Mol Reprod Dev, 2002 Jan, 61(1), 126 - 34 Characterization and potential function of a novel testis-specific nucleoporin BS-63; Cai Y et al.; A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambdaZAPII cDNA library, designated as BS-63 and assigned GenBank accession number: U64675 . By applying the methods of rapid amplification of cDNA ends (5' RACE) and PCR, a full-length BS-63 cDNA composed of 5475 bp was obtained . BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs . These repetitive motifs are structural characteristic of nucleoporins . BS-63 cDNA has high homology with Nup358/Ran BP2 . A 1599 bp fragment, corresponding to the C-terminus of BS-63 cDNA, was prepared and expressed in E . coli BL21(DE3) . The recombinant product was purified by affinity chromatography and SDS-PAGE and polyclonal antibodies raised . In rat testis section, the BS-63 protein was localized at the sites of nuclear pores in spermatids by immuno-gold transmission electron microscopy and on the nuclear membrane of Triton X-treated sperm by colloidal silver immuno-gold scanning electron microscopy . The recombinant BS-63 protein can be phosphorylated in vitro with PKC and p34(cdc2) . A yeast two-hybrid system was used to screen a mouse testis cDNA library to identify proteins capable of interacting with BS-63 . Using the 1.6 kb cDNA fragment as bait, the following interacting proteins were identified: Ran, transportin (karyopherin beta2), two proteins related to the nucleocytoplasmic transporter and aF10 protein . The latter protein is a putative transcriptor containing a cysteine-rich N-terminus, a LAP/PHD finger, a leucine zipper domain and a glutamine-rich C-terminus . Also it is highly expressed in murine testis and is located in the cell nucleus and cytoplasm . The interaction of BS-63 with aF10 (696-1001aa) was validated by surface plasmon resonance and by affinity precipitation combined with Western blot . aF10 (696-1001aa) interacted in vitro with BS-63 extracted from rat testis germ cells . It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a cotransporter of Ran and transportin . The complex performs the task of a carrier system in transporting aF10 into the nucleus of germ cells during spermiogenesis . Environ Mol Mutagen, 2001, 38(4), 292 - 6 Point mutations induced by 1,2-epoxy-3-butene N1 deoxyinosine adducts; Rodriguez DA et al.; The National Toxicology Program has recently classified 1,3-butadiene (BD) as a human carcinogen . BD is metabolized to the intermediates 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-dihydroxy-3,4-epoxybutane . All three metabolites have been implicated in producing specific types of DNA damage and as genotoxic agents in mice, rat, and human cells . This study has focused on EB-induced N1 deoxyinosine lesions that are formed by deamination of deoxyadenosine following reaction of the epoxide at the N(1) position . The R and S stereoisomers of this lesion were incorporated site-specifically within the context of an 11-mer oligodeoxynucleotide, incorporated into M13mp7L2 single-stranded DNA, and transfected into E . coli . Both stereoisomers modestly reduced plaque-forming ability, indicating that neither lesion presents a base modification that cannot be bypassed . The resulting plaques were assessed for point mutations using differential hybridization and DNA sequence analyses . The overall mutagenic spectrum revealed that the N1 adducts were highly mutagenic (approximately 90% per replication cycle), causing a predominance of A --> G transitions . Biopolymers, 2001, 60(3), 220 - 8 Phage-display evolution of tyrosine kinases with altered nucleotide specificity; Ting AY et al.; The problem of identifying downstream targets of kinase phosphorylation remains a challenge despite technological advances in genomics and proteomics . A recent approach involves the generation of kinase mutants that can uniquely use "orthogonal" ATP analogs to phosphorylate substrates in vivo . Using structure-based design, mutants of several protein kinase superfamily members have been found; robust and general methods are needed, however, for altering the nucleotide specificity of the remaining kinases in the genome . Here we demonstrate the application of a new phage display technique for direct functional selection to the identification of a tyrosine kinase mutant with the ability to use N6-benzyl-ATP . Our method produces, in five rounds of selection, a mutant identical to the best orthogonal Src kinase found to date . In addition, we isolate from a larger library of kinase mutants a promiscuous clone capable of using many different ATP analogs . This approach to engineering orthogonal kinases, combined with others, will facilitate the mapping of phosphorylation targets of any kinase in the genome . Plant Cell Physiol, 2001 Dec, 42(12), 1295 - 302 Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form phosphoenolpyruvate carboxylase from maize; Saze H et al.; The activity of phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) for the C4 photosynthesis is known to be regulated mainly in response to light/dark transitions through reversible phosphorylation by a specific protein kinase (PK) . PEPC-PK with an M(r) of 30 kDa was purified about 1.4 million-fold to homogeneity from maize leaves and characterized . The purified PEPC-PK was readily inactivated under mild oxidative conditions, but the activity could be recovered by dithiothreitol (DTT) . The recovery by DTT was strongly accelerated by thioredoxin (Trx) from E . coli . Trxs of plant origin such as Trx-m from spinach chloroplast and Trx-h from rice cytoplasm were also effective . These results suggest the possibility of PEPC-PK being redox-regulated via Trx in vivo. J Clin Microbiol, 2002 Jan, 40(1), 294 - 7 Characterization of enteropathogenic and enteroaggregative Escherichia coli isolated from diarrheal outbreaks; Yatsuyanagi J et al.; Virulence characteristics of diarrheal outbreak-associated Escherichia coli O55:NM, O126:NM, and O111:NM were examined . The E . coli O55:NM strains were atypical enteropathogenic E . coli (EPEC), while the E . coli O126:NM and O111:NM strains should be classified as enteroaggregative E . coli (EAggEC) . The contributions of EPEC and EAggEC to the human disease burden in Japan might be significantly greater than is currently appreciated. J Clin Microbiol, 2002 Jan, 40(1), 271 - 4 Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa; Paton AW et al.; We recently described a novel megaplasmid-encoded adhesin produced by certain Shiga toxigenic Escherichia coli (STEC) strains that lack the locus for enterocyte effacement (LEE) pathogenicity island . This adhesin, designated Saa (STEC autoagglutinating adhesin), may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic diseases in humans . In this study, we developed a pentavalent PCR assay for the detection of saa as well as other proven and putative STEC virulence genes (stx1, stx2, eae, and ehxA) . The five primer pairs used in the assay do not interfere with each other and generate amplification products of 119, 180, 255, 384, and 534 bp. J Clin Microbiol, 2002 Jan, 40(1), 61 - 7 Antigenic heterogeneity of the hepatitis C virus NS5A protein; Dou XG et al.; The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins . A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313 . Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other . The results of this analysis showed that the primary structure of this strong antigenic region is highly variable . Percent homology between different genotype sequences varied from 40.4 to 72.5% . Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region . These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase . All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n = 91) obtained from patients infected with HCV genotypes 1 through 6 . All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples . Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype . On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies . Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions . This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens. J Clin Microbiol, 2002 Jan, 40(1), 44 - 51 Immune reactivity of human sera to the glycoprotein B of human herpesvirus 7; Franti M et al.; The glycoprotein B (gB) is highly conserved among distinct human herpesvirus 7 (HHV-7) strains . Similarly to other herpesvirus glycoproteins, gB has been assumed to induce a specific human immune response . However, it did not appear as an immunodominant protein in conventional immunoblot assays . Recombinant gB, obtained from either Escherichia coli or baculovirus expression systems, did react specifically with HHV-7-seropositive sera, and the main corresponding epitopes were located in its N-terminal part . A 24-amino-acid peptide, corresponding to a predicted hydrophilicity peak and presenting no extensive homology with other betaherpesvirus glycoproteins, was selected in this region at positions 129 to 152 of the gB sequence . When tested by enzyme-linked immunosorbent assay (ELISA), this peptide specifically reacted with HHV-7-seropositive sera . This reactivity was significantly inhibited by the preincubation of sera with the peptide itself, lysates of gB-expressing cells, or lysates of HHV-7-infected cells . The reactivity was not significantly modified when sera were preincubated with lysates of either human cytomegalovirus (HCMV)- or HHV-6-infected cells . In cross-sectional studies including both children and adults, 49 out of 61 serum samples (80%) were found to be positive by HHV-7 ELISA, independent of their reactivity to HCMV . A longitudinal serological study of 17 children during the first 4 years of life showed that the level of ELISA-detected antibodies significantly decreased within a few weeks after birth and then increased in the following months, likely reflecting, respectively, the loss of maternal antibodies and the occurrence of seroconversion . These results demonstrate that gB peptide ELISA might be a useful tool for the serological study of HHV-7 infection. J Biol Chem, 2002 Mar 8, 277(10), 7633 - 6 Epub 2001 Dec 31. Analysis of DsRed Mutants . Space around the fluorophore accelerates fluorescence development; Terskikh AV et al.; Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A . V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P . (2000) Science 290, 1585--1588) . Further mutagenesis generated variants with novel and improved fluorescent properties . The mutant called AG4 exhibits only green fluorescence . The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed . Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis . Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely. J Biol Chem, 2002 Mar 8, 277(10), 8749 - 54 Epub 2001 Dec 28. Biochemical analysis of chromatin containing recombinant Drosophila core histones; Levenstein ME et al.; To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones . To this end, we synthesized the Drosophila core histones in Escherichia coli . The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues . The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP . With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones . We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones . In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template . Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications. Gut, 2002 Jan, 50(1), 13 - 8 Helicobacter pylori stimulates pepsinogen secretion from isolated human peptic cells; Lorente S et al.; BACKGROUND: Different acid and peptic related gastroduodenal diseases are associated with both increased gastric secretion and Helicobacter pylori infection . Patients with H pylori associated gastritis or duodenal ulcer have increased serum pepsinogen levels which decrease after eradication . The mechanisms of H pylori induced gastric mucosal damage are not completely understood . AIM: To determine the effects of H pylori on pepsinogen secretion from isolated human peptic cells . METHODS: Dispersed human peptic cells were prepared from endoscopically obtained biopsy specimens after collagenase digestion, mechanical disruption, and density gradient centrifugation . H pylori was obtained from gastric biopsies (antrum and body), and cultured in non-selective and selective media . Isolates of H pylori were used at different concentrations (1 - 20 x 10(6) colony forming units (cfu)) . RESULTS: H pylori (10(6) - 2 x 10(7) cfu) increased basal pepsinogen secretion in a concentration dependent manner . This stimulus was not observed with Escherichia coli . The increased secretion was in addition to that observed with 0.1 mM histamine and 0.1 mM dibutyryl-cyclic adenosine monophosphate . However, H pylori did not affect either carbamylcholine (0.1-10 microM) or cholecystokinin (1 microM) stimulated pepsinogen secretion . Addition of the nitric oxide synthase inhibitor N(w)-monomethyl-L-arginine (1 mM) inhibited H pylori induced cGMP generation and pepsinogen secretion, which were also reduced in the absence of extracellular calcium . H pylori induced pepsinogen secretion was not affected by the absence/presence of the cagA gene . CONCLUSIONS: H pylori increases pepsinogen secretion from human peptic cells through a calcium and nitric oxide mediated intracellular pathway . This effect is independent of the H pylori virulent cagA gene, and may be a mechanism of H pylori induced gastric mucosal damage. Appl Environ Microbiol, 2002 Jan, 68(1), 430 - 3 Hyperthermostable endoglucanase from Pyrococcus horikoshii; Ando S et al.; An endoglucanase homolog from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, and its enzymatic characteristics were examined . The expressed protein was a hyperthermostable endoglucanase which hydrolyzes celluloses, including Avicel and carboxymethyl cellulose, as well as beta-glucose oligomers . This enzyme is the first endoglucanase belonging to glycosidase family 5 found from Pyrococcus species and is also the first hyperthermostable endoglucanase to which celluloses are the best substrates . This enzyme is expected to be useful for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products. Biochem J, 2002 Jan 15, 361(Pt 2), 347 - 54 Characterization of monoclonal antibodies against Escherichia coli core RNA polymerase; Rouby J et al.; Multiple interactions with DNA, RNA and transcription factors occur in a transcription cycle . To survey the proximity of some of these factors to the Escherichia coli RNA polymerase surface, we produced a set of nine monoclonal antibodies (mAbs) against the enzyme . These mAbs, located at different places on the surface of the enzyme, were used in a co-immunopurification assay to investigate interference with the binding of NusA, sigma70, GreB and HepA to core RNA polymerase . One of these mAbs turned out to be the first antibody inhibitor of the binding of NusA and sigma70; it did not affect GreB and HepA interactions . Its epitope was located on the beta' subunit at the C-terminus of region G . The properties of this mAb reinforce the idea that the mutually exclusive binding of NusA and sigma70 to core RNA polymerase is due to, at least partially, overlapping binding sites, rather than allosteric interaction between two distant binding sites . This mAb is also useful to understand the occupancy of sigma70, NusA and Gre proteins on core RNA polymerase. Biochem J, 2002 Jan 15, 361(Pt 2), 327 - 35 Characterization and spatiotemporal expression of orchestin, a gene encoding an ecdysone-inducible protein from a crustacean organic matrix; Testeniere O et al.; We report the characterization of a new gene encoding an acidic protein named Orchestin . This protein is a component of the organic matrix of calcium storage structures (calcareous concretions) elaborated during the moulting cycles of the terrestrial crustacean Orchestia cavimana . The deduced molecular mass of Orchestin is estimated to be 12.4 kDa and the pI to be 4.4, whereas the native protein extracted from the calcium deposits migrates as a 23 kDa band on SDS/PAGE . This discrepancy is probably due to the richness of this protein in acidic amino acids (approx . 30%) . The protein obtained by expressing the Orchestin cDNA in Escherichia coli presents an electrophoretic mobility of 25 kDa . Antibodies raised against the recombinant protein recognize the 23 kDa native protein exclusively among the organic-matrix components . Spatiotemporal analysis of the expression of the orchestin gene shows that it is expressed only in the storage organ cells when the concretions are elaborated during the premoult period and also, to a smaller extent, during the postmoult period . The translation products are expressed in accordance with the transcript expression during both the premoult and postmoult periods . Study of the hormonal stimulation of orchestin reveals that 20-hydroxyecdysone induces this gene as a secondary-response or late-response gene. Biochemistry, 2002 Jan 8, 41(1), 415 - 21 15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine; Snider MJ et al.; 15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures . The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3) . Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction . Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining . The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage . This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme . The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate . These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step . The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C . These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction. Biochemistry, 2002 Jan 8, 41(1), 406 - 14 Transposable dual reporters for studying the structure-function relationships in membrane proteins: permissive sites in R . prowazekii ATP/ADP translocase; Alexeyev MF et al.; A new approach to studying membrane topology and permissive sites in membrane proteins expressed in Escherichia coli is described . The method is based on in vitro transposition of mini-Tn5 derivatives bearing dual pho-lac reporters {Alexeyev, M . F., and Winkler, H . H . (1999) J . Mol . Biol . 285, 1503-1513} . Two mini-Tn5 transposons, Tnpholac1 and Tnpholac2, were designed in such a way that their insertions can be converted either by restriction-ligation or by in vivo Cre-lox recombination into either sandwich reporter fusions or short amino acid (aa) tags (25 or 42 aa long) . A set of 48 unique insertions in the gene coding for the Rickettsia prowazekii ATP/ADP translocase (Tlc) was generated using Tnpholac2 . The topological information generated by these insertions was found in to be in good agreement with the existing topological model . Subsequently, these insertions were converted into both 25 and 42 aa tags, and the activity of the resulting mutants was determined . Also, site-directed mutagenesis was used to construct insertions in the loops, where no transposon hops were discovered . Of 13 extramembrane domains in Tlc, only 3 (loops 7, 10, and 13) were found to be permissive, which is in marked contrast to previous observations in the E . coli lactose permease (LacY), where most insertions in extramembrane domains were demonstrated to be permissive . The permissiveness of the insertion after I368 in TM IX lead us to reconsider the boundaries for this TM by placing I368 on the interface between TM IX and loop 10 . Interestingly, the 25 aa insertions consistently have 2-fold higher activity than the corresponding 42 aa insertions, which is also in contrast with observations made on LacY . Finally, in this study we report, for the first time, the frequency of 10 base pair target duplications generated by in vitro Tn5 transposition. Biochemistry, 2002 Jan 8, 41(1), 297 - 305 The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin; Bera S et al.; An autosomal dominant congenital cataract in humans is associated with mutation of Arg-116 to Cys in alphaA-crystallin (alphaA-R116C) . The chaperone activity and biophysical properties of reconstituted alpha-crystallin from different proportions of wild-type alphaB-crystallin (alphaB-wt) and alphaA-R116C-crystallin were studied by gel permeation chromatography, SDS-polyacrylamide gel electrophoresis, and fluorescence and circular dichroism spectroscopy and compared with those of reconstituted alpha-crystallin from alphaB-wt and wild-type alphaA-crystallin (alphaA-wt) . The reconstituted alpha-crystallin containing alphaA-R116C and alphaB-wt had a higher molecular mass, a higher thermal sensitivity to exposition of Trp side chains, fewer available hydrophobic surfaces, and lower chaperone activity than the alpha-crystallin containing alphaA-wt and alphaB-wt . The secondary structure exhibited very small changes, whereas the tertiary structure was distinctly different for alpha-crystallin formed from alphaA-R116C and alphaB-wt . Most importantly, subunit exchange studies by fluorescence resonance energy transfer showed that alphaA-R116C forms heteroaggregates faster than alphaA-wt with alphaB-wt, and the reconstituted alpha-crystallins were true heteroaggregates of two interacting subunits . These findings suggest that the molecular basis for the congenital cataract with the alphaA-R116C mutation is the formation of highly oligomerized heteroaggregates of alpha-crystallin with modified structure . However, contrary to the earlier conclusions based on the studies of homoaggregates, the loss in chaperone activity of the heteroaggregates having alphaA-R116C does not appear to be large enough to become the main factor in initiating cataract development in the affected individuals. Biochemistry, 2002 Jan 8, 41(1), 215 - 25 Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase; Numao S et al.; Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family involved in the degradation of starch . Some members of this family, including HPA, require chloride for maximal activity . To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced . Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes . X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes . However, the introduction of these mutations did alter the kinetic properties of the enzyme . Mutations to residue R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH . Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA . In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity . We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue. Biochemistry, 2002 Jan 8, 41(1), 205 - 14 Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters; Wolthers KR et al.; The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated . The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized . The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively . (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0 . Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding . In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction . The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis . Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity . The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc) . These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction. Biochemistry, 2002 Jan 8, 41(1), 162 - 9 Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu; Gromadski KB et al.; The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide . Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined . EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively . The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect . Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes . Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1) . At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell. Biochemistry, 2002 Jan 8, 41(1), 94 - 110 Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data; DeRose EF et al.; The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli . The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex . epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243) . Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs) . NMR studies were also performed on triple-lableled {U-(2)H,(13)C,(15)N}epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs . Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure . H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices . Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices . The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology . On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E . coli Exonuclease I . A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints . Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure . Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role. J Mol Biol, 2002 Jan 4, 315(1), 53 - 62 Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes; Zhang W et al.; By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N(7)/C(5) split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A) . Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired . Furthermore, cross-linking the two positions inactivates the protein . Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices . Thus, helices VII and X are in close proximity in the middle of the membrane . In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases . In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices . The results are consistent with a recently proposed mechanism for lactose/H(+) symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H(+) from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X) . Naturwissenschaften, 2001 Nov, 88(11), 482 - 5 Innate immune reactions stimulated by a lipopolysaccharide-like component of the alga Prototheca (strain 289); Bedick JC et al.; We report on the influence of an LPS-like molecule (aLPS) from the pathogenic alga, Prototheca (strain 289) on insect and murine innate immune reactions . Insect innate reactions to infection include nodule formation, a process of entrapping bacterial cells in aggregates of hemocytes . We recorded eicosanoid-dependent, dose-related nodulation reactions to aLPS in hornworms (Manduca sexta) . The insect reaction was attenuated by pre-incubating the aLPS with polymyxin-B . Conversely, the murine macrophages reacted to challenge with Escherichia coli LPS by secreting cytokines, but did not react to aLPS . We infer that, while highly conserved with respect to intracellular mechanisms of interaction, insect and mammalian immune surveillance systems differ in recognition of LPS molecular types. Methods Enzymol, 2002, 344, 186 - 93 Coexpression of proteins with methionine aminopeptidase and/or N-myristoyltransferase in Escherichia coli to increase acylation and homogeneity of protein preparations; Van Valkenburgh HA et al.; New plasmid constructs described in this article allow the coexpression in bacteria of any protein with several different NMT proteins, including the recently cloned full-length human NMT1 and 2, and with increased expression of bacterial Met-AP . Through the use of these plasmids in different combinations it should be possible to improve the homogeneity of a large number of recombinant protein preparations by the complete removal of the initiating methionine and increased extent of N-myristoylation . The new reagents described in this article are available upon request. Prikl Biokhim Mikrobiol, 2001 Nov-Dec, 37(6), 669 - 73 {Characteristics of Triticale glutaminase}; Sidel'nikova LI et al.; The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+) . A monovalent anion (Cl-) and a multivalent anion (phosphate) were shown to activate the glutaminase . Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli. Biofizika, 2001 Nov-Dec, 46(6), 1022 - 6 {Characteristics of electrostatic interaction of Escherichia coli RNA polymerase with promoters of T4 phage DNA}; Dzheliadin TR et al.; A comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken . The data obtained indicate that there are some particular elements in the patterns of electrostatic potential distribution of promoter DNA specific for promoter groups differing by their functional response to ADP-ribosylation of the alpha-subunit as well as to rpoB403- or rpoB409 mutationals of the beta-subunit of RNA-polymerase. Orthopedics, 2001 Dec, 24(12), 1151 - 4 A 4- to 10-year follow-up study of the Tricon-M noncemented total knee replacement; Faraj AA et al.; This study reviews the clinical and radiographic results of 207 consecutive noncemented Tricon-M (Smith & Nephew Inc, Memphis, Tenn) total knee replacements (TKRs) in 189 patients . The patella was surfaced in 119 cases, and mean follow-up was 8 years (range: 4-10 years) . At final follow-up, mean Hospital for Special Surgery score improved 45 points in 187 cases . Survivorship, with failure defined as the need for revision, was 98% at 4 years, 97% at 7 years, 94% at 8 years, and 90% at 10 years . Twenty-one (11.3%) patients went on to revision . Results for overweight and obese patients did not differ significantly from normal-weight patients . The noncemented Tricon-M TKR prosthesis yields acceptable results; however, patella surfacing and the use of a tibial polyethylene insert > or = 12 mm thick are recommended. J Endourol, 2001 Nov, 15(9), 915 - 7 Gas-containing renal stones treated with percutaneous nephrolithotomy: case report; Nilsen FS et al.; We present a rare case of E . coli emphysematous pyelonephritis with sepsis . The radiologic presentation consisted of multiple radiolucent gas-filled, free-floating uric acid calculi in a hydronephrotic renal pelvis . The infection was treated by intravenous fluids and antibiotics and percutaneous nephrostomy drainage . The patient was rendered stone free by percutaneous nephrolithotomy and ultrasound lithotripsy. J Bioenerg Biomembr, 2000 Aug, 32(4), 413 - 21 Insights into ATP synthase structure and function using affinity and site-specific spin labeling; Vogel PD; A variety of different approaches has been used during the last couple of decades to investigate structure and function relationships within the catalytic portion of the F0F1-ATP synthase and of its interactions with the proton-translocator F0 . In our group, we employ ESR spectroscopy with the use of stable organic radicals, so-called spin labels, as reporter groups . The radicals are either attached to substrates/ligands or specifically inserted into the protein structure by "site-specific spin labeling." Both approaches bear intrinsic advantages for their special uses and result in the specific information that is available through ESR, e.g., structural changes due to binding of effector molecules (e.g., Mg2+ ions), conformational transitions during catalytic turnover, distance information on radicals bound at 20 A or less, and information on the binding characteristics of labeled substrates . This review summarizes the results of a variety of different approaches we have used during the last years to study, with the help of ESR spectroscopy, the structure of the nucleotide binding sites of F1-ATPases of different origins as well as interactions with F0 subunits. J Bioenerg Biomembr, 2000 Aug, 32(4), 401 - 11 The structural and functional connection between the catalytic and proton translocating sectors of the mitochondrial F1F0-ATP synthase; Papa S et al.; The structural and functional connection between the peripheral catalytic F1 sector and the proton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed . The observations examined show that the N-terminus of subunit gamma, the carboxy-terminal and central region of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateral stalk, which connects the peripheries of F1 to F0 and surrounds the central element of the stalk, constituted by subunits gamma and delta . The ATPase inhibitor protein (IF1) binds at one side of the F1F0 connection . The carboxy-terminal segment of IF1 apparently binds to OSCP . The 42L-58K segment of IF1, which is per se the most active domain of the protein, binds at the surface of one of the three alpha/beta pairs of F1, thus preventing the cyclic interconversion of the catalytic sites required for ATP hydrolysis. J Bioenerg Biomembr, 2000 Aug, 32(4), 391 - 400 Partial assembly of the yeast mitochondrial ATP synthase; Mueller DM; The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation of ADP to ATP . The yeast mitochondrial ATP synthase is composed of at least 19 different peptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, one of which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor . Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis that the yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, of the polypeptides that are thought to comprise the rotor . However, the enzyme complex assembled in the absence of the rotor is thought to be uncoupled, allowing protons to freely flow through F0 into the mitochondrial matrix . Left uncontrolled, this is a lethal process and the cell must eliminate this leak if it is to survive . In yeast, the cell is thought to lose or delete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encoding essential components of F0 . Recent biochemical studies in yeast, and prior studies in E . coli, have provided support for the assembly of a partial ATP synthase in which the ATP synthase is no longer coupled to proton translocation. J Bioenerg Biomembr, 2000 Aug, 32(4), 373 - 81 Structural changes during ATP hydrolysis activity of the ATP synthase from Escherichia coli as revealed by fluorescent probes; Turina P; F1F0-ATPase complexes undergo several changes in their tertiary and quaternary structure during their functioning . As a possible way to detect some of these different conformations during their activity, an environment-sensitive fluorescence probe was bound to cysteine residues, introduced by site-directed mutagenesis, in the gamma subunit of the Escherichia coli enzyme . Fluorescence changes and ATP hydrolysis rates were compared under various conditions in F1 and in reconstituted F1F0 . The results are discussed in terms of possible modes of operation of the ATP synthases. J Bioenerg Biomembr, 2000 Aug, 32(4), 357 - 64 Subunit organization of the stator part of the F0 complex from Escherichia coli ATP synthase; Greie JC et al.; Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalytic mechanism utilizing the energy of an electrochemical ion gradient . The transmembrane potential is supposed to propel rotation of a subunit c ring of F0 together with subunits gamma and epsilon of F1, thereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complex functions as a stator for compensation of the torque generated during rotation . This review focuses on our recent work on the stator part of the F0 complex, e.g., subunits a and b . Using epitope insertion and antibody binding, subunit a was shown to comprise six transmembrane helixes with both the N- and C-terminus oriented toward the cytoplasm . By use of circular dichroism (CD) spectroscopy, the secondary structure of subunit b incorporated into proteoliposomes was determined to be 80% alpha-helical together with 14% beta turn conformation, providing flexibility to the second stalk . Reconstituted subunit b together with isolated ac subcomplex was shown to be active in proton translocation and functional