Escherichia coli

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 Nopp44/46, an abundant nucleolar phosphoprotein of Trypanosoma brucei, is associated with a protein kinase . In many organisms multiple nucleolar proteins are phosphorylated by the protein kinase CK2, formerly known as casein kinase II . Here we report the identification of two T . brucei genes, CK2a1and CK2a2, which encode protein kinases bearing signature motifs common to CK2 catalytic subunits . The protein specified by CK2a1, designated CK2alpha, was capable of associating with Nopp44/46 as assessed by yeast two-hybrid analysis . An epitope-tagged version of CK2alpha expressed in T . brucei colocalized with Nopp44/46, with a largely nucleolar localization . This localization contrasts with the predominantly nuclear localization of mammalian CK2 . When expressed in Escherichia coli, TbCK2alpha was catalytically active and phosphorylated Nopp44/46 . Together these data demonstrate that TbCK2alpha is a Nopp44/46-associated kinase . Competition assays revealed that, unlike most CK2s, TbCK2alpha discriminates highly between ATP and GTP . This distinction may be associated with the substitution of glutamic acid and alanine for the di-asparagine motif thought to participate in purine interaction.

Mol Biochem Parasitol, 2002 Jan, 119(1), 63 - 73
Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax; Leartsakulpanich U et al.; The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli . The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined . Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme . The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were approximately 60 to >4000 times less active against the mutant enzymes . In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred approximately 4000- and approximately 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR . The S58R+S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Km value than the single mutant . The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants . Data on kinetic parameters and inhibitory constant suggest that the wild-type P . vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P . vivax are responsible for antifolate resistance.

Neurobiol Aging, 2002 Jan-Feb, 23(1), 47 - 53
Elevated levels of oxidative DNA damage in lymphocytes from patients with Alzheimer's disease; Morocz M et al.; Previous studies have provided evidence of the involvement of oxidative damage in the pathogenesis of Alzheimer's disease (AD) . Although the role of oxidative stress in the aetiology of the disease is still not clear, the detection of an increased damage status in the cells of patients could have important therapeutic implications . The level of oxidative damage and repair capacity in peripheral lymphocytes of AD patients and of age-matched controls was determined by the Comet assay applied to freshly isolated blood samples with oxidative lesion-specific DNA repair endonucleases . This is less prone to errors arising from oxidative artifacts than chemical analytical methods; and is therefore a relatively reliable, as well as rapid method for assay of oxidative DNA damage in cells . Statistically significant elevations (P < 0.05) of oxidized purines were observed in nuclear DNA of peripheral lymphocytes from AD patients, compared to age matched control subjects, both at basal level and after oxidative stress induced by H(2)O(2.) AD patients also showed a diminished repair of H(2)O(2) -induced oxidized purines.

J Biochem (Tokyo), 2002 Jan, 131(1), 145 - 51
Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli; Fujihira E et al.; AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC . In this study, acrAB genes were cloned from the E . coli W3104 chromosome . To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced . The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance . We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB . The binding of {(14)C}N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated . The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane . The results supported the 12-membrane-spanning structure of AcrB . Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.

J Biochem (Tokyo), 2002 Jan, 131(1), 121 - 9
Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans; Satoh K et al.; RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans . However, it is still unclear how RecA contributes to the resistance . In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670 . The properties of the gene products from the recA mutants were compared . recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670 . In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity . RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not . The intracellular LexA level in KI696 was decreased following gamma-irradiation . However, the LexA level in strain rec30 was constant irrespective of irradiation . These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not . While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive . Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D . radiodurans.

J Biochem (Tokyo), 2002 Jan, 131(1), 59 - 69
Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase . The catalytic and structural roles of Gly313 and Thr317; Setoyama C et al.; According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD {Miura et al . (1997) J . Biochem . 122, 825-833} . We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type . The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type . The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase . The T317A mutant shows a decreased affinity for FAD . The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids . The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.

J Nat Prod, 2001 Dec, 64(12), 1568 - 71
Antifungal alkyl amino alcohols from the tropical marine sponge Haliclona n . sp; Clark RJ et al.; Three new amino alcohols presumably deriving from L-alanine were isolated from the tropical marine sponge Haliclona n . sp . and characterized by 2D NMR, while a fourth amino alcohol was characterized as an acetamide derivative . Relative stereochemistry was deduced from the NMR characteristics of oxazolidinone derivatives and absolute stereochemistry secured by preparation and analysis of an MPA ester . The amino alcohol fraction from Haliclona n . sp . acts as an antifungal agent and inhibits the development of larvae of the ascidian Herdmania curvata.

Biotechnol Bioeng, 2002 Feb 5, 77(3), 329 - 39
Characterization of aqueous two-phase partition systems by distribution analysis of radiolabeled analytes (DARA): application to process definition and control in biorecovery; Lebreton B et al.; In this article, we describe a characterization method applicable to aqueous two-phase systems (ATPS) heavily loaded with complex biological feed-stocks . We also studied the partition behavior of mixtures of traceable and quantifiable radiolabeled amino acids, selected on the basis of their relative hydrophobicity A unique linear relation was established between the tie-line length (TLL: commonly determined by graphical methods) and the hydrophobic factor (HF) for ATPS comprising potassium phosphate and PEG alone, and validated for polymer molecular weights from 300 to 8000 Da in systems operated at an apparent pH value of 7.5 . Radiolabeled amino acids were subsequently applied to the characterization of ATPS loaded with whole bovine blood by the determination of effective tie-line lengths (TLL(e)) . The addition of biomass to ATPS increased TLL(e) relative to that of blank ATPS of equivalent original composition of PEG and phosphate . In addition, an increase of biomass loading (variously sourced from blood, yeast, and E . coli) contributed to phase formation and stabilization of loaded ATPS in respect of system sensitivity toward operational conditions . The controlled application of sensitive ATPS (adjacent to the binodal curve) could thus be reconsidered for further application of aqueous two-phase partitioning as a primary purification process . The application of effective tie-line determinations by distribution analysis of radiolabeled analytes (DARA) as a process-aid in the design and operation of ATPS in biorecovery is discussed .

Biotechnol Bioeng, 2002 Jan 20, 77(2), 142 - 7
Stabilizing plasmid copy number to improve recombinant protein production; Grabherr R et al.; The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield . If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield . Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell . The transcriptional and translational machineries are extremely overstrained . By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system . Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization .

Zentralbl Gynakol, 2001 Oct, 123(10), 595 - 8
{Paravaginal abscess in pregnancy}; Hasbargen U et al.; Paravaginal abscess in pregnancy.We report the diagnosis and treatment of an infected Gartner's duct cyst during pregnancy . The patient presented with lower abdominal pain, fever (38.5 degrees C) and an elevated C-reactive Protein level . Pelvic examination revealed a painful paravaginal mass . Sonography was not able to detect the cranial border of the tumor . Magnetic resonance imaging (MRI) revealed fluid accumulation laterodorsal to the vagina without evidence of a connection with the retroperitoneal space . An infected Gartner's duct cyst with consecutive abscess formation along the mesonephric duct system, was diagnosed . Following transvaginal drainage, the remainder of the pregnancy was uneventful and the patient was delivered vaginally at 40 + 5 weeks without complications . - The rare clinical finding of a paravaginal abscess in pregnancy was treated without termination of the pregnancy . Preoperative planning of the surgical approach using MRI can be easier for pelvic processes extending out of the pelvis than using ultrasound and is less painful for the patient.

Nat Genet, 2002 Jan, 30(1), 31 - 9 Epub 2001 Dec 20.
Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step; Zhang P et al.; We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs . By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form . This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods . In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct . Conditional targeting constructs can be generated easily with minimal additional manipulation.

Nat Biotechnol, 2002 Jan, 20(1), 76 - 81
Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs; Murakami H et al.; A general method was developed for the construction of a library of mutant genes . The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position . The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene . In addition, the randomly selected three bases were replaced by a mixture of 20 codons . These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected . A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected . Several mutants of the DsRed protein that show different fluorescence properties were also obtained.

J Endotoxin Res, 2001, 7(6), 451 - 5
Complement-dependent platelet degradation and anaphylactoid shock in mice induced by lipopolysaccharide carrying the mannose homopolymer; Endo Y et al.; Intravenous injection of specified bacterial lipopolysaccharides (LPSs) induced anaphylactoid shock in mice of various strains, including LPS-resistant C3H/HeJ . The reaction was accompanied by occasional mortality of mice within 1 h . Prior to shock, rapid accumulation of blood platelets in the lungs and liver followed by degradation of platelets (or release of their contents) and tissue destruction were observed . In this study, LPS specimens carrying mannose-homopolymer (MHP), which markedly activate the human complement system through the lectin pathway, induced marked platelet degradation and anaphylactoid shock in BALB/c mice . In contrast, in C5-deficient DBA/2 mice, the platelet degradation and anaphylactoid reactions did not occur . Anti-complement agent K-76 COOH (C5 inhibitor) protected BALB/c mice from mortality in the anaphylactoid reaction . K-76 COOH also inhibited platelet degradation, but not accumulation, induced by LPS in mice . Based on these findings, we postulated that strong complement activation by specified LPS preparations induced degradation of platelets that have accumulated in the lungs and liver, resulting in acute inflammation accompanied by severe tissue destruction, especially in the lungs, which in turn leads to anaphylactoid reaction.

J Endotoxin Res, 2001, 7(5), 385 - 8
Human models of innate immunity: local and systemic inflammatory responses; Fiuza C et al.; We review human studies where different body sites (e.g . systemic--intravenous and local--skin or lung) are exposed to small amounts of bacterial components as a means to study innate immunity in vivo . Intravenous endotoxin administration is widely used to assess systemic inflammatory responses, and these have many similarities to those seen in early sepsis . While blood levels of cytokines, activated inflammatory cells, and stress hormones rise acutely, the alveolar space remains relatively protected from these inflammatory responses . Skin blister windows provide a means to study local neutrophil exudation without systemic inflammatory responses, and has been used to characterize defects in neutrophil transmigration . Recently, skin blister windows have been adapted to study phagocytic cell function in response to bacterial antigens in patients with cirrhosis . Inhalation of endotoxin leads to pulmonary inflammation with increases in broncho-alveolar lavage neutrophils and cytokines and mild systemic responses . Whole lung exposure to endotoxin provides a means to study the pathogenesis of occupational lung disease . These three models are important methods to study innate immune responses and their regulatory mechanisms in normal and diseased states.

