolytic activity, thus confirming the importance of the N-terminal amphiphilic alpha-helix for its cytolytic activity. Biochim Biophys Acta, 2000 Mar 16, 1478(1), 1 - 8 Cloning of the bovine leukemia virus proteinase in Escherichia coli and comparison of its specificity to that of human T-cell leukemia virus proteinase; Zahuczky G et al.; The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein . The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies . The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration . Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1 . Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced. Injury, 2000 May, 31(4), 253 - 6 Double traumatic abdominal wall hernia and colon laceration due to a pelvic fracture; Walcher F et al.; A traumatic abdominal wall hernia was observed in association with a colonic laceration due to a pelvic fracture . The presence of this specific combination of injuries is a rare clinical entity . CT evaluation with intravenous and bowel contrast media identified the traumatic abdominal wall hernia and the bowel entrapment. Mutagenesis, 2000 Mar, 15(2), 121 - 5 Spontaneous and osmium tetroxide-induced mutagenesis in an Escherichia coli strain deficient in both endonuclease III and endonuclease VIII; Najrana T et al.; Thymine glycol, uracil glycol, 5-hydroxycytosine and 5-hydroxyuracil are common base lesions produced by cellular metabolism as well as ionizing radiation and environmental carcinogens . Escherichia coli DNA glycosylase, endonuclease III and endonuclease VIII recognize and remove these lesions from DNA . In this study, we assessed the mutagenic potential of these lesions in the supF gene as a forward mutation target in double-stranded plasmid DNA using an E.coli strain deficient in both endonuclease III and endonuclease VIII . These lesions were introduced into pTN89 DNA by the chemical oxidant osmium tetroxide . Spontaneous supF mutations occurred at a frequency of 3.03x10(-7) and osmium tetroxide-induced at a frequency of 8 . 25x10(-7) . Sequence analysis of supF mutants revealed that mutations occurred at cytosine sites rather than thymine sites, suggesting that thymine glycol is not the principal premutagenic lesion . In contrast, G:C-->A:T transitions were dominantly detected in the spontaneous and osmium tetroxide-induced mutations in the endonuclease III and endonuclease VIII double defective host . In this case, products of cytosine oxidation such as 5-hydroxycytosine, which are the substrate for endonuclease III and endonuclease VIII, were the principal mutagenic lesions. Shock, 2000 Mar, 13(3), 244 - 50 CXC chemokine suppression of polymorphonuclear leukocytes apoptosis and preservation of function is oxidative stress independent; Dunican AL et al.; Interleukin 8 (IL-8) and growth-related oncogene alpha (Gro-alpha) delay neutrophil apoptosis, which is thought to be important for the resolution of inflammation . We hypothesized that (IL-8) and Gro-alpha interfere with extracellular death receptor signaling or intracellular caspase activation to suppress neutrophil apoptosis . In addition, we sought to determine if prolonged neutrophil half-life was associated with preservation of function . Polymorphonuclear leukocytes (PMN) were cultured with IL-8 or Gro-alpha (0-100 ng/mL) in normoxia or hypoxia, and the extent of apoptosis was assessed by histology and TdT-mediated dUTP nick end labeling (TUNEL) . Subsequently, to determine the role of apoptotic-associated receptors, PMN were cultured with IL-8 and neutralizing monoclonal antibody to Fas (CD95), TNFR55, and TNFR75 . To establish the effect of IL-8 or Gro-alpha on pro-apoptotic caspase activity, the cleavage of specific colorimetric substrates was assessed . Functional changes in PMN included the capacity to produce superoxide anion and phagocytosis of Escherichia coli . At the 100 ng/mL dose, the addition of IL-8 and Gro-alpha maximally suppressed PMN apoptosis from 54% (untreated) to 5% and 6%, respectively . The addition of neutralizing antibodies to Fas, TNFR55, or R75 caused no change in IL-8 suppression of apoptosis . Caspase 3 activity was markedly suppressed at 24 h by the inclusion of either IL-8 and Gro-alpha . IL-8 and Gro-alpha-stimulated PMN released more superoxide anion and had an increased phagocytic index vs . control PMN . IL-8 and Gro-alpha suppress neutrophil apoptosis to a similar level that is not influenced by oxygen tension at high doses . The effect of IL-8 and Gro-alpha does not depend on activation of the Fas, TNFR55, or R75 receptor pathways but involves suppression of caspase 3 activity . IL-8 or Gro-alpha extends the functional half-life of neutrophils and may explain their role in disease states such as acute respiratory distress syndrome. Shock, 2000 Mar, 13(3), 224 - 9 Cardiopulmonary and splanchnic blood flow during 48 hours of a continuous infusion of endotoxin in conscious pigs: a model of hyperdynamic shock; Murphey ED et al.; Hyperdynamic shock