F1 binding revealing the native conformation of the polypeptide chain . Chemical crosslinking in everted membrane vesicles led to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32C could be crosslinked to subunit a, indicating a close proximity of subunits a and b near the membrane . Further evidence for the proposed direct interaction between subunits a and b was obtained by purification of a stable ab2 subcomplex via affinity chromatography using His tags fused to subunit a or b . This ab2 subcomplex was shown to be active in proton translocation and F1 binding, when coreconstituted with subunit c . Consequences of crosslink formation and subunit interaction within the F1F0 complex are discussed. J Bioenerg Biomembr, 2000 Aug, 32(4), 347 - 55 The b subunit of Escherichia coli ATP synthase; Dunn SD et al.; The b subunit of ATP synthase is a major component of the second stalk connecting the F1 and F0 sectors of the enzyme and is essential for normal assembly and function . The 156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensively through mutagenesis, deletion analysis, and biophysical characterization . The two copies of b exist as a highly extended, helical dimer extending from the membrane to near the top of F1, where they interact with the delta subunit . The sequence has been divided into four domains: the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, and the C-terminal delta-binding domain . The dimerization domain, contained within residues 60-122, has many properties of a coiled-coil, while the delta-binding domain is more globular . Sites of crosslinking between b and the a, alpha, beta, and delta subunits of ATP synthase have been identified, and the functional significance of these interactions is under investigation . The b dimer may serve as an elastic element during rotational catalysis in the enzyme, but also directly influences the catalytic sites, suggesting a more active role in coupling. J Bioenerg Biomembr, 2000 Aug, 32(4), 341 - 6 Structural and functional features of the Escherichia coli F1-ATPase; Gruber G; The structural organization and overall dimensions of the Escherichia coli F1-ATPase in solution has been analyzed by synchroton X-ray scattering . Using an independent ab initio approach, the low-resolution shape of the hydrated enzyme was determined at 3.2 nm resolution . The shape permitted unequivocal identification of the volume occupied by the alpha3beta3gamma complex of the atomic model of the ECF1-ATPase . The position of the delta and epsilon subunits were found by interactive fitting of the solution scattering data and by cross-linking studies . Laser-induced covalent incorporation of 2-azido-ATP established a direct relationship between nucleotide binding affinity and the different interactions between the stalk subunits gamma and epsilon with the three catalytic subunits (beta) of the F1-ATPase . Mutants of the ECF1-ATPase with the introduction of Trp-for-Tyr replacement in the catalytic site of the complex made it possible to monitor the activated state for ATP synthesis (ATP conformation) in which the gamma and epsilon subunits are in close proximity to the alpha subunits and the ADP conformation, with the stalk subunits are linked to the beta subunit. J Bioenerg Biomembr, 2000 Aug, 32(4), 333 - 9 F1F0-ATP synthase-stalking mind and imagination; Wilkens S; Electron microscopy together with image analysis has been used to study the structure of the intact F1F0-ATPsynthase from Escherichia coli . A procedure has been developed which allows preparation of detergent-free enzyme . Aside from the well known two-domain structure, images of F1F0 prepared by this procedure show a number of additional features, including a second stalk, which can be seen extending all the way from the F0 to the top of the F1 in some images, and a small protein on the very top of the F1, which has been identified as the delta subunit by decoration with a monoclonal antibody . In light of these results, a refined model of the subunit arrangement of the complex is presented. J Bioenerg Biomembr, 2000 Aug, 32(4), 325 - 32 ATP synthases in the year 2000: evolving views about the structures of these remarkable enzyme complexes; Pedersen PL et al.; This introductory article briefly summarizes how our views about the structural features of ATP synthases (F0F1) have evolved over the past 30 years and also reviews some of our current views in the year 2000 about the structures of these remarkably unique enzyme complexes . Suffice it to say that as we approach the end of the first year of this new millinium, we can be conservatively confident that we have a reasonably good grasp of the overall "low-resolution" structural features of ATP synthases . Electron microscopy techniques, combined with the tools of biochemistry, molecular biology, and immunology, have played the leading role here by identifying the headpiece, basepiece, central stalk, side stalk, cap, and in the mitochondrial enzyme, the collar around the central stalk . We can be reasonably confident also that we have a fairly good grasp of much of the "high-resolution" structural features of both the F1 moiety comprised of fives subunit types (alpha, beta, gamma, delta, and epsilon) and parts of the F0 moiety comprised of either three (E . coli) or at least ten (mitochondria) subunit types . This information acquired in several different laboratories, either by X-ray crystallography or NMR spectroscopy, includes details about the active site and subunit relationships . Moreover, it is consistent with recently reported data that the F1 moiety may be an ATP driven motor, which, during ATP synthesis, is driven in reverse by the electrochemical proton gradient generated by the electron transport chain . The real structural challenges of the future are to acquire at high resolution "complete" ATP synthase complexes representative of different stages of the catalytic cycle during ATP synthesis and representative also of key regulatory states. Dig Dis Sci, 2001 Dec, 46(12), 2555 - 66 Small bowel review: diseases of the small intestine; Thomson AB et al.; In the past year there have been many advances in the area of small bowel physiology and pathology and therapy . In preparation for this review, over 1500 papers were assessed . The focus is on presenting clinically useful information for the practicing gastroenterologist . Selected important clinical learning points include the following: (1) glutamine may restore the AIDs-associated increased intestinal permeability to normal; (2) substance P is a major mediator of diarrhea caused by Costridium difficile toxin A, acting by binding to a G-protein-coupled receptor, and represents a possible 2therapeutic target; (3) the serological diagnosis of celiac disease has been greatly enhanced with the use of anti-endomysial antibody testing, and the recent antitransglutaminase; (4) a quarter of patients with celiac disease may have secondary pancreatic insufficiency and require enzyme replacement therapy; (5) in the patient with unexplained elevation in the serum transaminase concentration, consider celiac disease as an obscure possibility; (6) bosentan and endothelin receptor agonist may prove to be useful in reducing gut ischemia in patients with septic shock; and (7) the administration of recombinant human fibroblast growth factor-2 may prove to be useful to prevent radiation damage to the gastrointestinal tract. Can J Vet Res, 2001 Oct, 65(4), 233 - 40 Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha; Denis F et al.; Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response . As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale . In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha . The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig . The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively . Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer) . The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors . The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera. Can J Vet Res, 2001 Oct, 65(4), 206 - 12 Cloning and characterization of the gene coding for NADPH-sulfite reductase hemoprotein from Actinobacillus pleuropneumoniae and use of the protein product as a vaccine; Willson PJ et al.; An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate and screened with serum produced in pigs that had been vaccinated with the anionic fraction of a sodium chloride extract . One E . coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67,000 molecular mass . The A . pleuropneumoniae-derived DNA encoding the protein was localized and characterized by restriction enzyme digestion and nucleotide sequence analysis which showed strong homology with the cysI gene of E . coli . One open reading frame of 1764 bases in length was detected which encoded a cysI protein from serotype 1, with a calculated molecular mass of 66,678 . The DNA encoding the protein was labeled with radio-isotope and the homologous gene was isolated from an A . pleuropneumoniae serotype 5a library . The serotype 5a gene was the same length, but the cysI protein from serotype 5a was slightly larger (66,849) due to 8 substitutions in the amino acid sequence . Expression plasmids containing cysI from either serotype of A . pleuropneumoniae complemented an E . coli cysI mutant . Pigs vaccinated with the recombinant cysI were protected from challenge with A . pleuropneumoniae of the homologous serotype. Ross Fiziol Zh Im I M Sechenova, 2001 Oct, 87(10), 1362 - 9 {Changes in the afferent activity of the vagus nerve and the rectal temperature in rats following Escherichia coli endotoxin administration}; Lapsha VI et al.; In anaesthetised rats, i.p . administration of the Echerichia coli lipopolysaccharide in doses 5 mcg/kg (LPS) increased afferent activity of the cervical vagus, whereas 100 and 1000 mcg/kg doses inhibited the afferent discharges . Pyrogen-free saline (PFS) did not alter the activity . Rectal temperature (RT) was decreased by the PFS and by large doses of the LPS . Sodium salicylate administration prevented the effects. Biol Pharm Bull, 2001 Dec, 24(12), 1356 - 61 Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei); Sanda A et al.; A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei) . The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology . SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da . SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues . The active site Lys residue in BPTI is replaced by an Arg residue in SETI . SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin . SETI was expressed by E . coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained . The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods. Vet Res Commun, 2001 Dec, 25(8), 615 - 22 Feline ubiquitin fusion protein genes; Kano R et al.; Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes . Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids) . The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs . The recombinant glutathione S-transferase (GST)-feline ubiquitin fusion proteins were produced in Escherichia coli and purified . The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum . The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis . The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay . These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells. Res Microbiol, 2001 Dec, 152(10), 873 - 81 Loss of lambda prophage recombinogenicity in UV-irradiated Escherichia coli: the role of host genes ruvA, ruvB, ruvC, and recG; Zahradka K et al.; Earlier studies have revealed a radiation-induced process leading to the loss of lambda prophage recombinogenicity . The process takes place in UV-irradiated Escherichia coli cells, and renders the prophage incapable of site-specific recombination with the host chromosome, and of general recombination with an infecting homologous phage . It was found that the inhibition of prophage recombinogenicity depends on functional RecBCD enzyme of E . coli . In this work, the role of ruvABC and recG genes in the inhibitory process was assessed . The products of these genes are known to act at the last step of homologous recombination and recombinational DNA repair by catalyzing the resolution of recombination intermediates (the Holliday junctions) . Irradiated prophage retained its ability to recombine in ruvA, ruvB, ruvC, and recG mutants . These results suggest that in addition to RecBCD enzyme, RuvABC and RecG proteins are also involved in the inhibition of prophage recombinogenicity . We infer that RuvABC and RecG act in this process before RecBCD, probably by processing the Holliday junctions formed upon replication arrest, and thereby providing double-stranded DNA breaks as substrate for RecBCD-mediated recombinational repair of UV-damaged bacterial chromosome. Ying Yong Sheng Tai Xue Bao, 2000 Feb, 11(1), 146 - 8 {Ecological effect of CdCl2 on plasmid and role of plasmid in Cd-tolerance of its host}; Meng L et al.; After treating the plasmid of Escherichia coli HB101 by CdCl2 in vitro or in vivo, the effect of Cd on the structure of plasmid pWH58 DNA was studied through argarose gel electrophesis and restriction fragment length polymorphism analysis . By comparing the growth of E . coli with and without plasmid cultured in the medium with Amp LB and non-anti LB under different concentrations of CdCl2, the effect of Cd on plasmid E . coli of in vivo and the role of plasmid in Cd-tolerance of its host were studied . Cd treatments of E . coli in vitro or in vivo didn't cause an obvious change of the structure of plasmid pWH58 DNA . Plasmid pWh58 could replicate for regeneration and gene express under Cd stress . Plasmid pWH58 of E . coli in vivo lowered Cd-tolerance of its host form 75 mg.L-1 to 50 mg.L-1 . Cd-tolerance of E . coli ascended markedly after Cd taming, but had a trend of reversing to original level after restoration, indicating that Cd-tolerance of E . coli could be tamed, but the tamed Cd-tolerance still had no genetic basis. Prep Biochem Biotechnol, 2001 Nov, 31(4), 341 - 54 Purification of soluble recombinant human FcgammaRII (CD32); Gruel N et al.; The present study describes the methodology used to purify human recombinant low-affinity FcgammaRIIa2 produced in E . coli and to evaluate its binding to surface IgG . The recombinant molecule was purified by a two-step chromatographic procedure, including affinity chromatography using IV.3 anti-FcgammaRIIa1/2 immunosorbent, followed by gel filtration chromatography . Using this method, the purified recombinant FcgammaRIIa2 was 99% pure . It exhibited an isoeletric point of 5.2 . Binding studies demonstrated a specific binding of the purified recombinant molecule to surface IgG expressed by human B cells . Thus, we have set up a method which allows to purify functional human recombinant FcgammaRIIa2 for further characterization of its biological activities. Adv Exp Med Biol, 2001, 500, 687 - 96 Dose dependent induction of DNA adducts, gene mutations, and cell proliferation by the antiandrogenic drug cyproterone acetate in rat liver; Wolff T et al.; 1 . CPA does not only induce the formation of DNA adducts but also of mutations in female rat liver . 2 . The mutation frequency exhibited a characteristic time course . Within a period of 3 days post administration, a tremendous increase was noted, which remained at a high level until 2 weeks post exposure . Thereafter, most mutation-carrying cells were eliminated within a period of 2 weeks leaving a cell population remaining at a constant level for another 4 weeks . Thus, the length of the observation period post exposure, i . e . the manifestation time, seems to be a critical factor for the strength of the mutagenic response . The highest as observed between 1 and 2 weeks post exposure . Correspondingly, the dose response curve recorded 2 weeks post exposure showed a higher mutagenic response than the curve after 6 weeks of exposure recorded previously . 3 . When CPA-induced mutations were recorded as a function of the dose, mutation frequencies at the lower dose range were found that did not differ from those of controls . The non-effective dose recorded 2 weeks post exposure was much lower than that recorded after 6 weeks of exposure indicating that it is a function of the manifestation time . Since DNA adducts were formed in high amounts at the non-effective doses, we assume that the mitogenic activity required for the conversion of DNA adducts into mutations was not sufficiently strong . The liver of adult animals exhibits a very low endogeneous proliferation rate, which is not likely to contribute significantly to the expression of mutations . We conclude that it is the mitogenic activity of CPA itself, which stimulates the expression of mutations. Vet Rec, 2001 Nov 24, 149(21), 643 - 6 Effect of selenium and vitamin E on antibody production by dairy cows vaccinated against Escherichia coli; Panousis N et al.; Sixty clinically healthy Holstein cows were randomly assigned to one of four groups according to their age and parity and vaccinated in late pregnancy (day 190) with a multivalent vaccine against Escherichia coli . The 15 cows in the first group (SeE) were injected intramuscularly with a solution of sodium selenite (0.1 mg Se/kg bodyweight) and vitamin E (alpha-tocopherol acetate, 8 U/kg bodyweight), the cows in the second group (Se) received only selenium and the cows in the third group (E) received only vitamin E at the same doses and by the same route of administration; the cows in the fourth group were used as controls . The vaccination and the injections of selenium and vitamin E were repeated 42 days later . The concentration of selenium in whole blood and of vitamin E in serum was determined by fluorometric methods . Specific antibody titres against E coli were determined in serum samples by ELISA . The results showed that the injection of selenium either alone or in combination with vitamin E significantly improved the production of specific antibodies against E coli, and that the production of specific antibodies was greater after the administration of selenium alone. J Rheumatol, 2001 Dec, 28(12), 2579 - 82 Expression of CD44 in synovium of rabbits with chronic arthritis induced by immunization with Escherichia coli; Takagi T et al.; OBJECTIVE: To investigate the expression of CD44 and its role in experimental chronic arthritis in rabbits . METHODS: Rabbits were immunized with Escherichia coli 0:14 for short (4 mo) and long (8-10 mo) periods . Immunohistochemical staining was performed on knees, using anti-CD44 antibodies . RESULTS: Lymphocyte infiltration in the synovium was found in 30.0% of rabbits after short term immunization, and the rate increased to 58.3% after longterm immunization . CD44 was present in synovial lining cells in 30.0% of rabbits after short term immunization, and it increased significantly (p < 0.05) after longterm immunization (66.7%) . CD44 was also observed in lymphocytes in knee synovium after longterm immunization (25.0%) . CONCLUSION: CD44 in lining cells might play a role in promoting chronic arthritis in rabbits immunized with E . coli. J Neuropathol Exp Neurol, 2001 Dec, 60(12), 1170 - 80 Oligodendroglial tumors frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes; Wolter M et al.; We investigated 34 oligodendroglial tumors (7 oligodendrogliomas, 11 anaplastic oligodendrogliomas, 8 oligoastrocytomas, and 8 anaplastic oligoastrocytomas) for deletion, mutation, hypermethylation, and expression of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes at 9p21 . One anaplastic oligoastrocytoma carried a homozygous deletion including all 3 genes . None of the tumors demonstrated point mutations in any of the genes . Methylation-specific polymerase chain reaction (MSP) analysis and sequencing of bisulfite-modified DNA, however, revealed frequent hypermethylation of the 5'-CpG islands in CDKN2A, p14ARF, and CDKN2B . Partial or complete methylation of the majority of CpG sites analyzed from each gene was detected in 32% of the tumors at the CDKN2A gene and at a similar percentage (41%) of the tumors at the p14ARF gene and the CDKN2B gene . Most tumors with CDKN2A, p14ARF, and/or CDKN2B hypermethylation either lacked detectable transcripts from these genes or had lower mRNA levels than those determined for non-neoplastic brain tissue . There was a significant correlation between hypermethylation of these genes and the presence of allelic losses on chromosomal arms 1p and 19q . In addition, p14ARF hypermethylation was predominantly found in tumors without a demonstrated TP53 mutation . Taken together, our results indicate that hypermethylation of CDKN2A, p14ARF, and CDKN2B is an important epigenetic mechanism by which oligodendroglial tumors may escape from p53- and pRb-dependent growth control. Parasitol Res, 2001 Dec, 87(12), 1029 - 30 Blastocystis hominis modulates immune responses and cytokine release in colonic epithelial cells; Long HY et al.; An experimental in vitro model has been developed in order to determine whether Blastocystis hominis is able to trigger inflammatory cytokine response in colonic epithelial cells . After 24 h incubation of B . hominis with the cell lines HT-29 and T-84, B . hominis cells were not able to cause cytopathic effects, but significant increases in the release of the cytokines IL-8 and GM-CSF could be observed . However, after the first 6 h of co-incubation, the production of IL-8 was not increased in HT-29 cells, and even reduced when Escherichia coli (bacteria or lipopolysaccharide) was present during co-incubation . Similar effects were observed using supernatants of B . hominis culture . These data indicate that B . hominis induces as well as modulates the immune response in intestinal epithelial cells, and we conclude that different pathophysiological events may occur during B . hominis infection. Biomaterials, 2002 Jan, 23(1), 161 - 6 Generation of mesoscopic patterns of viable Escherichia coli by ambient laser transfer; Ringeisen BR et al.; We have generated mesoscopic patterns of viable Escherichia coli on Si(1 1 1), glass, and nutrient agar plates by using a novel laser-based transfer process termed matrix assisted pulsed laser evaporation direct write (MAPLE DW) . We observe no alterations to the E . coli induced by the laser-material interaction or the shear forces during the transfer . Transferred E . coli patterns were observed by optical and electron microscopes, and cell viability was shown through green fluorescent protein (GFP) expression and cell culturing experiments . The transfer mechanism for our approach appears remarkably gentle and suggests that active biomaterials such as proteins, DNA and antibodies could be serially deposited adjacent to viable cells . Furthermore, this technique is a direct write technology and therefore does not involve the use of masks, etching, or other lithographic tools. Planta, 2001 Nov, 214(1), 75 - 84 Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum; Yamazaki Y et al.; Psilotum nudum Griseb is a pteridophyte and belongs to the single family (Psilotaceae) of the division, Psilophyta . Being the only living species of a once populated division, P . nudum is the most primitive vascular plant . Chalcone synthase (CHS; EC 2.3.1.74) superfamily enzymes are responsible for biosyntheses of diverse secondary metabolites, including flavonoids and stilbenes . Using a reverse transcription-polymerase chain reaction strategy, four CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P . nudum, and heterologously expressed in Escherichia coli . These four enzymes of 396-406 amino acids showed sequence identity of > 50% among themselves and to other higher-plant CHS-superfamily enzymes . PnJ and PnP preferred p-coumaroyl-CoA and isovaleryl-CoA respectively, as starter CoA and catalyzed CHS-type ring formation, indicating that they are CHS and phlorisovalerophenone synthase, respectively . On the other hand, PnI and PnL preferred cinnamoyl-CoA as starter CoA and catalyzed stilbene synthase-type cyclization and thus were determined to be pinosylvin synthases (EC 2.3.1.146) . In addition, PnE, which uniquely contains a glutamine in place of otherwise strictly conserved histidine, had no apparent in vitro catalytic activity . Phylogenetic analysis indicated that these P . nudum clones form a separate cluster together with Equisetum arvense CHS . This cluster of pteridophytes is located next to the cluster formed by pine (gymnosperm) enzymes, in agreement with their evolutionary relationships . Psilotum nudum represents a plant with the most diverse CHS-superfamily enzymes and this ability to diverge may have provided a survival edge during evolution. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2001 Sep, 15(5), 257 - 60 {Construction of eukaryotic expression plasmid for mouse myogenic regulatory factor MyoD gene}; Qin RF et al.; OBJECTIVE: To construct eukaryotic expression plasmid of mouse myogenic regulatory factor MyoD gene for further study on MyoD gene function in molecular regulatory mechanism in skeletal muscle repair . METHODS: The plasmids PEMMBC2 beta 5 containing full cDNA length of MyoD inserted in EcoRI restriction site, were first propagated in Escherichia coli DH5a, then extracted and purified with the Wizard Plus Minipreps DNA Purification System (Promega, USA) . The coding sequence of MyoD in PEMMBC2 beta 5 was confirmed by agarose gel electrophoresis and DNA sequence analysis . After plasmids PEMMBC2 beta 5 and plasmids pcDNA3-neo were prepared by digestion with EcoRI, the MyoD cDNA fragment was inserted into EcoRI site in pcDNA3-neo eukaryotic expression vector, and pcDNA3/MyoD was formed . The pcDNA3/MyoD, digested with restriction enzymes, was found to contain the MyoD cDNA sequence by agarose gel electrophoresis analysis . RESULTS: The extracted and purified PEMMBC2 beta 5 contained the correct nucleotide sequence for the full length of MyoD cDNA fragment . The MyoD cDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA3-neo, which formed the pcDNA3/MyoD . CONCLUSION: The pcDNA3/MyoD, a eukaryotic expression plasmid, for MyoD is constructed successfully. Przegl Epidemiol, 2001, 55(3), 287 - 97 {Adherence patterns of Escherichia coli strains isolated from children with diarrhea.}; Sobieszczanska BM et al.; Among enteropathogenic E . coli strains (EPEC) there are different patterns of adherence to the culture cells in vitro assay: localized, localized-like and diffuse . The adherence pattern is dependent on the ability of E . coli strains to cause of diarrhea . The strains locally adhering possess a 60 MDa plasmid--E . coli adherence factor (EAF), and produce characteristic histopathologic intestinal lesions linked with the presence of chromosomal eae gene . The pathogenicity of diffusely adherent as well as cells detaching E . coli (CDEC) remains controversial . The aim of the study was to identify the adherence patterns of E . coli strains isolated from children with diarrhea and to compare that patterns with the serotypes and the presence of EAF and/or pO157 plasmids, fimbriae and eae, stx1, and stx2 specific sequences . Nine out of examined E . coli strains showed the localized pattern of adherence . About half (46.8%) of strains were diffusely adherent and six isolates were cells detaching E . coli (CDEC) . A total of 22 (23%) examined strains showed the presence of specific for verocytotoxins sequences . The results showed that many strains recognized on the ground of agglutination with specific EPEC antisera as unpathogenic could be an etiologic agents of diarrhea which are able to produce histopathologic lesions in the intestinal epithelium . In turn, many strains classified as EPEC could be unpathogenic on the basis of diffuse pattern of adherence. DNA Seq, 2001, 12(2), 97 - 106 Cloning and characterization of 5'-upstream sequence of the M32 gene for a mouse homologue of Drosophila heterochromatin protein 1 (HP1); Sato M et al.; M32 {also termed chromatin modifier protein 2 (MOD2)} is a nuclear protein consisting of the condensed chromatin structure (heterochromatin) and considered one of the mammalian homologues of heterochromatin protein 1 (HP1), first isolated as one of the components of heterochromatin in Drosophila . This report presents the isolation and characterization of the 5'-upstream region of the mouse M32 gene containing a promoter region and 5'-untranslated region (5'-UTR) exon . The 5'-upstream region (approximately 0.27 kb starting from the 5' end of the 5'-UTR exon) of the M32 gene contained neither a TATA box nor a CCAAT box, but possessed potential binding sites for transcription factors such as Sp1, H4TF-1, PEA2, PEA3, GSG element and Egr-1, and was highly G/C-rich . The promoter activity of this 5'-upstream region was demonstrated by transfecting its fusion-construct with the E . coli beta-galactosidase gene into the F9 mouse teratocarcinoma cell line . The 5' ends of the mRNA were mapped to at least two positions in the 5'-upstream region . Interestingly, the 5'-upstream region exhibited a high degree of similarity to a portion of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 gene, which is thought to play a role in RNA processing, located in the reverse orientation to the M32 gene, and also to several known ESTs and cDNAs . These findings suggest that the 5'-upstream region of the M32 gene consists of a multiple regulatory complex which probably plays important roles in nuclear function such as chromatin organization and RNA processing. Antioxid Redox Signal, 2001 Oct, 3(5), 867 - 79 The FAD-PAS domain as a sensor for behavioral responses in Escherichia coli; Taylor BL et al.; Aer, the aerotaxis receptor in Escherichia coli, is a member of a novel class of flavoproteins that act as redox sensors . The internal energy of the cell is coupled to the redox state of the electron transport system, and this status is sensed by Aer(FAD) . This is a more versatile sensory response system than if E . coli sensed oxygen per se . Energy-depleting conditions that decrease electron transport also alter the redox state of the electron transport system . Aer responds by sending a signal to the flagellar motor to change direction . The output of other sensory systems that utilize redox sensors is more commonly transcriptional regulation than a behavioral response . Analysis in silico showed Aer to be part of a superfamily of PAS domain proteins that sense the intracellular environment . In Aer, FAD binds to the PAS domain . By using site-specific mutagenesis, residues critical for FAD binding and sensory transduction were identified in the PAS domain . The PAS domain appears to interact with a linker region in the C-terminus . The linker region is a member of a HAMP domain family, which has signal transduction roles in other systems. J Chromatogr A, 2001 Nov 30, 936(1-2), 59 - 69 Chromatographic performance on a C30-bonded stationary phase of monohydroxycarotenoids with variable chain length or degree of desaturation and of lycopene isomers synthesized by various carotene desaturases; Breitenbach J et al.; Selectivity towards geometric isomers is a superior feature of a C30 polymeric stationary phase . Therefore, lycopene isomers synthesized in Escherichia coli transformants by catalysis of divers carotene desaturases were separated on this stationary phase . Due to their spectral characteristics and by co-chromatography with nuclear magnetic resonance-characterized carotene standards, some of them could be identified . Most of the lycopene isomers were cyclized by lycopene cyclase yielding mainly 9Z, 13Z and all-E beta-carotene . In contrast, 7,9,7',9'Z prolycopene is accumulating since it cannot be converted by this enzyme . Finally several acyclic hydroxycarotenoids with a chain of 30, 40 and 45 carbon atoms differing in the length of the polyene chain from 9 to 13 were separated on the C30 stationary phase . Longer retention times were observed when the length of the molecule increased and also when the conjugated double bond system was extended . Corresponding monocyclic carotenoids were less retained on the C30 stationary phase and derivatives with an epsilon-ionone end group eluted earlier than with a beta-end group. Neurol Res, 2001 Dec, 23(8), 862 - 8 N(omega) -nitro-L-arginine methyl ester (L-NAME) attenuates the acute inflammatory responses and brain injury during the early phase of experimental Escherichia coli meningitis in the newborn piglet; Park WS et al.; We evaluated the anti-inflammatory and neuroprotective effect of nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), in experimental bacterial meningitis in the newborn piglet . Meningitis was induced by intracisternal injection of 10(8) colony forming units of Escherichia coli . L-NAME 10 mg kg(-1) was given intravenously 30 min before induction of meningitis . L-NAME significantly attenuated the increase in intracranial pressure and decrease in cerebrospinal fluid glucose concentration observed in the meningitis group . Systemic and cerebral perfusion pressure were even higher compared to the control and meningitis groups . However, the meningitis-induced increase in tumor necrosis factor-alpha level, leukocyte numbers and lactate level in the cerebrospinal fluid was not significantly attenuated with L-NAME administration . Reduced cerebral cortical cell membrane Na+, K+ -ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain cell membrane dysfunction, were significantly improved with L-NAME treatment . Decreased brain glucose and ATP levels were also significantly improved with L-NAME treatment . These findings suggest that L-NAME was effective in attenuating the acute inflammatory responses and brain injury in neonatal bacterial meningitis. Clin Exp Rheumatol, 2001 Sep-Oct, 19(5 Suppl 24), S59 - 61 Behçet's disease complicated by pylephlebitis and hepatic abscesses; Gelber AC et al.; A 22 year old man presented with fever, abdominal pain, weight loss and diarrhea . Past medical history revealed recurrent aseptic meningitis, uveitis, and erythema nodosum . Further inquiry unveiled a prominent history of oral aphthous ulcers; all features of Behcet's disease . Imaging revealed mesenteric arteritis and pylephlebitis, septic thrombophlebitis of the portal vein, a previously unrecognized complication of Behcet's disease, with multiple intrahepatic abscesses . Portal venography demonstrated an extensively diseased, expanded, and obstructed portal venous system . Blood cultures and portal vein aspirate yielded polymicrobial flora . Percutaneous intraportal thrombolytic therapy and mechanical thrombectomy were attempted to restore flow to the portal venous system . This distinctly rare manifestation of Behcet's Disease, pylephlebitis, may result from ischemic injury and structural compromise of the bowel mucosa, resulting from underlying vasculitis. Clin Exp Rheumatol, 2001 Sep-Oct, 19(5 Suppl 24), S19 - 24 Neutrophil activation in Behçet's disease; Eksioglu-Demiralp E et al.; OBJECTIVE: Neutrophils are implicated in the pathogenesis of Behcet's disease (BD) . Various functions of neutrophils are studied to clarify this role . METHODS: The oxidative burst and phagocytic functions of neutrophils and surface molecules associated with neutrophil activation (CD10, CD14 and CD16) were investigated in BD patients by flow cytometric methods . Patients with inflammatory arthropathies, sepsis and healthy controls were also studied . RESULTS: In the oxidative burst experiments, after fMLP and PMA stimulation, stimulation index was found to be significantly decreased in patients both with BD and sepsis compared to healthy controls and inflammatory arthropathies (p < 0.001 and p < 0.01, respectively) . The phagocytosis of labelled E . coli particles in patients with BD was not different from that of the healthy controls, while it was decreased in diseased controls (p < 0.001) . The surface density of neutral endopeptidase (CD10) and the mean percentage of LPS receptor (CD14) was found to be significantly higher in both BD patients and diseased controls (p < 0.001) . The mean percentage of CD16 expression was only low in patients with sepsis (p < 0.001), whereas CD16 intensity on cells was found to be lower in patients with BD as well as in sepsis (p < 0.01) . CONCLUSION: These findings indicate the presence of in vivo pre-activated neutrophils in BD . A similar activation was also a feature of severe inflammatory disorders. Appl Microbiol Biotechnol, 2001 Oct, 57(3), 363 - 7 Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme; Goh LL et al.; Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA . Escherichia coli is known to possess two forms of malate synthase, A and G respectively . The recent elucidation of the E . coli malate synthase G crystal structure suggested two residues, Arg338 and Asp631, are essential for catalysis . Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13-18%) . The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy . Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis . Heterologous expression in E . coli followed by purification of the constructed mutant proteins, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality. Nutr Cancer, 2001, 39(2), 259 - 66 Modulation of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine-induced mutation in the cecum and colon of big blue rats by conjugated linoleic acid and 1,2-dithiole-3-thione; Yang H et al.; 2-Amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) is a potent mutagen and suspected human carcinogen present in cooked protein-rich food . It preferentially induced colon tumors in male rats and mammary tumors in female rats . In the present study, the in vivo antimutagenic efficacy of two dietary compounds, conjugated linoleic acid (CLA) and 1,2-dithiole-3-thione (DTT), against PhIP was explored using 1acI transgenic Big Blue rats . Five- or six-week-old male Big Blue rats were fed a diet containing CLA (0.5%, wt/wt) or DTT (0.005%, wt/wt) starting one week before exposure to 200 ppm PhIP for 61 days . PhIP treatment induced a approximately 8- to 16-fold increase in the mutation frequency (MF) in the colon . The induced MF was significantly lower in the cecum than in the proximal and distal colon (approximately 52 x 10(-5) vs . 100 x 10(-5), p < 0.008) . CLA and DTT significantly reduced the PhIP-induced MF in the distal colon (p < 0.05) by 14% and 24%, respectively . The frequency of -1 frameshift mutations was lower in the distal colon of CLA- or DTT-treated rats . This protective effect was not observed in the cecum or in the proximal colon . In contrast, the PhIP-induced MF in the cecum (specifically, the frequency of -1 frameshifts and GC-->TA transversions) was elevated by 43% after treatment with CLA . In conclusion, CLA and DTT modulate PhIP-induced mutagenesis in a tissue-specific manner, and different modulation pathways are employed by CLA and DTT. Fish Shellfish Immunol, 2001 Nov, 11(8), 697 - 709 Rainbow trout (Oncorhynchus mykiss) recombinant IL-1beta and derived peptides induce migration of head-kidney leucocytes in vitro; Peddie S et al.; The present work provides the first information concerning the chemoattractant activity of trout recombinant IL-1beta and its derived peptides, referred to as P1, P2 and P3 . The predicted rainbow trout mature interleukin-1beta peptide was produced as a recombinant protein in Escherichia coli . The first peptide, P1, corresponded to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and had an MW of 137 kDa . It was equivalent to a region known to be part of the receptor binding domain from the mammalian crystal structure of IL-1beta complexed to its receptor . P2 was used as control peptide, consisting of the same 12 amino acids as P1, but arranged in a random sequence (VVEEYIRAPPTT) . P3 was synthesised to complex with an adjacent region of the IL-1 receptor, and corresponded to fragment 207-216 (YRRNTGVDIS) of the trout sequence, with an MW of 1.18 kDa . Migration was stimulated when leucocytes were exposed to concentrations of > or = 10 ng ml(-1) rIL-1beta . Peptide P3 also induced leucocyte migration, with an optimal dose of 0.25 mM being recorded . While P1 had no effect on cell migration when used alone, synergism was evident as a consequence of combining P1 with a suboptimal dose (0.01 mM) of P3 . No synergism occurred when cells were exposed to a combination of P3 and the control peptide P2. Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2259 - 64 Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae; Inanaga H et al.; Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds . Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively . We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua . When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed . Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor . In contrast, BGIT had little effect on the growth of the S . exigua larvae . This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S . exigua larvae . Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance. Zhonghua Jie He He Hu Xi Za Zhi, 2001 Sep, 24(9), 548 - 50 {Construction and immunogenicity of DNA vaccine encoding secreted form of Ag85B protein of Mycobacterium tuberculosis}; Fan X et al.; OBJECTIVE: To construct the recombinant eukaryotic plasmid DNA expression vector encoding Mycobacterium tuberculosis antigen 85B (Ag85B) and to investigate its immunogenicity . METHODS: The gene encoding secreted form of Ag85B was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Ra strain, and was inserted into sites cut with Hind III plus EcoR I of eukaryotic expression vector pcDNA3 after restriction endonuclease digestion . The gene fragment encoding secreted form of Ag85B was inserted into the vector of E . coli JM109 strain and was confirmed by restriction endonuclease digestion . After 4 weeks since BALB/c mice were vaccinated by recombinant eukaryotic expressing vector, dot blotting and ELISA were used to detect the serum antibody against Ag85B and its titer . RESULTS: Recombinant eukaryotic expressing vector, namely pTB30s, constructed successfully based on the gene encoding secreted form of Mycobacterium tuberculosis Ag85B; pTB30s induced high titer specific antibody against Ag85B in immunized mice . CONCLUSION: pTB30s as DNA vaccine should be further studied to confirm its stimulating role in cell-mediated immune responses in TB prevention. Curr Opin Investig Drugs, 2001 Jul, 2(7), 896 - 9 Helivax Antex Biologics; Giudice GD; Antex Biologics is developing an oral vaccine against Helicobacter pylori infection as a potential treatment and prophylaxis for gastric ulcers . The vaccine incorporates a mucosal adjuvant and is in phase II trials {376332} . Enrollment for the trial was completed in September 2000 and results are expected in 2001 {382128} . In July 2000, Antex started a two-part phase Ib/II clinical trial of the vaccine . The first part was an open-label study to assess the general safety of the vaccine in uninfected and asymptomatic Helicobacter pylori-infected individuals . The second part was an expanded placebo-controlled study to evaluate safety and immunogenicity of the vaccine . The vaccine was generally well-tolerated and it generated an immune response in both infected and non-infected individuals {312681}. J Infect Dis, 2002 Jan 1, 185(1), 74 - 84 Epub 2001 Dec 14. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms; Friedrich AW et al.; Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection . Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS . Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004) . The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%) . The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea . None of the 626 STEC isolates contained stx2f . All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c . stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS. J Biol Chem, 2002 Mar 8, 277(10), 8260 - 6 Epub 2001 Dec 26. Activation of human MutS homologs by 8-oxo-guanine DNA damage; Mazurek A et al.; The DNA lesion 8-oxo-guanine (8-oxo-G) is a highly mutagenic product of the interaction between reactive oxygen species and DNA . To maintain genomic integrity, cells have evolved mechanisms capable of removing this frequently arising oxidative lesion . Mismatch repair (MMR) appears to be one pathway associated with the repair of 8-oxo-G lesions (DeWeese, T . L., Shipman, J . M., Larrier, N . A., Buckley, N . M., Kidd, L . R., Groopman, J . D., Cutler, R . G., te Riele, H., and Nelson, W . G . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 11915-11920; Ni, T . T., Marsischky, G . T., and Kolodner, R . D . (1999) Mol . Cell 4, 439-444) . Here we report the effect of double-stranded DNA oligonucleotides containing a single 8-oxo-G on the DNA binding affinity, ATPase, and ADP right arrow ATP exchange activities of hMSH2-hMSH6 and hMSH2-hMSH3 . We found that hMSH2-hMSH6 binds the oligonucleotide DNA substrates with the following affinities: 8-oxo-G/T > 8-oxo-G/G > 8-oxo-G/A > 8-oxo-G/C approximately G/C . A similar trend was observed for DNA-stimulated ATPase and ADP --> ATP exchange activities of hMSH2-hMSH6 . In contrast, hMSH2-hMSH3 did not appear to bind any of the 8-oxo-G containing DNA substrates nor was there enhanced ATPase or ADP --> ATP exchange activities . These results suggest that only hMSH2-hMSH6 is activated by recognition of 8-oxo-G lesions . Our data are consistent with the notion that post-replication MMR only participates in the repair of mismatched 8-oxo-G lesions. J Biol Chem, 2002 Mar 15, 277(11), 8790 - 6 Epub 2001 Dec 26. Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family; Pirch T et al.; To elucidate the functional importance of transmembrane domain II in the Na(+)/proline transporter (PutP) of Escherichia coli we analyzed the effect of replacing Ser-54 through Gly-58 . Substitution of Asp-55 or Met-56 dramatically reduces the apparent affinity for Na(+) and Li(+) in a cation-dependent manner . Conversely, Cys in place of Gly-58 significantly reduces only the apparent proline affinity while substitution of Ser-57 results in a dramatic reduction of the apparent proline and cation affinities . Interestingly, upon increasing the proline concentration the apparent Na(+) affinity of Ser-57 replacement mutants converges toward the wild-type value, indicating a close cooperativity between cation and substrate site(s) . This notion is supported by the fact that Na(+)-stimulated site-specific fluorescence labeling of a single Cys at position 57 is completely reversed by the addition of proline . Similar results are obtained upon labeling of a Cys at position 54 or 58 . Taken together, these results indicate that Asp-55 and Met-56 are located at or close to the ion-binding site while Ser-54, Ser-57, and Gly-58 may be close to the proline translocation pathway . In addition, the data prod at an involvement of the latter residues in ligand-induced conformational dynamics that are crucial for cation-coupled transport. J Biol Chem, 2002 Apr 5, 277(14), 12396 - 405 Epub 2001 Dec 26. Structure and function of threonine synthase from yeast; Garrido-Franco M et al.; Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate . Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target . The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction . The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body . All three domains show the typical open alpha/beta architecture . The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule . Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis . Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form. J Biol Chem, 2002 Mar 1, 277(9), 7239 - 45 Epub 2001 Dec 27. The nucleic acid melting activity of Escherichia coli CspE is critical for transcription antitermination and cold acclimation of cells; Phadtare S et al.; Members of bacterial Csp (cold-shock protein) family promote cellular adaptation to low temperature and participate in many other aspects of gene expression regulation through mechanisms that are not yet fully elucidated . Csp proteins interact with single-stranded nucleic acids and destabilize nucleic acid secondary structures . Some Csp proteins also act as transcription antiterminators in vivo and in vitro . Here, we selected a mutation in the cloned cspE gene that abolished CspE-induced transcription antitermination . In vitro, mutant CspE showed RNA binding activity similar to that of the wild-type CspE but was unable to destabilize nucleic acid secondary structures . Thus, nucleic acid melting ability of CspE and its transcription antitermination activity are correlated . In vivo, mutant cspE was functional with respect to up-regulation of expression of rpoS, but, unlike the wild-type cspE, it did not complement the cold-sensitive phenotype of the quadruple DeltacspADeltacspBDeltacspGDeltacspE deletion strain . Thus, the nucleic acid-melting activity of Csp is critical for its prototypical function of supporting low temperature survival of the cell. J Biol Chem, 2002 Mar 1, 277(9), 7231 - 8 Epub 2001 Dec 27. Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases; Saeed-Kothe A et al.; Carbamoyl phosphate synthetases (CPSs) utilize either glutamine or ammonia for the ATP-dependent generation of carbamoyl phosphate . In glutamine-utilizing CPSs (e.g . the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at a triad-type glutamine amidotransferase domain . Non-glutamine-utilizing CPSs (e.g . rat and human CPS I), lacking the catalytic cysteine residue, can generate carbamoyl phosphate only in the presence of free ammonia . Frog CPS I (fCPS I), unlike mammalian CPS Is, retains most of the glutamine amidotransferase residues conserved in glutamine-utilizing CPSs, including an intact catalytic triad, and could therefore be expected to use glutamine . Our work with native fCPS I provides the first demonstration of the inability of this enzyme to bind/utilize glutamine . To determine why fCPS I is unable to utilize glutamine, we compared sequences of glutamine-using and non-glutamine-using CPSs to identify residues that are present or conservatively substituted in all glutamine-utilizing CPSs but absent in fCPS I . We constructed the site-directed mutants Q273E, L270K, Q273E/N240S, and Q273E/L270K in E . coli CPS and have determined that simultaneous occurrence of the two substitutions, Gln-->Glu and Leu-->Lys, found in the frog CPS I glutamine amidotransferase domain are sufficient to eliminate glutamine utilization by the E . coli enzyme. J Biol Chem, 2002 Mar 8, 277(10), 8579 - 87 Epub 2001 Dec 27. A molecular basis for the selective recognition of 2-hydroxy-dATP and 8-oxo-dGTP by human MTH1; Sakai Y et al.; MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-dATP, 2-hydroxy-dATP, and 2-hydroxy rATP to monophosphates, and thus avoids errors caused by their misincorporation during DNA replication or transcription, which may result in carcinogenesis or neurodegeneration . This substrate specificity for oxidized purine nucleoside triphosphates was investigated by mutation analyses based on the sequence comparison with the Escherichia coli homolog, MutT, which hydrolyzes only 8-oxo-dGTP and 8-oxo-rGTP but not oxidized forms of dATP or ATP . Neither a replacement of the phosphohydrolase module of MTH1 with that of MutT nor deletions of the C-terminal region of MTH1, which is unique for MTH1, altered the substrate specificity of MTH1 . In contrast, the substitution of residues at position Trp-117 and Asp-119 of MTH1, which showed apparent chemical shift perturbations with 8-oxo-dGDP in NMR analyses but are not conserved in MutT, affected the substrate specificity . Trp-117 is essential for MTH1 to recognize both 8-oxo-dGTP and 2-hydroxy-dATP, whereas Asp-119 is only essential for recognizing 2-hydroxy-dATP, thus suggesting that origins of the substrate-binding pockets for MTH1 and MutT are different. J Biotechnol, 2002 Feb 28, 93(3), 283 - 8 Enhancing the sensitivity of a two-stage continuous toxicity monitoring system through the manipulation of the dilution rate; Gu MB et al.; Optimization of the dilution rates has been studied to provide an enhanced sensitivity to toxicity by several recombinant bioluminescent Escherichia coli strains, TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE) and DPD2540 (fabA::luxCDABE), in the two-stage continuous toxicity monitoring system . It was found that the sensitivity of both TV1061 and DPD2794 to a pulse injection of phenol and mitomycin C increased with a decrease in the dilution rate . The sensitivity, however, for all the strains to step injections of the toxic chemicals was found to increase with an increase in the dilution rate up to a certain dilution rate and then decreased, mainly due to the rapid washing out of the injected chemicals . The response kinetics of the strains were explained by evaluating the mode of action of the recombinant bioluminescent bacteria to toxicity with the dilution rate, the operating parameter of minibioreactors under consideration in this study. FEBS Lett, 2002 Jan 2, 510(1-2), 83 - 8 Anti-angiogenic activity of a novel multi-substrate analogue inhibitor of thymidine phosphorylase; Liekens S et al.; 7-Deazaxanthine (7-DX) was recently identified as the first purine derivative with pronounced inhibitory activity against Escherichia coli thymidine phosphorylase (TP) and angiogenesis . In order to "freeze" the enzyme in an open, inactive conformation, a novel multi-substrate analogue inhibitor of TP, containing an alkyl phosphonate moiety covalently linked to 7-DX, was synthesized . The prototype compound TP65 (9-(8-phosphonooctyl)-7-deazaxanthine) (at 250 microM) completely inhibited TP-induced formation of microvascular sprouts from endothelial cell aggregates in a three-dimensional fibrin gel . In the chick chorioallantoic membrane assay, TP caused a dose-dependent stimulation of angiogenesis, which was completely inhibited by 250 nmol TP65 . This dose proved to be non-toxic for the developing chick embryo . TP65 thus emerges as a potent and specific inhibitor of TP and TP-induced angiogenesis, which opens new perspectives for multi-substrate analogue inhibitors of TP as potential anti-cancer agents and as inhibitors of angiogenesis and of diseases with enhanced expression of TP. FEBS Lett, 2002 Jan 2, 510(1-2), 17 - 21 The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity; de Keyzer J et al.; SecYEG forms the protein-conducting channel of the Escherichia coli translocase . It binds the peripheral ATPase SecA that drives the preprotein translocation reaction . PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence . The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes . SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4 . This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity . SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity . These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity. FEBS Lett, 2002 Jan 2, 510(1-2), 10 - 2 Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase; Muranaka N et al.; Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system . Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity . The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form . On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form . In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained. Acta Trop, 2002 Jan, 81(1), 1 - 5 Protozoon infections and intestinal permeability; Dagci H et al.; Intestinal permeability (IP) studies using some macromolecules have been assumed to demonstrate the intactness of intestinal mucosa . The aim of the present study is to determine the changes in IP among patients with protozoan infections . Thirty nine patients with protozoan infections and ten healthy controls were enrolled in the study . Protozoa were diagnosed by Native-lugol, Richie and Trichrome staining of faeces . IP was evaluated by diethyl triamine penta acetic acid labeled with 99m Technetium (99mTc labeled DTPA) assay . The IP was found to have increased in patients with protozoan infections compared with control patients (7.20+/-5.52 vs . 4.47+/-0.65%, P=0.0017) . The IP values were 9.91+/-10.05% in Giardia intestinalis group, 6.81+/-2.25% in Blastocystis hominis group, 5.78+/-2.84% in Entamoeba coli group . In comparison with the control group, the IP was significantly higher in G . intestinalis and B . hominis patients (P=0.0025, P=0.00037, respectively), but not in E . coli patients . In conclusion, the IP increases in patients with G . intestinalis and B . hominis but not with E . coli infection . This finding supports the view that IP increases during the course of protozoan infections which cause damage to the intestinal wall while non-pathogenic protozoan infections have no effect on IP . The increase in IP in patients with B . hominis brings forth the idea that B . hominis can be a pathogenic protozoan. Biochim Biophys Acta, 2001 Dec 19, 1541(3), 170 - 8 In situ detection of ALA-stimulated porphyrin metabolic products in Escherichia coli B by fluorescence line narrowing spectroscopy; Szocs K et al.; In a recent work {Photochem . Photobiol . B: Biol . 50 (1999) 8} the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on delta-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation . In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques . Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed . We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium . Incorporation of Zn ions originating from the glassware could also be supposed . In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins . The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins . The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population. Biochim Biophys Acta, 2001 Dec 17, 1550(2), 164 - 74 DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein; Kawaguchi M et al.; The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs . It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases . In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule . A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity . We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75 . AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity . The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity . Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA . In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA . These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription . The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs. Mol Biochem Parasitol, 2002 Jan, 119(1), 97 - 106 Molecular cloning of Trypanosoma brucei CK2 catalytic subunits: the alpha isoform is nucleolar and phosphorylates the nucleolar protein Nopp44/46; Park JH et al.; We have demonstrated previously that N