J Endotoxin Res, 2001, 7(5), 359 - 64
Vagotomy attenuates the effect of lipopolysaccharide on body temperature of rats in a dose-dependent manner; Azab AN et al.; There is a growing body of evidence suggesting that vagal afferents play a major role in peripheral-neural communication . This study was undertaken to determine whether a dose-dependent effect of lipopolysaccharide (LPS) on vagotomy-induced febrile unresponsiveness exists, and to examine the effect of vagotomy on LPS-induced increase in hypothalamic prostaglandin E2 (HT PGE2) production . Vagotomized and sham-operated rats were subjected to two experimental protocols . In the first, vagotomized and sham-operated rats were injected intraperitoneally with different doses of LPS (200, 500 and 1000 microg/kg) in order to examine the dose-dependent effect of LPS on the biphasic febrile response of the rats . In the second protocol, vagotomized and sham-operated rats were injected intraperitoneally with LPS (500 microg/kg) . Two hours post injection, body temperature was measured, the rats were decapitated and blood was collected . Simultaneously, the rats' hypothalami were excised and incubated for 1 h in a Krebs-Henseleit buffer . Next, HT PGE2 was determined by radioimmunoassay . Vagotomy-induced gastric enlargement was then measured to examine the correlation between the magnitude of the enlargement and that of the vagotomy-related febrile unresponsiveness . It was found that vagotomized-induced febrile unresponsiveness is a dose-dependent effect . Subdiaphragmatic resection of the vagus prevented the biphasic febrile response caused by the lowest dose (200 microg/kg) of LPS, whereas the highest dose of LPS (1000 microg/kg) caused a similar biphasic febrile response in both vagotomized and sham-operated rats . Indeed, vagotomy attenuates LPS-induced increase in HT PGE2, and blocks the hypothermic phase of the febrile response . On the other hand, no correlation between gastric enlargement and febrile unresponsiveness was found . The results of the present study may cast further light on the crucial role of the vagus nerve as a peripheral-neural pathway in the mediation of the febrile response.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 160 - 2 Epub 2001 Dec 21.
Purification and crystallization of the respiratory complex formate dehydrogenase-N from Escherichia coli; Jormakka M et al.; A membrane-protein complex, formate dehydrogenase-N from Escherichia coli, has been purified and crystallized . This molybdenum-containing enzyme, composed of alpha, beta and gamma subunits, is the major electron donor to the nitrate respiratory chain of E . coli . The formate dehydrogenase-N crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 203 A . An asymmetric unit of the crystals is assumed to contain one formate dehydrogenase-N monomer (MW 170 kDa) . One data set to 1.6 A resolution, with 342 711 independent observations (94.4% complete) and an R(merge) of 0.08, has been collected from a single crystal . This is the highest resolution data set reported for a membrane-protein complex to date.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 157 - 9 Epub 2001 Dec 21.
Crystallization and preliminary X-ray diffraction studies of catalase-peroxidase from Synechococcus PCC 7942; Wada K et al.; The recombinant catalase-peroxidase of Synechococcus PCC 7942 overexpressed in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using sodium formate as a precipitant . The crystals belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.3, c = 202.0 A . The calculated V(M) value based on a dimer in the asymmetric unit was 1.9 A(3) Da(-1) . A native data set was collected to 2.3 A resolution from a frozen crystal using synchrotron radiation at SPring-8.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 130 - 2 Epub 2001 Dec 21.
Crystallization and preliminary crystallographic studies of five crystal forms of Escherichia coli L-asparaginase II (Asp90Glu mutant); Kozak M et al.; L-Asparaginase II from Escherichia coli with an Asp90Glu mutation in the active site has been crystallized in five polymorphic forms . Crystals of all polymorphs suitable for X-ray diffraction experiments were obtained by the vapour-diffusion method . Crystals of form I belong to the monoclinic system (space group C2), have unit-cell parameters a = 73.1, b = 133.1, c = 62.6 A, beta = 108.8 degrees and diffract to 2.27 A resolution . Three of the crystal forms are orthorhombic, with unit-cell parameters a = 225.4, b = 128.0, c = 62.6 A (form II, P2(1)2(1)2), a = 59.9, b = 71.2, c = 130.6 A (form III, primitive cell) and a = 73.8, b = 122.1, c = 124.2 A (form IV, P2(1)2(1)2(1) or P2(1)2(1)2); the crystals diffract to 2.33, 3.5 and 1.7 A, respectively . Polymorph V is trigonal, space group P3(1)21, with unit-cell parameters a = 123.1, c = 83.8 A; the crystals diffract to 2.65 A resolution.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 127 - 9 Epub 2001 Dec 21.
Crystallization and preliminary crystallographic analysis of a metastasis-inducing protein, human S100A4; Zhang H et al.; A metastasis-inducing protein, human S100A4, cDNA was overexpressed in Escherichia coli . The recombinant protein was purified by three chromatographic procedures . Crystals were obtained using polyethylene glycol as a precipitant . The native S100A4 crystals did not diffract sufficiently well for data collection, but the SeMet S100A4 crystal, grown using similar conditions as for the native, was found to be stable during exposure to X-rays and diffracted to 4 A resolution in-house . The crystal belongs to space group P6 or P3 . The unit-cell parameters are a = b = 47.1, c = 175.6 A, alpha = beta = 90, gamma = 120 degrees . Complete data will be collected using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 60 - 9 Epub 2001 Dec 21.
Low-resolution detergent tracing in protein crystals using xenon or krypton to enhance X-ray contrast; Sauer O et al.; Xenon and krypton show different solubilities in polar versus apolar solvents . Therefore, these noble gases should accumulate in apolar regions of protein crystals . Specifically, they should accumulate in lipid and detergent solvent regions within crystals of membrane proteins, which can be used as a basis for contrast-variation experiments to distinguish such apolar solvent regions from the aqueous phase by a low-resolution X-ray diffraction experiment . This possibility was explored with the OmpF porin, one of the general diffusion pores of the Escherichia coli outer membrane . Trigonal crystals were exposed to elevated pressures of the two noble gases (up to 10(7) Pa) for several minutes and subsequently flash-cooled to liquid-nitrogen temperatures . Both rare gases bind to a number of 'specific' sites, which can be classified as 'typical' noble-gas binding sites . Compared with a representative water-soluble protein, they are however much more abundant in OmpF . In addition, a very large number of weakly populated sites are observed which accumulate in the region of the 'detergent belt' for crystals exposed to xenon . After application of a Fourier-filtering protocol, low-resolution images of the detergent belt can be obtained . The resulting maps are similar to maps obtained from low-resolution neutron diffraction experiments on contrast-matched crystals.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 29 - 38 Epub 2001 Dec 21.
The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'; Cobessi D et al.; Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 A resolution . This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point-group symmetry like ferritin and bacterioferritin from Escherichia coli . Crystallized in the I422 space group, its structural analysis reveals for the first time the non-symmetric heme molecule located on a twofold crystallographic symmetry axis . Other hemes of the protomer are situated on twofold noncrystallographic axes . Apparently, both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal . Five water molecules are situated close to the heme, which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues . Its ferroxidase center, in which Fe(II) is oxidized to Fe(III), is empty or fractionally occupied by a metal ion . Two positions are observed for the coordinating Glu18 side chain instead of one in the E . coli enzyme in which the site is occupied . This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 10 - 20 Epub 2001 Dec 21.
Structural flexibility, an essential component of the allosteric activation in Escherichia coli glucosamine-6-phosphate deaminase; Rudino-Pinera E et al.; A new crystallographic structure of the free active-site R conformer of the allosteric enzyme glucosamine-6-phosphate deaminase from Escherichia coli, coupled with previously reported structures of the T and R conformers, generates a detailed description of the heterotropic allosteric transition in which structural flexibility plays a central role . The T conformer's external zone {Horjales et al . (1999), Structure, 7, 527-536} presents higher B values than in the R conformers . The ligand-free enzyme (T conformer) undergoes an allosteric transition to the free active-site R conformer upon binding of the allosteric activator . This structure shows three alternate conformations of the mobile section of the active-site lid (residues 163-182), in comparison to the high B values for the unique conformation of the T conformer . One of these alternate R conformations corresponds to the active-site lid found when the substrate is bound . The disorder associated with the three alternate conformations can be related to the biological regulation of the K(m) of the enzyme for the reaction, which is metabolically required to maintain adequate concentrations of the activator, which holds the enzyme in its R state . Seven alternate conformations for the active-site lid and three for the C-terminus were refined for the T structure using isotropic B factors . Some of these conformers approach that of the R conformer in geometry . Furthermore, the direction of the atomic vibrations obtained with anisotropic B refinement supports the hypothesis of an oscillating rather than a tense T state . The concerted character of the allosteric transition is also analysed in view of the apparent dynamics of the conformers.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14837 - 42
Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein; Hecht S et al.; Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with (13)C-labeled 1-deoxy-D-xylulose . Cell extracts were analyzed directly by NMR spectroscopy . (13)C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF . The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway . Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 19 - 24 Epub 2001 Dec 18.
Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation; Kiick KL et al.; The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies . One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus . Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures . We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents . Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

Nucleic Acids Res, 2002 Jan 1, 30(1), 56 - 8
The EcoCyc Database; Karp PD et al.; EcoCyc is an organism-specific pathway/genome database that describes the metabolic and signal-transduction pathways of Escherichia coli, its enzymes, its transport proteins and its mechanisms of transcriptional control of gene expression . EcoCyc is queried using the Pathway Tools graphical user interface, which provides a wide variety of query operations and visualization tools . EcoCyc is available at http://ecocyc.org/.

Mol Biol Evol, 2002 Jan, 19(1), 58 - 67
Protein structure, neighbor effect, and a new index of amino acid dissimilarities; Xia X et al.; Amino acids interact with each other, especially with neighboring amino acids, to generate protein structures . We studied the pattern of association and repulsion of amino acids based on 24,748 protein-coding genes from human, 11,321 from mouse, and 15,028 from Escherichia coli, and documented the pattern of neighbor preference of amino acids . All amino acids have different preferences for neighbors . We have also analyzed 7,342 proteins with known secondary structure and estimated the propensity of the 20 amino acids occurring in three of the major secondary structures, i.e., helices, sheets, and turns . Much of the neighbor preference can be explained by the propensity of the amino acids in forming different secondary structures, but there are also a number of intriguing association and repulsion patterns . The similarity in neighbor preference among amino acids is significantly correlated with the number of amino acid substitutions in both mitochondrial and nuclear genes, with amino acids having similar sets of neighbors replacing each other more frequently than those having very different sets of neighbors . This similarity in neighbor preference is incorporated into a new index of amino acid dissimilarities that can predict nonsynonymous codon substitutions better than the two existing indices of amino acid dissimilarities, i.e., Grantham's and Miyata's distances.

J Virol, 2002 Jan, 76(2), 560 - 8
CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G); McNeal MM et al.; Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM {epizootic diarrhea of infant mice virus}) shedding for at least 1 year . The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate . Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected . Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection . Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection . As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM . Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days . Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required . Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).

J Biol Chem, 2002 Mar 1, 277(9), 7178 - 82 Epub 2001 Dec 21.
The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA; Nonekowski ST et al.; tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA . The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs . Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F . L., Nonekowski, S., and Garcia, G . A . (2000) RNA 6, 233-244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S . T., and Garcia, G . A . (2001) RNA 7, 1432-1441) . The TGT reaction involves the breakage and re-formation of a glycosidic bond . To rule out a potential chemical mechanism involving the 2'-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2'-deoxy-G at 34 . The high level of activity exhibited by this analogue indicates that the 2'-hydroxyl of G(34) is not required for catalysis . Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2'-deoxyuridines replace the thymidines in the DNA . The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT . However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide.