can be modeled in pigs by chronic administration of a continuous, low-dose infusion of endotoxin . Lipopolysaccharide (LPS, E . coli 0111:B4, 80 ng/kg/min i.v.) initially resulted in a hypodynamic shock state with significant decreases in mean arterial pressure (112+/-3 mmHg at baseline to 94+/-4 mmHg at 8 h) and cardiac index (5.35+/-0.32 L/min/m2 at baseline to 4.07+/-0.32 L/min/m2 at 4 h) . Eight hours after the initiation of the LPS infusion, cardiac index rose to above baseline values (5.82+/-0.4 L/min/m2 at 8 h) and remained elevated for the duration of the 48-h study (6.54+/-0.39 L/min/m2 at 48 h) . Similarly, systemic vascular resistance fell significantly by 8 h (1640+/-100 dyne sec cm(-5) at baseline to 1239+/-80 dyne sec cm(-5) at 8 h) and remained decreased for the duration of the study . Blood flow in major abdominal vessels, including the left renal artery, the cranial mesenteric artery, and the portal vein, paralleled changes in the cardiac index . Serum concentrations of tumor necrosis factor were increased after 2 h of LPS infusion, but did not remain elevated above baseline concentrations for more than about 4 h despite continuous LPS challenge . Concentrations of tumor necrosis factor did not differ between arterial and portal venous samples . This model of hyperdynamic shock should be useful to assess potential therapies for septic shock. Shock, 2000 Mar, 13(3), 209 - 16 Phagocytic and intestinal endothelial and epithelial barrier function during the early stage of small intestinal ischemia and reperfusion injury; Sun Z et al.; The effects of intestinal ischemia and reperfusion (I/R) on small intestinal mucosal endothelial and epithelial barrier integrity and phagocytic function were assessed in rats subjected to 20- or 40-min mesenteric ischemia and a 3-h reperfusion . The results showed that human serum albumin (125I-HSA) flux through the endothelial layer to the interstitial space increased as did 125I-HSA clearance from blood to the gut lumen and 131I-HSA flux from the gut lumen to the interstitial space in rats with I/R . E . coli adhering to microvilli, invading and passing into the microvessels, were noted on the small intestinal mucosa in animals subjected to 40-min ischemia and a 3-h reperfusion . Phagocytic function increased, especially in the small intestinal wall, lungs, liver, and spleen in the groups with I/R, correlating with the length of ischemia . The results imply that both endothelial and epithelial barrier integrity is impaired in the early phase after I/R and that the epithelial barrier more effectively restricts macromolecular leakage compared with the endothelial barrier . I/R impairs the intestinal barrier not only by causing tissue hypoxia but also by activating the phagocytic system and aggravating barrier damage, which finally may result in bacterial translocation and remote organ dysfunction. DNA Res, 2000 Feb 28, 7(1), 19 - 26 Random PCR-based genome sequencing: a non-divide-and-conquer strategy; Nishigaki K et al.; We propose a genome sequencing strategy, which is neither divide-and-conquer (clone by clone) nor the shotgun approach . Random PCR-based and PCR relay sequencing constitute the basis of this novel strategy . Most of the genome is sequenced by the former process that requires only a set of non-specific primers and a template DNA . Random PCR-based sequencing reduces redundancy in sequencing by exploiting known sequence information . The number of primers required for random PCR was significantly diminished by using a combination of primers . The former process can be partially replaced by the shotgun method, if necessary . The gap-filling process can be effectively performed by way of PCR relay . The feasibility of this strategy was demonstrated using the Escherichia coli genome . This strategy enhances the global effort towards genome sequencing by being available through the Internet and by allowing the use of preexisting sequence data. Microbes Infect, 2000 Jan, 2(1), 45 - 53 Potential effect of cattle diets on the transmission of pathogenic Escherichia coli to humans; Russell JB et al.; Grain feeding seems to promote the growth and acid resistance of Escherichia coli in fattening beef cattle, and acid-resistant E . coli are more likely to survive the human gastric stomach . When cattle were fed hay for only five days, the number and acid resistance of E . coli decreased dramatically. Microbes Infect, 2000 Jan, 2(1), 5 - 16 In vivo interactions of rabbit enteropathogenic Escherichia coli O103 with its host: an electron microscopic and histopathologic study; Heczko U et al.; A family of human and animal pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC), trigger formation of 'attaching and effacing' lesions on cultured and intestinal epithelial surfaces . However, our understanding of these events in vivo is incomplete . To further study these interactions in a natural infection model, weaned rabbits were infected with rabbit enteropathogenic E . coli