J Biol Chem, 2002 Mar 1, 277(9), 7282 - 6 Epub 2001 Dec 21.
Residues 137 and 153 at the N terminus of the XylS protein influence the effector profile of this transcriptional regulator and the sigma factor used by RNA polymerase to stimulate transcription from its cognate promoter; Ruiz R et al.; The 321-residue XylS and XylS1 proteins, encoded by the pWW0 and pWW53 plasmids respectively, differ in only 5 residues at positions 4, 53, 90, 137, and 153 . As a result, the effector profile of XylS is wider than that of XylS1, and XylS mediates higher levels of transcription from its cognate-regulatable promoter than does XylS1 . We generated a series of XylS-pWW0 mutants and found that the single mutants Asp-137-->Glu and His-153-->Asn exhibited an activation pattern different from that of the wild-type regulator . In the double-mutant XylSD137E,H153N the effector profile for benzoates was similar to that of XylS1 . This suggests that these two residues are crucial for effector recognition and regulator activation to stimulate transcription . XylS-dependent transcription from its cognate promoter is mediated by RNA polymerase with sigma(32) or sigma(38), whereas XylS1 uses RNA polymerase with sigma(32) or sigma(70) . We also found that point mutations at positions 137 and 153 of XylS led RNA polymerase to mediate transcription with sigma(70) rather than with sigma(38), as demonstrated by primer extension analysis in a sigma(70)-thermosensitive background proficient and deficient in sigma(38) . This suggests that a positive transcriptional regulator can choose the RNA polymerase complex that mediates transcription from a given promoter.

J Biol Chem, 2002 Mar 8, 277(10), 7882 - 8 Epub 2001 Dec 20.
Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling; Zhao R et al.; PZR is an immunoglobulin superfamily cell surface protein containing a pair of immunoreceptor tyrosine-based inhibitory motifs . As a glycoprotein, PZR displays a strong association with concanavalin A (ConA), a member of the plant lectin family . Treatment of several cell lines with ConA caused tyrosine phosphorylation of a major cellular protein . Immunoblotting and immunoprecipitation studies indicated that this protein corresponded to PZR . Tyrosine phosphorylation of PZR was accompanied by recruitment of SHP-2 and was inhibited by PP1, a selective inhibitor of the Src family tyrosine kinases . Furthermore, c-Src was constitutively associated with PZR and was activated upon treatment of cells with ConA . Moreover, tyrosine phosphorylation of PZR was markedly enhanced in v-Src-transformed NIH-3T3 cells and was predominant in Escherichia coli cells co-expressing c-Src . Expression of an intracellular domain-truncated form of PZR in HT-1080 cells affected cell morphology and had a dominant negative effect on ConA-induced tyrosine phosphorylation of PZR, activation of c-Src, and agglutination of the cells . Together, the data indicate that PZR is a major receptor of ConA and has an important role in cell signaling via c-Src . Considering the various biological activities of ConA, the study of PZR may have major therapeutic implications.

J Biol Chem, 2002 Mar 1, 277(9), 7127 - 35 Epub 2001 Dec 20.
Tyrosine phosphorylation of protein kinase Wzc from Escherichia coli K12 occurs through a two-step process; Grangeasse C et al.; In bacteria, several proteins have been shown to autophosphorylate on tyrosine residues, but little is known on the molecular mechanism of this modification . To get more information on this matter, we have analyzed in detail the phosphorylation of a particular autokinase, protein Wzc, from Escherichia coli K12 . The analysis of the hydropathic profile of this protein indicates that it is composed of two main domains: an N-terminal domain, including two transmembrane alpha-helices, and a C-terminal cytoplasmic domain . The C-terminal domain alone can undergo autophosphorylation and thus appears to harbor the protein-tyrosine kinase activity . By contrast, the N-terminal domain is not phosphorylated when incubated either alone or in the presence of the C-domain, and does not influence the extent of phosphorylation of the C-domain . The C-domain contains six different sites of phosphorylation . Among these, five are located at the C-terminal end of the molecule in the form of a tyrosine cluster (Tyr(708), Tyr(710), Tyr(711), Tyr(713), and Tyr(715)), and one site is located upstream, at Tyr(569) . The Tyr(569) residue can autophosphorylate through an intramolecular process, whereas the tyrosine cluster cannot . The phosphorylation of Tyr(569) results in an increased protein kinase activity of Wzc, which can, in turn, phosphorylate the five terminal tyrosines through an intermolecular process . It is concluded that protein Wzc autophosphorylates by using a cooperative two-step mechanism that involves both intra- and interphosphorylation . This mechanism may be of biological significance in the signal transduction mediated by Wzc.

J Biol Chem, 2002 Mar 8, 277(10), 7670 - 5 Epub 2001 Dec 20.
Direct interaction of YidC with the Sec-independent Pf3 coat protein during its membrane protein insertion; Chen M et al.; YidC is a newly defined translocase component that mediates the insertion of proteins into the membrane bilayer . How YidC functions in the insertion process is not known . In this study, we report that the Sec-independent Pf3 coat protein requires the YidC protein specifically for the membrane translocation step . Using photocrosslinking techniques and ribosome-bound Pf3 coat derivatives with an extended carboxyl-terminal region, we found that the transmembrane region of the Pf3 coat protein physically interacts with YidC and the bacterial signal recognition particle Ffh component . We also find that in the insertion pathway, Pf3 coat interacts strongly with YidC only after its transmembrane segment is fully exposed outside the ribosome tunnel . Interaction between Pf3 coat and YidC occurs even in the absence of the proton motive force and with a Pf3 coat mutant that is defective in transmembrane insertion . Our study demonstrates that YidC can directly interact with a Sec-independent membrane protein, and the role of YidC is at the stage of folding the Pf3 protein into a transmembrane configuration.

J Biol Chem, 2002 Mar 1, 277(9), 7108 - 17 Epub 2001 Dec 20.
Structural plasticity in influenza virus protein NS2 (NEP); Lommer BS et al.; The cellular nuclear transport machinery relies on the assembly of specialized transport complexes between soluble transport receptors, transport substrates, and additional accessory proteins . This study focuses on the structural characteristics of influenza virus protein NS2 (NEP), which interacts with the nuclear export machinery during viral replication, and has been proposed to act as an adapter molecule between the nuclear export machinery and the viral ribonucleoprotein complex . For this purpose, we have purified recombinant NS2 under nondenaturing conditions, and have investigated its structure and aggregation state using optical spectroscopy, differential scanning calorimetry, as well as hydrodynamic techniques . Our results indicate that isolated NS2 exists as a monomer in solution, and adopts a compact, but very flexible conformation, which shows characteristics of the molten globule state under near physiological conditions . Proteolytic sensitivity suggests that, despite its overall plasticity, the structure of NS2 is heterogeneous . While the C terminus of the protein adopts a relatively rigid conformation, its N terminus, which is recognized by the nuclear export machinery, exists in a highly mobile and exposed state . It is proposed that the flexibility observed in the nuclear export domain of NS2 is an important element in the recognition of substrate proteins by the nuclear export machinery.

J Biol Chem, 2002 Mar 1, 277(9), 7092 - 8 Epub 2001 Dec 20.
Binding and invasion of liver cells by Plasmodium falciparum sporozoites . Essential involvement of the amino terminus of circumsporozoite protein; Rathore D et al.; Plasmodium sporozoites display circumsporozoite (CS) protein on their surface, which is involved in the attachment of sporozoites to liver cells . CS protein is a member of the thrombospondin type I repeat (TSR) domain family and possess a single copy of TSR domain toward its carboxyl terminus . We show by a direct measurement the correlation between the binding activity of various segments of the CS protein and their ability to inhibit the invasion of liver cells by the sporozoites . We made eight truncated versions of Plasmodium falciparum CS protein to elucidate the role of various regions in the binding and invasion process . Deletion of the TSR domain actually enhanced binding activity by 2-3-fold without the loss of receptor specificity, indicating that TSR may not be the only domain in defining the specificity of binding . These same deletions blocked invasion of live sporozoites more efficiently than proteins that include the TSR domain . Deletion of as little as six amino acids from amino terminus of the protein, however, renders it incapable of binding to liver cells and as an inhibitor of sporozoite invasion . Hence, the binding of CS protein to liver cells and its ability to inhibit the invasion process are affected in a parallel manner, both positively and negatively, by sequence changes in the encoded CS gene . This indicates that both assays are measuring interrelated phenomenon and points to the essential involvement for the amino-terminal portion of the CS protein in these processes.

J Biol Chem, 2002 Mar 8, 277(10), 7664 - 9 Epub 2001 Dec 19.
Erythroid expression and oligomeric state of the AQP3 protein; Roudier N et al.; Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used . Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea . At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells . Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3 . By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure . Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure . Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.

J Biol Chem, 2002 Mar 8, 277(10), 8172 - 7 Epub 2001 Dec 18.
Flavorubredoxin, an inducible catalyst for nitric oxide reduction and detoxification in Escherichia coli; Gardner AM et al.; Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects . In Escherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification . Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification . Mutation of either ygaA or ygaK eliminated inducible anaerobic NO metabolism, whereas ygbD disruption partly impaired the activity . NO-sensitive {4Fe-4S} (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK or ygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions . flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E . coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions . We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVW modulon for NO reduction and detoxification.

J Biol Chem, 2002 Mar 1, 277(9), 7059 - 65 Epub 2001 Dec 18.
Negative regulation of toll-like receptor-mediated signaling by Tollip; Zhang G et al.; Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity and disease resistance . The adaptor protein Tollip was identified initially as an intermediate in interleukin (IL)-1 signaling . Here we report that Tollip also associates directly with TLR2 and TLR4 and plays an inhibitory role in TLR-mediated cell activation . Inhibition by Tollip is mediated through its ability to potently suppress the activity of IL-1 receptor-associated kinase (IRAK) after TLR activation . In addition, we show for the first time that Tollip is a bona fide substrate for IRAK and is phosphorylated by IRAK upon stimulation with lipopolysaccharide or IL-1 . Negative regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory mediators during inflammation and infection.

J Bacteriol, 2002 Jan, 184(2), 494 - 502
Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences; Mawn MV et al.; We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli . These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs . An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA . Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs . Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient . Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin . The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA . We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.

J Bacteriol, 2002 Jan, 184(2), 479 - 87
Molecular evolution of the intimin gene in O111 clones of pathogenic Escherichia coli; Tarr CL et al.; Intimin is an important virulence factor in two groups of enteric pathogens: enteropathogenic Escherichia coli (EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagic E . coli (EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries . Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE) . At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages . We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing . The sequences from both O111:H8 and O111:H9 differed from the Int-beta that is typical of other clones in the same evolutionary lineage . The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-alpha, Int-beta, or Int-gamma . The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-alpha . The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives . Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein . An N-terminal domain that presumably functions in the periplasm may also be under positive selection.

J Bacteriol, 2002 Jan, 184(2), 444 - 51
Genes required for plasmid R64 thin-pilus biogenesis: identification and localization of products of the pilK, pilM, pilO, pilP, pilR, and pilT genes; Sakai D et al.; We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation . In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing . Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed . Overproduced proteins were purified and used to raise specific antibodies . Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively . The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody . Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins . PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins . Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein . We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.

J Bacteriol, 2002 Jan, 184(2), 363 - 9
Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli; Kneidinger B et al.; The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated . In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography . We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core . This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose . Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate . A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

J Antimicrob Chemother, 2002 Jan, 49(1), 193 - 6
Therapeutic efficacy of intraperitoneal polymyxin B and polymyxin-like peptides alone or combined with levofloxacin in rat models of septic shock; Giacometti A et al.; The efficacy of two polymyxin-like peptides, KFFKFFKFF and IKFLKFLKFL, alone and combined with levofloxacin, was investigated in a rat model of septic shock . Rats were given an ip injection of 2 x 10(10) cfu of Escherichia coli and randomized to receive ip isotonic sodium chloride solution, 7 mg/kg levofloxacin, 1 mg/kg polymyxin B and 1 mg/kg of each polymyxin-like peptides alone or combined with 7 mg/kg levofloxacin . Polymyxins achieved a significant reduction in plasma endotoxin and tumour necrosis factor alpha (TNF-alpha) concentration . Levofloxacin significantly reduced the bacterial growth and TNF-alpha concentration . The combinations of polymyxin-like peptides and levofloxacin demonstrated the highest efficacy.

EMBO Rep, 2002 Jan, 3(1), 39 - 44 Epub 2001 Dec 19.
Methylation at arginine 17 of histone H3 is linked to gene activation; Bauer UM et al.; The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro . The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays . However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones . We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1 . Using this antibody we show that methylated R17 exists in vivo . Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated . Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene . Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.

EMBO Rep, 2002 Jan, 3(1), 45 - 9 Epub 2001 Dec 19.
The processivity factor beta controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo; Lenne-Samuel N et al.; The dinB-encoded DNA polymerase IV (Pol IV) belongs to the recently identified Y-family of DNA polymerases . Like other members of this family, Pol IV is involved in translesion synthesis and mutagenesis . Here, we show that the C-terminal five amino acids of Pol IV are essential in targeting it to the beta-clamp, the processivity factor of the replicative DNA polymerase (Pol III) of Escherichia coli . In vivo, the disruption of this interaction obliterates the function of Pol IV in both spontaneous and induced mutagenesis . These results point to the pivotal role of the processivity clamp during DNA polymerase trafficking in the vicinity of damaged-template DNA.

Chem Senses, 2002 Jan, 27(1), 39 - 44
Odorants of different chemical classes interact with distinct odorant binding protein subtypes; Lobel D et al.; The ligand profile for three odorant binding proteins (OBPs) of the rat have been determined using a large number of odorous compounds from different chemical classes . To evaluate the binding spectra of distinct subtypes, all OBPs were produces in Escherichia coli as recombinant His-tagged fusion proteins . The individual binding properties of each OBP subtype were analysed using a large array of organic compounds, representing derivatives of aliphatic and aromatic compounds, as well as terpenes, pyrazines and thiazoles, in a competitive spectroscopic binding assay with various fluorescence chromophores as the specific interacting partner for the OBPs . Most of the compounds were identified to interact only with one OBP subtype . But interestingly, a small change, for example in the 2-methyl or 2-ethoxy side chain in the pyrazine and thiazole derivatives to a 2-isobutyl group, caused overlapping binding affinities to rat-OBP1 and rat-OBP3 . However, the data strongly support the notion that each OBP subtype displays a characteristic ligand binding profile and interacts with a different subset of exogenous organic compounds in a micromolar range.

Cell Growth Differ, 2001 Dec, 12(12), 581 - 9
14-3-3 binding regulates catalytic activity of human Wee1 kinase; Rothblum-Oviatt CJ et al.; The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15 . Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15 . Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions . Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function . We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01 . A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo . These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.

Cancer Res, 2001 Dec 15, 61(24), 8737 - 42
Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma; Huhn M et al.; A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy . Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin . The completely human fusion gene was inserted into a pET-based expression plasmid . Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes . After isopropyl beta-D-thiogalactoside induction, the M(r) 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography . The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells . Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein . Ang-CD30L showed RNase activity in vitro . The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC(50) = 8 ng/ml) as determined by cell proliferation assays . CD30 specificity was confirmed by competitive toxicity assays . This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.

Biol Reprod, 2002 Jan, 66(1), 241 - 50
Tektin B1 demonstrates flagellar localization in human sperm; Wolkowicz MJ et al.; The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined . Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry . The resulting peptides did not match any protein in the (then current) protein databases . Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)(+) mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library . The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1 . Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney . Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins . Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.

Biol Reprod, 2002 Jan, 66(1), 50 - 6
Synaptotagmin VIII is localized to the mouse sperm head and may function in acrosomal exocytosis; Hutt DM et al.; The acrosome is a large secretory granule that undergoes exocytosis when receptors on the sperm surface bind ligands in the egg extracellular matrix . Acrosomal exocytosis resembles stimulated secretion in neurons in that it is triggered by a rise in intracellular Ca(2+) . Synaptotagmins (Syt) comprise proteins thought to transduce this Ca(2+) signal to the fusion machinery . In this study, we showed that Syt VIII is present in spermatogenic cDNA libraries . Antiserum raised against a Syt VIII-specific peptide, which recognizes Syt VIII but does not cross-react with other Syt isoforms, labeled a single prominent band on Western immunoblots of mouse sperm homogenate . Syt VIII was restricted to the sperm membrane fraction enriched in markers associated with the mouse sperm head . Fluorescent immunocytochemistry on intact mouse sperm showed that Syt VIII is localized to the acrosomal crescent and is lost upon acrosome reaction . Moreover, the amount of Syt VIII remaining with the sperm decreased proportionately with the extent of acrosome-reacted sperm . Thus, Syt VIII is a candidate for the Ca(2+) sensor that regulates acrosomal exocytosis in mammalian sperm.

Bioinformatics, 2001 Dec, 17(12), 1224 - 5
Evolution of amino acid biosynthesis and enzymes with broad substrate specificity; Nishida H; I selected 82 proteins that were related to amino acid biosynthesis in the genome of Escherichia coli . I then searched the extensive sequence homology for each of the selected proteins from among the proteins of E.coli . The result showed that 30 proteins of the selected proteins had extensive sequence homology within the selected proteins, and 21 proteins had extensive sequence homology to proteins outside the selected proteins . In addition, the enzymes with broad substrate specificity play an important role in the amino acid biosynthesis . I demonstrate here that some substrate-specific enzymes evolved from an ancestor enzyme with broad substrate specificity . CONTACT: hnishida@iam.u-tokyo.ac.jp

Am J Respir Crit Care Med, 2001 Dec 15, 164(12), 2206 - 12
Genistein prevents nuclear factor-kappa B activation and acute lung injury induced by lipopolysaccharide; Kang JL et al.; Protein tyrosine kinase (PTK) inhibitors have been proposed to reduce lung injury and lethal toxicity . The mechanisms responsible for the effects of PTK inhibitors remain obscure . The purpose of the present study was to examine whether genistein, a specific inhibitor of PTK, inhibits nuclear factor-kappa B (NF-kappaB) activation during acute lung injury induced by lipopolysaccharide (LPS) and, if so, to enumerate the effects of inhibition of NF-kappaB activation on LPS-induced proinflammatory gene products, such as cytokine-inducible neutrophil chemoattractant (CINC) and matrix metalloproteinase-9 (MMP-9), as well as neutrophil influx into the lungs . Intratracheal treatment of rats with LPS (6 mg/kg) resulted in increases in total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid and activated DNA-binding activity of NF-kappaB in alveolar macrophages and lung tissue . A 2-h pretreatment with genistein (50 mg/kg, intraperitoneally) inhibited the LPS-induced changes in lung injury parameters and the induction of NF-kappaB activation . Furthermore, these inhibitory effects of genistein correlated with a depression of LPS-induced protein tyrosine phosphorylation (approximately molecular masses of 46, 48, and 54 kD) and phosphorylation of Jun N-terminal kinase (JNK) in lung tissue . Genistein also substantially reduced the LPS-induced CINC production and MMP-9 activity and suppressed neutrophil recruitment . These results suggest that genistein attenuates LPS-induced acute lung responses through inhibition of NF-kappaB activation . In addition, NF-kappaB activation appears to be an important mechanism mediating LPS-induced CINC production and MMP-9 activity and resulting neutrophil recruitment associated with acute lung inflammation and injury.

Eur J Endocrinol, 2002 Jan, 146(1), 113 - 9
Epitope mapping of cytochrome P450 cholesterol side-chain cleavage enzyme by sera from patients with autoimmune polyglandular syndrome type 1; Liiv I et al.; OBJECTIVE: Autoimmune polyglandular syndrome type 1 (APS-1) is a disease associated with defects of the autoimmune regulator gene and is characterized by autoimmune lesions of several tissues, predominantly endocrine glands, with multiple autoantibodies . In this study we describe autoantigenic epitopes on cholesterol side-chain cleavage enzyme (P450scc) using sera from Finnish and Sardinian patients with APS-1, and analyze the epitope reactivities during disease follow-up . METHODS: A series of P450scc cDNA fragments were expressed in E . coli and tested by immunoblotting assay using the patients' sera . RESULTS: Epitope regions were found over the whole P450scc molecule except the last N- (amino acids (aa) 1-40) and C-termini (aa 456-521) . The strongest reactivity with patients' sera was found with central and C-terminal regions of the P450scc protein . All studied patients had IgG1 subclass antibodies . CONCLUSIONS: The results show that Finnish and Sardinian patients with APS-1 have similar, polyclonal immune reactions against P450scc, and that epitope reactivities did not change during the disease course . These results support the opinion that autoantibodies against P450scc and their epitope reactivity pattern are formed at an early stage of steroidogenic autoimmunity.