O103 (REPEC O103), followed clinically, and infected tissues were evaluated by electron and light microscopy . Of the 36 rabbits challenged, morbidity and mortality were 65 and 23%, respectively . Twenty-four hours after infection, expression of fimbriae-like organelles was observed on the bacterial surface . Microvilli of ileal Peyer's patches (PP) became disorganized, and intestinal mucus secretion increased which coincided with intraluminal binding of the pathogen in the proximal colon . Forty-eight hours after infection, there was conspicuous lack of fimbriae-like organelle expression, while bacterial adherence preferentially occurred at the domed villi of PP . Seventy-two hours after infection, broad morphological heterogeneity was noted within pedestals beneath attached bacteria, including extended pseudopods . We conclude that REPEC O103 express surface organelles during initial exposure to the host, that the initial target sites of adherence are the domed villi of ileal PP, and that increased mucus secretion occurs during REPEC O103 infection . As well, extended pseudopod formation was demonstrated in vivo. Biochimie, 2000 Jan, 82(1), 19 - 24 Repair and replication of oxidized DNA bases using modified oligodeoxyribonucleotides; Gasparutto D et al.; Modified oligodeoxyribonucleotides (ODNs) are powerful tools to assess the biological significance of oxidized lesions to DNA . For this purpose, we developed original synthetical pathways for the site-specific insertion of several oxidized bases into DNA fragments . Thus, the chemical solid-phase synthesis of ODNs using original strategies of protection and mild conditions of deprotection, as well as a specific post-oxidation approach of an unique nucleoside residue within the sequence have been applied . These two approaches of preparation allowed us to have access to a set of modified ODNs that contain a single modified nucleoside, i.e., N-(2-deoxy-beta-D-erythro-pentofuranosyl)formylamine (dF), 5-hydroxy-2'-deoxycytidine (5-OHdCyd), thymidine glycol (dTg), 5,6-dihydrothymidine (DHdThd), 2,2-diamino-4-{(2-deoxy-beta-D-erythro-pentofuranosyl)-amino}-5(2H)- oxazolone (dZ), N-(2-deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (dY), 5',8-cyclo-2'-deoxyguanosine (cyclodGuo) and 5',8-cyclo-2'-deoxyadenosine (cyclodAdo) . The substrates were used to investigate recognition and removal of the lesions by bacterial DNA N-glycosylases, including endonuclease III (endo III) and Fapy glycosylase (Fpg) . In addition, the DNA polymerase-mediated nucleotide incorporation opposite the damage was determined using modified ODNs as templates. Biochimie, 2000 Jan, 82(1), 5 - 17 Mechanisms and consequences of replication fork arrest; Hyrien O; Chromosome replication is not a uniform and continuous process . Replication forks can be slowed down or arrested by DNA secondary structures, specific protein-DNA complexes, specific DNA-RNA hybrids, or interactions between the replication and transcription machineries . Replication arrest has important implications for the topology of replication intermediates and can trigger homologous and illegitimate recombination . Thus, replication arrest may be a key factor in genome instability . Several examples of these phenomena are reviewed here. Vaccine, 2000 Apr 28, 18(21), 2257 - 65 Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages; Huang JA et al.; cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn(2+) . The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide libraries . Polyclonal antibodies were then used to pan the SGRP libraries for antibody-binding phages . Analysis of the phage isolates revealed that the majority (86%) of them only had a single insert . However, phages displaying composite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isolated . Inserts containing A or C region peptide were found to contain extra sequences from the C region, while the B region epitope was linear and had additional sequence from either upstream or downstream . In addition a dominant and possibly non-neutralising VP7 epitope was identified around amino acids 263-270 . One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maintain its antigenicity . The results in this study may have significant implication for recreation of conformational epitopes and vaccine development. Mol Biochem Parasitol, 2000 Mar 15, 107(1), 45 - 55 Cyclin H activation and drug susceptibility of the Pfmrk cyclin dependent protein kinase from Plasmodium falciparum; Waters NC et al.; The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs) . Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully . To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity . Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity . Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro . Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity . This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs . CDKs are attractive drug targets due to their role in cellular proliferation . Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target . Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds . A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk. Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2785 - 90 A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography; Gambhir SS et al.; We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy . We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene . The HSV1-sr39tk mutant was identified from a library of site-directed mutants . Accumulation (net uptake) of the radioactively labeled substrates {8-(3)H}penciclovir ({8-(3)H}PCV), and 8-{(18)F}fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of approximately 2.0 when compared with C6 cells expressing wild-type HSV1-tk . The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors . The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression in vivo. Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2538 - 43 Extension of transverse relaxation-optimized spectroscopy techniques to allosteric proteins: CO- and paramagnetic fluoromet-hemoglobin {beta (15N-valine)}; Nocek JM et al.; We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism . Each beta-chain of the hemoglobin (Hb) tetramer has 17 valine residues . We have (15)N-labeled the beta-chain Val residues and detected 16 of the 17 (1)H-(15)N correlation peaks for beta-chain Val of the R state CO-Hb structure by using the TROSY technique . Sequence-specific assignments are suggested, based mainly on analysis of the (1)H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form . When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional {(1)H,(1)H}-NOESY-TROSY experiment (NOESY, NOE spectroscopy) . We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample . This change induces substantial increments in the (1)H and (15)N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments . These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb . More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone (15)N/(13)C resonances upon nonselective labeling. Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2474 - 9 Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit; Chabes A et al.; Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides . Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)) . The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors . The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical . Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding . Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex . This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient . In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits . The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity . This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy . Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit. Exp Neurol, 2000 Mar, 162(1), 189 - 93 Mammalian-cell-produced neurturin (NTN) is more potent than purified Escherichia coli-produced NTN; Hoane MR et al.; Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor . Both factors promote the survival of dopaminergic (DA) neurons . We investigated the biological activity of mammalian-cell-produced NTN versus purified Escherichia coli-produced NTN . Baby hamster kidney cells were engineered to stably secrete mature human NTN . Mammalian-cell-derived NTN enhanced the activity of embryonic DA neurons in vitro, with greater potency (maximum effect achieved in the picogram range) than purified E . coli-produced NTN . Cell-based delivery of NTN (less than 10 ng/day) was also shown to be biologically active in vivo . These results suggest that mammalian-cell-derived NTN, synthesized de novo and delivered in small quantities to the parenchyma at the target site, may be as active as much larger quantities of purified, E . coli-produced NTN, delivered by other means . Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3136 - 41 An approach to gene-specific transcription inhibition using oligonucleotides complementary to the template strand of the open complex; Milne L et al.; The single-stranded region of DNA within the open complex of transcriptionally active genes provides a unique target for the design of gene-specific transcription inhibitors . Using the Escherichia coli lac UV5 and trp EDCBA promoters as in vitro models of open complex formation, we have identified the sites inside these transcription bubbles that are accessible for hybridization by short, nuclease-resistant, non-extendable oligoribonucleotides (ORNs) . Binding of ORNs inside the open complex was determined by linking the chemical nuclease bis(1,10-phenanthroline) cuprous chelate {(OP)(2)Cu(+)} to the ORN and demonstrating template-specific DNA scission . In addition, these experiments were supported by in vitro transcription inhibition . We find that the most effective inhibitors are 5 nt long and have sequences that are complementary to the DNA template strand in the region