Curr Opin Struct Biol, 2001 Dec, 11(6), 694 - 700
The ACT domain family; Chipman DM et al.; A novel ligand-binding domain, named the 'ACT domain', was recently identified by a PSI-BLAST search . The archetypical ACT domain is the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH), which folds with a ferredoxin-like betaalphabetabetaalphabeta topology . A pair of ACT domains form an eight-stranded antiparallel sheet with two molecules of the allosteric inhibitor serine bound in the interface . The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand binding fold . Rat phenylalanine hydroxylase has a regulatory domain with a similar fold, but different ligand-binding mode . Putative ACT domains in some proteins of unknown structure (e.g . acetohydroxyacid synthase regulatory subunits) may also fold like the 3PGDH regulatory domain . The regulatory domain of threonine deaminase, although not a member of the ACT sequence family, is similar in structure to the paired 3PGDH regulatory domains . Repeats of ACT-like domains can create nonequivalent ligand-binding sites with the potential for complex regulatory patterns . The structures and mechanisms of such systems have only begun to be examined.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 299 - 303
The genetic dependence of RecBCD-Gam mediated double strand end repair in Escherichia coli; Paskvan I et al.; The repair of double strand breaks after gamma-irradiation in wild-type Escherichia coli lysogenic for lambda cI857 red3 is more efficient when lambda Gam protein is present . This phenomenon, called gam dependent radioresistance, requires the interaction of RecBCD enzyme and Gam protein . We compared cell survival after gamma-irradiation in wild-type and mutant lysogens with and without induction of Gam by transient heat treatment of the cells (6 min, 42 degrees C) . The main conclusions are: (1) the RecBCD-Gam pathway of recombination repair is similar but not equivalent to RecBCD, a pathway operating in recD mutants; (2) the RecBCD-Gam pathway is dependent on recJ, recQ and recN gene products and it is proposed that the RecBCD-Gam complex has ability to load RecA protein onto single strand DNA.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 271 - 5
Escherichia coli utilizes methanesulfonate and L-cysteate as sole sulfur sources for growth; Eichhorn E et al.; Twenty-three Escherichia coli strains were tested for their ability to use taurine, methanesulfonate, L-cysteate and other alkanesulfonates as sole sulfur sources for growth . One strain was unable to use any of the alkanesulfonates offered as sole sulfur sources for growth but grew with sulfate . Seven strains (class I) used alkanesulfonates for this purpose, but not methanesulfonate or L-cysteate . A further seven strains (class II) grew with all compounds tested, except with L-cysteate, and eight strains (class III) utilized all compounds tested as sulfur sources . Sulfur assimilation from methanesulfonate and L-cysteate was absolutely dependent on the ssuEADCB operon that encodes an alkanesulfonate uptake system (SsuABC) and a two-component monooxygenase (SsuDE) involved in the release of sulfite from alkanesulfonates . Long-term exposure of class I strains to methanesulfonate and of class II strains to L-cysteate selected for derivatives that utilized these two sulfur sources as efficiently as sulfate . The nucleotide sequence of the ssuEADCB operon in the methanesulfonate- and L-cysteate-utilizing derivative EC1250Me+ was identical to that in the class I wild-type EC1250 . Gain of the ability to utilize methanesulfonate and L-cysteate as sulfur sources thus appears to result from increased expression of ssu genes rather than from a change in the quality of one or several of the Ssu proteins.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 191 - 6
Analysis of the regulatory region of the heat-shock gene rpoH of Escherichia coli strains isolated from non-human hosts; Solis-Guzman G et al.; The regulatory region of the gene for sigma32, rpoH, of Escherichia coli strains isolated from non-human hosts and different geographic regions, was sequenced and compared with that of E . coli K12 . The main nucleotide changes observed are localized to the right inverted octamer motif of the CytR box . The effect of these changes was evaluated using transcriptional fusions . The results presented could guide further studies on the transcription regulation of rpoH using E . coli K12 as a model.

Mol Immunol, 2002 Jan, 38(7), 515 - 25
Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01; Luttkopf D et al.; The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study.Total RNA was isolated from immature hazelnuts and transcribed into cDNA . Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E . coli ER2566 cells . Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins . IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays . Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975) . Cor a 1.0402 and 03 only differed in a C4S exchange . Cor a 1.0404 had a unique proline residue in position 99 . Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1 . EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404 . The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls . Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange . The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1 . IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant) . Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404 . It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen . Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Diagn Microbiol Infect Dis, 2001 Nov, 41(3), 93 - 8
Diarrhoeagenic Escherichia coli--an emerging problem?
Clarke SC.
Diarrhea remains one of the main sources of morbidity and morbidity in today's world and a large proportion is caused by diarrheagenic Escherichia coli . They are a particular problem in developed countries although traveler's diarrhea and hemorrhagic colitis are also a problem in developed countries . There are seven classes of diarrheagenic E . coli, namely enteropathogenic E . coli (EPEC), enterohaemorrhagic E . coli (EHEC), enteroinvasive E . coli (EIEC), enterotoxigenic E . coli (ETEC), enteroaggregative E . coli (EAggEC), diarrhea-associated hemolytic E . coli (DHEC) and cytolethal distending toxin (CDT)- producing E . coli . Many of their virulence determinants have been determined and some classes of diarrheagenic E . coli produce toxins . The virulence factors of some diarrhogenic E . coli have yet to be full determined and in the meantime they remain a large and emerging problem without the availability of effective vaccines.

Gene, 2001 Dec 27, 281(1-2), 71 - 9
Characterization of the yaeL gene product and its S2P-protease motifs in Escherichia coli; Kanehara K et al.; An Escherichia coli open reading frame, yaeL, encodes a predicted homolog of human site-2 protease (S2P), a putative membrane-bound zinc metalloproteinase involved in the proteolytic activation of regulatory factors for sterol biosynthesis and for stress responses . The potential importance of YaeL in processes analogous to the regulated intramembrane proteolysis in E . coli prompted us to characterize it . Cell fractionation and alkaline phosphatase fusion experiments established that YaeL has four transmembrane segments with both termini orienting toward the periplasm . A strain in which a chromosomal disruption of yaeL was combined with arabinose promoter-controlled yaeL on a plasmid enabled us to deplete this protein from the cell . The depletion was found to cause rapid loss of viability, cell elongation and growth cessation . Mutations affecting the HEXXH metalloproteinase motif and those affecting the LDG motif, conserved among S2Ps, abolished the ability of YaeL to support cell growth . These results indicate that YaeL is indispensable in E . coli, and probably functions as a metalloproteinase at the membrane.

Biochim Biophys Acta, 2001 Dec 3, 1522(2), 112 - 7
Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585; Soh BS et al.; The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source . The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized . In this study, the aceA gene, encoding ICL from S . clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR . The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far . The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated . Furthermore, the relationship between the carbon sources, growth and ICL activity in S . clavuligerus were investigated . Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate . However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S . clavuligerus.

Biochim Biophys Acta, 2001 Dec 3, 1522(2), 74 - 81
Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells; Wakatsuki S et al.; p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells . In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15 . The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity . From the poly(A)(+) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His(6)-tagged recombinant p15 was produced in Escherichia coli . The recombinant protein purified by a single step on Ni(2+) agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay . The effect of His(6)-p15 was blocked by nicardipine, suggesting that Ca(2+) influx through the L-type Ca(2+) channels is essential for its neurite-inducing activity . In addition, mutational analysis of His(6)-p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity.

Acta Pharmacol Sin, 2001 Sep, 22(9), 831 - 6
Anti-human hepatocellular carcinoma effects of tumor necrosis factor-related apoptosis-inducing ligand in vitro & in vivo; Guo BC et al.; AIM: To investigate the effect of over-expression of Bcl-2 protein on Trail protein-induced apoptosis in human hepatoma cells, and the cytotoxicity of Trail protein on human hepatoma cells in vitro and in vivo . METHODS: The Trail gene was cloned and expressed in E coli . The cytotoxicity of the recombinant Trail protein was assayed on human hepatoma cells in vitro and in vivo . The cell viability was assessed by trypan blue exclusion . The stable human hepatoma cells clone in which Bcl-2 protein over-expressed was established by transfecting eukaryotic expression plasmid pcDNA3-Bcl-2 into BEL-7404 human hepatoma cells, and was selected with G418 400 mg/L . RESULTS: The recombinant Trail protein actively killed human hepatoma cells tested in this study such as BEL-7404, BEL-7402, and SMMC-7721 . Over-expression of Bcl-2 protein could inhibit apoptosis induced by Trail in BEL-7404 human hepatoma cells in vitro . It was obvious that the purified recombinant Trail protein could inhibit tumor formation of BEL-7404 human hepatoma cells in nude mice . CONCLUSION: The recombinant Trail protein could kill human hepatoma cells in vitro and in vivo . Over-expression of Bcl-2 protein could inhibit Trail-induced apoptosis in BEL-7404 human hepatoma cells . The results suggested that Trail might be a potential agent for the liver cancer therapy.

Acta Pharmacol Sin, 2001 Jul, 22(7), 624 - 8
Gene construction, expression, and characterization of double-copy truncated form of human insulin-like growth factor I; Sun HY et al.; AIM: To increase the production of recombinant truncated form of insulin-like growth factor I {des(1-3)IGF-I}, purify the expressed product, and compare its bioactivity with that of standard IGF-I . METHODS: The second copy of des(1-3)IGF-I gene was inserted into the previously constructed pExSec1/IGF-I to form a pExSec1/2(IGF-I) expression plasmid, then the plasmid was transformed into a protease-deficient E coli strain BL21(DE3) . The engineered bacteria were cultured and induced by IPTG at 12 degrees C . The expressed product was purified through ultrafiltration and Sephadex G-50 gelfiltration . The bioactivity of the preliminarily purified protein was tested by MTT method and compared with standard IGF-I . RESULTS: The amount of des(1-3)IGF-I expressed by pExSec1/2(IGF-I) reached up to 20 % of the total soluble bacterial protein, which was higher than the amount (12 %) expressed by a single copy of pExSec1/IGF-I gene . The purity of recombinant des(1-3)IGF-I reached 49 % and 82 % after ultrafiltration and gelfiltration . The bioactivity of des(1-3)IGF-I after gelfiltration was about 77 % of standard IGF-I at the same concentration . CONCLUSION: The yield of recombinant des(1-3)IGF-I was increased about 8 % by construction of expression plasmid with two copies of des(1-3)IGF-I gene compared with only one copy of gene, and preliminarily purified des(1-3)IGF-I showed about 77 % bioactivity compared with standard IGF-I.

Acta Pharmacol Sin, 2001 Nov, 22(11), 1039 - 44
Dual function of human necrosis factor receptor 75 in cytotoxicity induced by human tumor necrosis factor alpha; Fang J et al.; AIM: To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFalpha-induced cytotoxicity . METHODS: HEp-2 cells were transfected with bicistronic expression vector of hTR75 gene, and HEp-2-A75 cells with intrinsic hTR55 and overexpressed hTR75 were obtained . Two hTNFalpha muteins with exclusive specificity for hTR55 or hTR75 were constructed, expressed in high-levels in E coli, and then purified . hTNFalpha-induced cytotoxicity was determined by crystal violet colorimetric method . RESULTS: The expression of hTR75 in HEp-2 cells was demonstrated by RT-PCR and indirect ELISA, and was quantified by binding of {125I}hTNFalpha and Scatchard analysis . The overexpressed hTR75 could markedly increase the susceptibility of HEp-2 cells to hTNFalpha . CONCLUSION: hTR75 could not only partially mediate hTNFalpha-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity . It played a dual function in hTNFalpha-induced cytotoxicity in HEp-2 cells.

Jpn J Cancer Res, 2001 Dec, 92(12), 1313 - 21
Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein; Itoh K et al.; The Fab gene of anti-CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system . The variable heavy and light chain genes of HBJ127 were found to be derived from VOx-1 and IgVk8-30 germline, respectively . Extensive somatic mutation was found in the heavy chain complementarity determining region 2 . rFab fragment was purified homogeneously from crude bacterial lysates by Ni-chelate chromatography in a yield of 71.4 mg from 100 ml of culture . rFab fragment was reactive with the cell surface of CD98-positive cells irrespective of tissues of origin, but not with CD98-negative cells . The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS(3) cells was completely inhibited by pretreatment with an excess of mAb . The relative affinity values of rFab fragment and mAb were found to be 0.11 x 10(8) and 0.35 x 10(8) M(-1), respectively . Three-fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation . Cell growth inhibition in vitro by rFab fragment preincubated with anti-Fab suggests that the rFab fragment produced by cloned gene-bearing Escherichia coli was identical to the Fab part of HBJ127 mAb . These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system . To our knowledge, this is a first report of the production of anti-CD98 h.c . rFab fragment.

Clin Sci (Lond), 2002 Jan, 102(1), 107 - 14
The response of liver albumin synthesis to infection in rats varies with the phase of the inflammatory process; Ruot B et al.; To discriminate between the effects of infection and of anorexia associated with infection, liver albumin synthesis was measured in well-fed rats, in rats injected with live Escherichia coli and in pair-fed rats at different stages of the inflammatory response (1, 6 and 10 days after infection) using a large dose of l-{1-(14)C}valine . Albuminaemia and albumin mRNA levels were unchanged following food restriction . However, absolute albumin synthesis was decreased in pair-fed rats compared with control animals after 1 day of food restriction, and had returned to normal values by day 10 when food intake was restored . Infection was characterized by a decrease in the plasma albumin concentration (35%, 45% and 28% as compared with pair-fed rats at 1, 6 and 10 days after infection respectively) . Albumin mRNA levels and relative albumin synthesis were reduced in infected rats as compared with both control and pair-fed animals at all stages of infection . However, during the early acute response, the albumin absolute synthesis rate was similar in infected rats and pair-fed rats, indicating no specific effect of infection at this stage . Later in the course of infection, the amount of albumin synthesized by the liver was lower in infected than in pair-fed rats, and hypoalbuminaemia was probably maintained due to a lack of stimulation of synthesis despite increased food intake.

Biomacromolecules, 2001 Summer, 2(2), 538 - 40
Effects of macromolecular crowding on the intrinsically disordered proteins c-Fos and p27(Kip1); Flaugh SL et al.; A number of biologically active proteins exhibit intrinsic structural disorder in vitro under thermodynamically ideal conditions . In vivo, however, proteins exist in a crowded, thermodynamically nonideal environment . We tested the hypothesis that intrinsically disordered proteins adopt stable structure under crowded conditions in which excluded volume is predicted to stabilize compact, native conformations . In the presence of macromolecular crowding agents, neither the intrinsically disordered C-terminal activation domain of c-Fos nor the kinase-inhibition domain of p27(Kip1) undergoes any significant conformational change that is detected by changes in either circular dichroism or fluorescence spectra . We conclude that molecular crowding effects are not necessarily sufficient to induce ordered structure in intrinsically disordered proteins.

Biomacromolecules, 2001 Spring, 2(1), 111 - 25
Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence; Zhou Y et al.; The flagelliform silk protein underlies the unique elastomeric properties displayed by the capture spiral of arachnid webs . To investigate molecular mechanism underlying the elastomeric recovery of the capture spiral, a model polypeptide based upon the elastomeric repeat sequence of Nephila clavipes flagelliform silk protein has been synthesized using recombinant DNA techniques . Polypeptide 1 contains 11 repeats of the 25 amino acid sequence {(Gly-Pro-Gly-Gly-Ser-Gly-Pro-Gly-Gly-Tyr)(2)-Gly-Pro-Gly-Gly-Lys} and was expressed in Escherichia coli strain BL21(DE3) as a C-terminal fusion to a decahistidine leader sequence . A combination of (1)H-(1)H COSY, DEPT, (1)H-(13)C HETCOR, and (1)H-(13)C HMBC NMR spectroscopy was employed on polypeptides 1 and the {1-(13)C}glycine-labeled analogue 1G to assign the (1)H and (13)C NMR resonances of the amino acid residues comprising the flagelliform silk repeat sequence . The conformational properties of 1 in aqueous solution were investigated using a combination of CD, FTIR, VT-NMR, and two-dimensional NOESY NMR . These techniques were consistent with the presence of small but detectable population of beta-turn conformers between Gly(1) and Gly(4) of the pentapeptide units of 1 . FTIR and CD studies of solid films of 1 indicated an increase in beta-turn population in the solid state, which coincided with the decrease in hydration level of the polypeptide . The spectroscopic information suggests that the pentapeptide segments of the flagelliform silk protein adopt a beta-turn conformation in the fiber and that the mechanism of elasticity may resemble that proposed for other beta-turn forming polypeptides including elastin.

J Biol Chem, 2002 Feb 22, 277(8), 5703 - 6 Epub 2001 Dec 03.
The HET-s prion protein of the filamentous fungus Podospora anserina aggregates in vitro into amyloid-like fibrils; Dos Reis S et al.; The HET-s protein of Podospora anserina is a fungal prion . This protein behaves as an infectious cytoplasmic element that is transmitted horizontally from one strain to another . Under the prion form, the HET-s protein forms aggregates in vivo . The specificity of this prion model compared with the yeast prions resides in the fact that under the prion form HET-s causes a growth inhibition and cell death reaction when co-expressed with the HET-S protein from which it differs by 13 residues . Herein we describe the purification and initial characterization of recombinant HET-s protein expressed in Escherichia coli . The HET-s protein self-associates over time into high molecular weight aggregates . These aggregates greatly accelerate precipitation of the soluble form . HET-s aggregates appear as amyloid-like fibrils using electron microscopy . They bind Congo Red and show birefringence under polarized light . In the aggregated form, a HET-s fragment of approximately 7 kDa is resistant to proteinase K digestion . CD and FTIR analyses indicate that upon transition to the aggregated state, the HET-s protein undergoes a structural rearrangement characterized by an increase in antiparallel beta-sheet structure content . These results suggest that the {Het-s} prion element propagates in vivo as an infectious amyloid.

J Biol Chem, 2002 Feb 8, 277(6), 4446 - 54 Epub 2001 Dec 03.
Resolving a fidelity paradox: why Escherichia coli DNA polymerase II makes more base substitution errors in AT- compared with GC-rich DNA; Wang Z et al.; The activity of DNA polymerase-associated proofreading 3'-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences . Unexpectedly, however, the opposite result was found for Escherichia coli DNA polymerase II (pol II) . Nucleotide misincorporation frequencies for pol II were found to be 3-5-fold higher in AT- compared with GC-rich DNA, both in the presence and absence of polymerase processivity subunits, beta dimer and gamma complex . In contrast, E . coli pol III holoenzyme, behaving "as expected," exhibited 3-5-fold lower misincorporation frequencies in AT-rich DNA . A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3'-exonuclease proofreading activity that preferentially degrades AT-rich compared with GC-rich DNA primer-template in the absence of DNA synthesis . Concomitant with a reduction in fidelity, pol II polymerization efficiencies were 2-6-fold higher in AT-rich DNA, depending on sequence context . Pol II paradoxical fidelity behavior can be accounted for by the enzyme's preference for forward polymerization in AT-rich sequences . The more efficient polymerization suppresses proofreading thereby causing a significant increase in base substitution error rates in AT-rich regions.

J Biol Chem, 2002 Feb 22, 277(8), 5749 - 55 Epub 2001 Dec 03.
The conserved active site asparagine in class I ribonucleotide reductase is essential for catalysis; Kasrayan A et al.; The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate . To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln) . All mutant proteins were incapable of enzymatic turnover but promoted rapid protein R2 tyrosyl radical decay in the presence of the k(cat) inhibitor 2'-azido-2'-deoxy-CDP with similar decay rate constants as the wild-type R1 . These results show that all Asn-437 mutants can perform 3'-H abstraction, the first substrate-related step in the reaction mechanism . The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP . The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2'-OH group in the form of water, a step that has been proposed to drive the 3'-H abstraction step . A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step . We conclude that Asn-437 is essential for catalysis but not for 3'-H abstraction . We propose that the suicidal N437A, N437S, and N437D mutants can also catalyze the water elimination step, whereas the inert N437Q mutant cannot . Our results suggest that Asn-437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.

J Biol Chem, 2002 Feb 1, 277(5), 3141 - 9 Epub 2001 Dec 03.
Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate; Burke C et al.; Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes . Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin . The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis . Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case . These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains . Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

J Biol Chem, 2002 Feb 15, 277(7), 5611 - 9 Epub 2001 Nov 30.
Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue; Kunz J et al.; Type I and type II phosphatidylinositol phosphate (PIP) kinases generate the lipid second messenger phosphatidylinositol (PtdIns) 4,5-bisphosphate and thus play fundamental roles in the regulation of many cellular processes . Although the two kinase families are highly homologous, they phosphorylate distinct substrates and are functionally non-redundant . Type I PIP kinases phosphorylate PtdIns 4-phosphate at the D-5 hydroxyl group and are consequently PtdIns 4-phosphate 5-kinases . By contrast, type II PIP kinases are PtdIns 5-phosphate 4-kinases that phosphorylate PtdIns 5-phosphate at the D-4 position . Type I PIP kinases, in addition, also phosphorylate other phosphoinositides in vitro and in vivo and thus have the potential to generate multiple lipid second messengers . To understand how these enzymes differentiate between stereoisomeric substrates, we used a site-directed mutagenesis approach . We show that a single amino acid substitution in the activation loop, A381E in IIbeta and the corresponding mutation E362A in Ibeta, is sufficient to swap substrate specificity between these PIP kinases . In addition to its role in substrate specificity, the type I activation loop is also key in subcellular targeting . The Ibeta(E362A) mutant and other mutants with reduced PtdIns 4-phosphate binding affinity were largely cytosolic when expressed in mammalian cells in contrast to wild-type Ibeta which targets to the plasma membrane . These results clearly establish the role of the activation loop in determining both signaling specificity and plasma membrane targeting of type I PIP kinases.

J Biol Chem, 2002 Mar 1, 277(9), 6874 - 80 Epub 2001 Nov 30.
Mitochondrial protein import: molecular basis of the ATP-dependent interaction of MtHsp70 with Tim44; Moro F et al.; Protein translocation across the mitochondrial inner membrane is driven by cycles of binding and release of mitochondrial heat shock protein 70 (mtHsp70) in the matrix . The peripheral inner membrane protein, Tim44, recruits mtHsp70 in an ATP-dependent manner to the import sites . We show that DnaK, the closely related Hsp70 of Escherichia coli, when targeted to the matrix of yeast mitochondria, interacts in a specific manner with Tim44 . The interaction is, however, not regulated by ATP, and DnaK cannot support protein translocation . We used truncated mtHsp70s and chimeric proteins consisting of segments of mtHsp70 and DnaK to analyze which portions of mtHsp70 bind and functionally interact with Tim44 . We show that Tim44 interacts with the beta-stranded core of the peptide binding domain of mtHsp70 and of DnaK . The alpha-helices A and B of the peptide binding domain of mtHsp70 are required to transmit the nucleotide state of the ATPase domain to the peptide binding domain . Tim44, by interacting in this way with the peptide binding domain, is proposed to coordinate the binding of mtHsp70 to the incoming preprotein and the subsequent release of the mtHsp70-preprotein complex from the TIM23 complex, the translocase of the inner membrane.

Biochemistry, 2001 Dec 11, 40(49), 14847 - 54
Geranylgeranylglyceryl phosphate synthase . Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum; Soderberg T et al.; Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate {(S)-GP} by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate {(S)-GGGP} . This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea . The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme . The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli . The recombinant GGGP synthase was purified to homogeneity . The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments . GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2) . V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM . These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M . thermoautotrophicum {Chen, A., Zhang, D., and Poulter, C . D . (1993) J . Biol . Chem . 268, 21701-21705} . Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.

Biochemistry, 2001 Dec 11, 40(49), 14781 - 94
A structural view of the action of Escherichia coli (lacZ) beta-galactosidase; Juers DH et al.; The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented . These complexes clarify and enhance previous proposals regarding the catalytic mechanism . The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety . Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role . A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl . The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket . For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding . In some cases this can be inhibited by the binding of additional ligands . The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.