near the transcription start site . The ORNs bind to the DNA template strand, forming an antiparallel heteroduplex inside the open complex . In this system, RNA polymerase is essential not only to melt the duplex DNA but also to facilitate hybridization of the incoming ORN . This paradigm for gene-specific inactivation relies on the base complementarity of the ORN and the catalytic activity and sequence specificity of RNA polymerase for the site- and sequence-specific recognition and inhibition of transcriptionally active DNA. Int J Biochem Cell Biol, 2000 Mar, 32(3), 327 - 37 Dihydrofolate influences the activity of Escherichia coli dihydrofolate reductase synthesised de novo; Mouat MF; The folding of proteins into their native structures is known to depend on molecular chaperones . However, other ligands or cofactors which still require characterisation are also likely to influence protein folding . The intention of this study was to reveal how the folding of an enzyme, Escherichia coli dihydrofolate reductase, was affected by a substrate ligand, i.e . dihydrofolate . The enzyme was synthesised by coupled transcription/translation in a bacterial cell-free system . Correct folding of the protein into its native structure was measured by its enzymatic activity . Synthesis of dihydrofolate reductase was found to be inhibited, at the level of translation, by dihydrofolate . The syntheses of other proteins were also inhibited by this compound and the reasons for this inhibition could not be determined . Most notably, the specific activity of the dihydrofolate reductase formed in the presence of the substrate dihydrofolate was increased and this effect was specific for dihydrofolate reductase since it was not observed with other proteins synthesised in the same system . The increase in dihydrofolate reductase specific activity could not be attributed to mere thermal stabilisation of the fully folded enzyme by dihydrofolate . The effects of dihydrofolate on dihydrofolate reductase synthesis and activity were similar to those of the molecular chaperone DnaJ which is known to promote the folding of newly synthesised proteins . It is suggested that dihydrofolate may interact with the newly synthesised dihydrofolate reductase polypeptide chain and promote its productive folding. Int J Biochem Cell Biol, 2000 Mar, 32(3), 289 - 301 Serine hydroxymethyltransferase and threonine aldolase: are they identical? Ogawa H, Gomi T, Fujioka M. Serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme, catalyses the interconversion of serine and glycine, both of which are major sources of one-carbon units necessary for the synthesis of purine, thymidylate, methionine, and so on . Threonine aldolase catalyzes the pyridoxal phosphate-dependent, reversible reaction between threonine and acetaldehyde plus glycine . No extensive studies have been carried out on threonine aldolase in animal tissues, and it has long been believed that serine hydroxymethyltransferase and threonine aldolase are the same, i.e . one entity . This is based on the finding that rabbit liver serine hydroxymethyltransferase possesses some threonine aldolase activity . Recently, however, many kinds of threonine aldolase and corresponding genes were isolated from micro-organisms, and these enzymes were shown to be distinct from serine hydroxymethyltransferase . The experiments with isolated hepatocytes and cell-free extracts from various animals revealed that threonine is degraded mainly through the pathway initiated by threonine 3-dehydrogenase, and there is little or no contribution by threonine aldolase . Thus, although serine hydroxymethyltransferase from some mammalian livers exhibits a low threonine aldolase activity, the two enzymes are distinct from each other and mammals lack the "genuine" threonine aldolase. Nature, 2000 Mar 2, 404(6773), 37 - 41 The importance of repairing stalled replication forks; Cox MM et al.; The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades . Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention . In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation . The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism . On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination. Protein Sci, 2000 Feb, 9(2), 387 - 94 Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA; Petrosian SA et al.; The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges . Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding . The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein . However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition . Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group . This amino group appears to be the N-terminal alpha-amino group of the CspA molecule . It undergoes a 1.6 U shift in pKa values between native and unfolded states . Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol . This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.
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