Anat Rec, 2002 Jan 1, 266(1), 5 - 9
Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice; Ueda J et al.; A 1.4-kb human alpha1-proteinase inhibitor (alpha1-PI) 5'-flanking sequence fused to the E . coli LacZ gene was used to generate transgenic mice . The 1.4-kb alpha 1-PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain . This finding implies that the alpha 1-PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments .

J Exp Med, 2001 Dec 17, 194(12), 1813 - 21
The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells; Lechmann M et al.; CD83 is an immunoglobulin (Ig) superfamily member that is upregulated during the maturation of dendritic cells (DCs) . It has been widely used as a marker for mature DCs, but its function is still unknown . To approach its potential functional role, we have expressed the extracellular Ig domain of human CD83 (hCD83ext) as a soluble protein . Using this tool we could show that immature as well as mature DCs bind to CD83 . Since CD83 binds a ligand also expressed on immature DCs, which do not express CD83, indicates that binding is not a homophilic interaction . In addition we demonstrate that hCD83ext interferes with DC maturation downmodulating the expression of CD80 and CD83, while no phenotypical effects were observed on T cells . Finally, we show that hCD83ext inhibits DC-dependent allogeneic and peptide-specific T cell proliferation in a concentration dependent manner in vitro . This is the first report regarding functional aspects of CD83 and the binding of CD83 to DCs.

J Biol Chem, 2002 Feb 22, 277(8), 6550 - 8 Epub 2001 Dec 17.
Activation of smooth muscle myosin light chain kinase by calmodulin . Role of LYS(30) and GLY(40); Van Lierop JE et al.; Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction . A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM . We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK . A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK . Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity . Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction . K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK . Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK . Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.

Infect Immun, 2002 Jan, 70(1), 323 - 34
Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regulated peripheral membrane protein; Matsunaga J et al.; Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents . We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa . Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library . In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated . We designated this putative lipoprotein LipL45 . Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45 . In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein . In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum . Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45) . Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein . Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated . Hamsters infected with L . kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection . Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L . kirschneri that colonized the renal tubule . These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

Infect Immun, 2002 Jan, 70(1), 315 - 22
Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells; Lee SH et al.; Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans . Leptospira sp . hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis . Previously, we cloned a hemolysin SphH of Leptospira interrogans serovar lai, a homologue of L . borgpetersenii sphingomyelinase (SphA), from a genomic library (S . H . Lee, K . A . Kim, Y . K . Kim, I . W . Seong, M . J . Kim, and Y . J . Lee, Gene 254:19-28, 2000) . Escherichia coli lysate harboring the sphH showed high hemolytic activities on sheep erythrocytes . However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity . Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane . In the present study, we have prepared the Leptospira hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells . SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy . Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations . Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities . In conclusion, these results suggest that the virulence of Leptospira SphH was due to the pore formation on mammalian cell membranes.

Infect Immun, 2002 Jan, 70(1), 268 - 76
Role for fimbriae and lysine-specific cysteine proteinase gingipain K in expression of interleukin-8 and monocyte chemoattractant protein in Porphyromonas gingivalis-infected endothelial cells; Nassar H et al.; Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease . Previously, we have established that the periodontal pathogen Porphyromonas gingivalis is capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC) . Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions of P . gingivalis with endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis . In this study we examined the consequences of P . gingivalis infection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) . HUVEC were found to constitutively produce low levels of IL-8 and MCP-1 . The addition of P . gingivalis fimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses . In contrast, coculture of HUVEC with live P . gingivalis strain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses . Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherent P . gingivalis fimA mutant DPG3 or when P . gingivalis was preincubated with fimbrillin peptide antisera prior to the addition to HUVEC . Furthermore, treatment of P . gingivalis-infected HUVEC with cytochalsin D, which prevented P . gingivalis invasion, also abolished the constitutive IL-8 and MCP-1 responses . Treatment of HUVEC with E . coli LPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with live P . gingivalis . Analysis of P . gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC . Pretreatment of P . gingivalis with protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production in P . gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated . Coculture of HUVEC with a P . gingivalis mutant deficient in lysine-specific cysteine proteinase (gingipain K {Kgp}) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with the P . gingivalis wild-type strain . These results indicate that P . gingivalis can temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.

Hum Gene Ther, 2001 Nov 20, 12(17), 2121 - 7
Efficient liposome-mediated gene transfection and expression in the intact human uterus; Daftary GS et al.; Although gene therapy has been used for correction of metabolic defects in diseases such as cystic fibrosis, as adjuvant treatment in cancer, and in the treatment of infectious diseases, there has been no report of gene transfer to the intact female reproductive tract . We assessed the ability to transfect the human uterus ex vivo and thereby evaluate the applicability of gene therapy to gynecology . The uterine lumen was accessed transcervically, using an intrauterine insemination catheter . pcDNA3.1 plasmid containing the Escherichia coli lacZ reporter gene was delivered to each uterus via liposome-mediated transfection . Control uteri were transfected with empty pcDNA3.1 . Immunohistochemical analysis revealed beta-galactosidase expression in the lacZ-treated uteri in endometrial epithelial cells, endometrial stromal cells, and myometrium to a depth of 1.75 cm from the endometrial-myometrial junction . Highest expression was seen in endometrial glandular epithelial cells, with significant expression in the stroma and adjacent myometrium . Each of these cell types in the control uteri showed no beta-galactosidase expression . Successful gene transfection and expression in the intact human uterus can be accomplished easily, rapidly, and efficiently . Gene therapy may have wide applicability in the treatment and study of gynecologic disease.

Int J Radiat Biol, 2001 Dec, 77(12), 1195 - 205
Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA . Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength; Milligan JR et al.; PURPOSE: Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN)2*- . Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG) . It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN)2*- is reversible . In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of one-electron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions . MATERIALS AND METHODS: Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137Cs gamma-irradiation . After irradiation, the plasmid was incubated with the E . coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG) . The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks . RESULTS: The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate . By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites . A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively) . CONCLUSIONS: It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN)2*- and DNA-G*+ . This leads to an estimate of the reduction potential at pH 7 for the couple DNA G*+/DNA of E7 = +1.39+/-0.01V.

Biochemistry, 2001 Dec 25, 40(51), 15650 - 9
Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery; Falcon CM et al.; The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA . This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding . Furthermore, this small region serves as a central element in communicating the allosteric response to inducer . Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for O(1) operator DNA compared to wild-type LacI and abolishes allosteric response to inducer {Falcon, C . M., Swint-Kruse, L., and Matthews, K . S . (1997) J . Biol . Chem . 272, 26818} . We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations . Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants . Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI . The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains . Moreover, the results confirm that DNA sequence/conformation can profoundly influence allostery for this prototypic regulatory protein.

Biochemistry, 2001 Dec 25, 40(51), 15464 - 70
Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations; Reeves CD et al.; Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues . As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced . Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding . Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains . Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B . Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4 . This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Arch Biochem Biophys, 2002 Jan 1, 397(1), 119 - 29
Ipso-substitution by cytochrome P450 with conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species; Vatsis KP et al.; Evidence for multiple functional active oxidants in cytochrome P450-catalyzed reactions was previously obtained in this laboratory with mutants in which proton delivery was perturbed by replacement of the highly conserved threonine residue in the active site by alanine, thus apparently interfering with the conversion of the peroxo-iron to the hydroperoxo-iron and the latter to the oxenoid-iron species . These enzymes have now been employed to examine the reaction in which cytochrome P450 in liver microsomes is known to effect ipso-substitution, the elimination of p-substituents in phenols to yield hydroquinone . As shown with purified NH(2)-truncated cytochromes in a reconstituted enzyme system, the reaction exhibits an absolute requirement for cytochrome P450 and NADPH-cytochrome P450 reductase . Under optimal conditions truncated cytochrome P450 2E1 is active with 10 of the p-substituted phenols examined . Of particular interest, the corresponding cytochrome with threonine-303 replaced by alanine is from 1.5- to 50-fold higher in activity with the p-chloro, -bromo, -nitro, -cyano, -hydroxymethyl, -formyl, and -acetyl derivatives, and the reaction with the p-benzoyl, -methyl, and -t-butyl compounds is catalyzed by the mutant enzyme only . The results implicate the hydroperoxo-iron species as an electrophilic active oxidant in cytochrome P450-catalyzed aromatic ipso-substitution.

Arch Biochem Biophys, 2002 Jan 1, 397(1), 57 - 68
Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase; Kolberg M et al.; In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1 . We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines . In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals . The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1 . In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton . Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.

Arch Biochem Biophys, 2002 Jan 1, 397(1), 1 - 10
Cysteine-reactive fluorescence probes of catalytic sites of ATP synthase; Weber J et al.; We searched for new fluorescent probes of catalytic-site nucleotide binding in F(1)F(0)-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F(1) with thiol-reactive fluorescent probes . Four suitable mutant/probe combinations were identified . beta F410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal changes in response to nucleotide, allowing facile measurement of fluorescence and nucleotide-binding parameters, not only in F(1) but also in F(1)F(0) . The results are consistent with the presence of three asymmetric catalytic sites of widely different affinities, with similar properties in both enzymes, and revealed a unique probe environment at the high-affinity site 1 . beta Y331C F(1) labeled by ABD-F gave a large signal which monitored catalytic site polarity changes that occur along the ATP hydrolysis pathway . Two other mutant/probe combinations with significant nucleotide-responsive signals were beta Y331C labeled by 5-((((2-iodoacetyl)amino)ethyl)amino)naphthaline-1-sulfonic acid and alpha F291C labeled by 2-4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid . The signal of the latter responds differentially to nucleoside diphosphate versus triphosphate bound in catalytic sites.

Arch Biochem Biophys, 2001 Dec 15, 396(2), 219 - 24
Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase; Lee LV et al.; L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values . The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose . In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates . The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates . In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates . The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose . L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial . These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred . (c)2001 Elsevier Science.

Rapid Commun Mass Spectrom, 2001, 15(22), 2186 - 92
Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques; Elviri L et al.; Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase . In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids . Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction ( approximately 20%) retaining the initial methionine . By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure . ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E . coli grown in different media . Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used . In particular, this technique was applied to identify and quantify fatty acids bound to CRBP . Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein . Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression .

Environ Mol Mutagen, 2001, 38(2-3), 235 - 43
Novel DNA repair alkyltransferase from Caenorhabditis elegans; Kanugula S et al.; O6-alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6-position of guanine . The search of the C . elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein . The predicted protein sequence of cAGT-2 contains the amino acid sequence -ProCysHisPro- at the presumed active site of the protein, whereas all other known AGTs have -ProCysHisArg- . A truncated version of the cAGT-2 protein was expressed in E . coli . This purified recombinant protein was able to repair O6-methylguanine and O4-methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6-benzylguanine . This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described . The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C . Expression of cAGT-2 in an E . coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N-methyl-N'-nitro-N-nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase .

Proteins, 2002 Jan 1, 46(1), 1 - 7
Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2; Adkins JN et al.; The cell cycle inhibitor p57Kip2 induces cell cycle arrest by inhibiting the activity of cyclin-dependent kinases . p57, although active as a cyclin A-CDK2 inhibitor, is largely unfolded or intrinsically disordered as shown by circular dichroism and fluorescence spectra characteristic of an unfolded protein and a hydrodynamic radius consistent with an unfolded structure . In addition, the N-terminal domain of p57 is both functionally independent as a cyclin A-CDK2 inhibitor and unstructured, as demonstrated by circular dichroism and fluorescence spectra indicative of unfolded proteins, a lack of 1H chemical shift dispersion and a hydrodynamic radius consistent with a highly unfolded structure . The amino acid compositions of full-length p57 and the excised QT domain of p57 exhibit significant deviations from the average composition of globular proteins that are consistent with the observed intrinsic disorder . However, the amino acid composition of the CDK inhibition domain of p57 does not exhibit such a striking deviation from the average values observed for proteins, implying that a general low level of hydrophobicity, rather than depletion or enrichment in specific amino acids, contributes to the intrinsic disorder of the excised p57 CDK inhibition domain .

Proteins, 2001 Dec 1, 45(4), 471 - 7
Identification of protein fold and catalytic residues of gamma-hexachlorocyclohexane dehydrochlorinase LinA; Nagata Y et al.; gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level and for which the evolutionary origin is unknown . We here propose that LinA is a member of a novel structural superfamily of proteins containing scytalone dehydratase, 3-oxo-Delta(5)-steroid isomerase, nuclear transport factor 2, and the beta-subunit of naphthalene dioxygenase-all known structures with different functions . The catalytic and the active site residues of LinA are predicted on the basis of its homology model . Nine mutants that carry substitutions of the proposed catalytic residues were constructed by site-directed mutagenesis . In addition to these, eight mutants that have a potential to make contact with the substrate were prepared by site-directed mutagenesis . These mutants were expressed in Escherichia coli, and their activities in crude extract were evaluated . Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity . We conclude that LinA catalyzes the proton abstraction via the catalytic dyad H73-D25 by a similar mechanism as described for scytalone dehydratase . The results suggest that LinA and scytalone dehydratase evolved from a common ancestor . LinA may have evolved from an enzyme showing a dehydratase activity .

Eur J Immunol, 2001 Dec, 31(12), 3475 - 83
Lack of specific receptors for C-reactive protein on white blood cells; Hundt M et al.; The classic acute-phase reactant C-reactive protein (CRP) plays an important role in innate immunity . Specific CRP receptors have been described on white blood cells and were further characterized as Fcgamma receptors I and II . Here, we used biotinylated, highly purified natural CRP and recombinant human CRP from E . coli to investigate binding to white blood cells . The structural integrity of recombinant CRP was demonstrated by proof of pentamer assembly using non-denaturing gel electrophoresis . Furthermore, the functional capability was confirmed by calcium-dependent ligand binding (phosphorylcholine-coupled BSA and nuclear constituents), and by complement activation (C3 deposition) . The monocytic cell line U937 expresses FcgammaRI and FcgammaRII--the proposed CRP receptors--in high density . Binding of biotinylated CRP was only detected by flow cytometry using a partially purified CRP preparation, that contained additional proteins, e.g . IgG as demonstrated by immunoblotting . Highly purified and recombinant CRP, free of IgG, were not bound . To exclude blocking of binding epitopes by labeling on recombinant CRP, biotinylation was performed at various biotin to protein ratios . In addition, competition assays demonstrated that binding of biotinylated, partially purified CRP was only inhibited by partially purified CRP and IgG, but not by highly purified and recombinant CRP . Recombinant CRP bound to U937 cells only after contamination with 0.5 microg IgG per 100 microg CRP before biotinylation . Therefore, we conclude that CRP itself is not bound to white blood cells and strongly suggest a reassessment of previous data.

Biotechnol Bioeng, 2002 Jan 5, 77(1), 27 - 36
Characterizing the metabolic phenotype: a phenotype phase plane analysis; Edwards JS et al.; Genome-scale metabolic maps can be reconstructed from annotated genome sequence data, biochemical literature, bioinformatic analysis, and strain-specific information . Flux-balance analysis has been useful for qualitative and quantitative analysis of metabolic reconstructions . In the past, FBA has typically been performed in one growth condition at a time, thus giving a limited view of the metabolic capabilities of a metabolic network . We have broadened the use of FBA to map the optimal metabolic flux distribution onto a single plane, which is defined by the availability of two key substrates . A finite number of qualitatively distinct patterns of metabolic pathway utilization were identified in this plane, dividing it into discrete phases . The characteristics of these distinct phases are interpreted using ratios of shadow prices in the form of isoclines . The isoclines can be used to classify the state of the metabolic network . This methodology gives rise to a "phase plane" analysis of the metabolic genotype-phenotype relation relevant for a range of growth conditions . Phenotype phase planes (PhPPs) were generated for Escherichia coli growth on two carbon sources (acetate and glucose) at all levels of oxygenation, and the resulting optimal metabolic phenotypes were studied . Supplementary information can be downloaded from our website .

Biotechnol Bioeng, 2001 Dec, 76(4), 333 - 40
On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation; Hoffmann F et al.; Human basic fibroblast growth factor (hFGF-2) was produced in high-cell density cultures of recombinant Escherichia coli using a temperature-inducible expression system . The synthesis rates of proteins were followed by two-dimensional gel electrophoresis of the (35)S-methionine-labeled proteom . After temperature induction of hFGF-2 synthesis, the rate of total protein synthesis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins . The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 gene but followed time profiles different from the producing strain . The energy demand for the extra synthesis of plasmid-encoded and heat-shock proteins resulted in an elevated respiratory activity and, consequently, in a reduction of the growth rate and the biomass yield . A procedure was developed to relate the energy demand for the additional synthesis of these proteins to the generation of energy in the respiratory pathway . Specific energy production was estimated based on on-line measurable rates of oxygen consumption, or carbondioxide evolution and growth, respectively . In this way, the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins was quantified from on-line accessible data .

Biotechnol Bioeng, 2001 Dec, 76(4), 318 - 24
Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp . with surface-expressed organophosphorus hydrolase; Shimazu M et al.; Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP) . The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation . A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E . coli and Moraxella sp . However, whole cell activity was 70-fold higher in Moraxella sp . than E . coli . The resulting Moraxella sp . degraded organophosphates as well as PNP rapidly, all within 10 h . The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively . The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future .

Biotechnol Bioeng, 2001 Dec 5, 75(5), 615 - 8
Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation; Nasseau M et al.; How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases . However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate . To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli . In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes .

J Biol Chem, 2002 Mar 1, 277(9), 7369 - 76 Epub 2001 Dec 14.
Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants; Chen YH et al.; Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP) . In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change . (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change . This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate . Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution . Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding . Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+) . However, during the binding of E.FsPP with IPP, only the fastest phase was observed . These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change . The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.

J Biol Chem, 2002 Mar 22, 277(12), 9645 - 54 Epub 2001 Dec 14.
Apolipoprotein A-I alpha -helices 7 and 8 modulate high density lipoprotein subclass distribution; Reschly EJ et al.; Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3) . Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile . Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution . The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8) . The recombinant proteins were examined for their association with human plasma HDL subclasses . The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially . T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI . Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association . Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed . Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I . These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.

J Biol Chem, 2002 Feb 22, 277(8), 6032 - 6 Epub 2001 Dec 13.
Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu . Contribution of contacts at the guanine base; Wieden HJ et al.; Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts) . Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu . In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis . The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP . Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation . The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later . This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.

J Appl Physiol, 2002 Jan, 92(1), 279 - 87
Lung albumin accumulation is spatially heterogeneous but not correlated with regional pulmonary perfusion; Gerbino AJ et al.; The contribution of pulmonary perfusion heterogeneity to the development of regional differences in lung injury and edema is unknown . To test whether regional differences in pulmonary perfusion are associated with regional differences in microvascular function during lung injury, pigs were mechanically ventilated in the prone position and infused with endotoxin (Escherichia coli 055:B5, 0.15 microg . kg(-1) . h(-1); n = 8) or saline (n = 4) for 4 h . Extravascular albumin accumulation and perfusion were measured in multiple approximately 0.7-ml lung regions by injecting pigs with radiolabeled albumin and radioactive microspheres, respectively . Extravascular albumin accumulation was spatially heterogeneous but not correlated with regional perfusion . Extravascular albumin accumulation was greater in dorsal than ventral regions, and regions with similar albumin accumulation were spatially clustered . This spatial organization was less evident in endotoxemic than control pigs . We conclude that there are regional differences in lung albumin accumulation that are spatially organized but not mediated by regional differences in pulmonary perfusion . We speculate that regional differences in microvascular pressure or endothelial function may account for the observed distribution of extravascular albumin accumulation.

J Inorg Biochem, 2001 Dec 15, 87(4), 245 - 51
Characterization of NO binding to human cystathionine beta-synthase: possible implications of the effects of CO and NO binding to the human enzyme; Taoka S et al.; Homocysteine is a key junction metabolite that can be converted to cystathionine in a reaction catalyzed by the heme and pyridoxal phosphate-dependent cystathionine beta-synthase . The heme has unusual spectroscopic properties and the axial ligands have been assigned as histidine and cysteine, respectively . Its role in the protein is not obvious from the chemistry of the beta-replacement reaction that is catalyzed . We have characterized the binding of the gaseous signaling molecule, NO, to cystathionine beta-synthase and examined its effect on the reactions catalyzed by the truncated dimeric form of the enzyme, W409X, which is a natural variant . Binding of NO appears to result in the formation of a five-coordinate ferrous nitrosyl species in which both endogenous ligands have been lost . This is in contrast to CO binding which is reported to displace the thiolate ligand and form a six-coordinate species . NO binds to the full-length enzyme with a K(d) of 281+/-50 microM and to the truncated enzyme with a K(d) of 350+/-44 microM . Binding of NO to the full-length enzyme inhibits activity with a K(i) of 320+/-60 microM . These studies demonstrate that as with CO, perturbation of the heme environment by NO is communicated to the active site with concomitant inhibition of enzyme activity, and suggests a regulatory role for heme in cystathionine beta-synthase.

J Inorg Biochem, 2001 Dec 15, 87(4), 175 - 84
Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method; Ivanov YD et al.; Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method . It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system) . Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes) . Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle . At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems . For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered . The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.

Cardiovasc Res, 2002 Jan, 53(1), 156 - 64
Endotoxin induces desensitization of cardiac endothelin-1 receptor signaling by increased expression of RGS4 and RGS16; Patten M et al.; OBJECTIVE: Endotoxin (LPS)-induced acute cardiac failure during sepsis is associated with alterations in G protein mediated signal transduction . We therefore examined the expression of the G proteins G(i), G(q), and G(s) and of four 'regulators of G protein signaling' (RGS) proteins, RGS1, RGS4, RGS5, and RGS16 in rat hearts . METHODS: For in vivo experiments, Wistar rats were treated with 600 microg/day E . coli LPS, intravenously) and hearts were excised after 6, 24 and 72 h . Cultured neonatal rat cardiomyocytes were treated with 4 microg/ml LPS for 24 and 72 h . Isolated membrane proteins were used for Western blot analysis and for evaluation of phospholipase C (PLC) activity . RGS16 mRNA was detected by RNAse protection . RESULTS: LPS induced G(i) protein 1.4-fold 72 h after in vivo administration of LPS, whereas expression of G(s) and G(q) was unaltered . After 6 h of LPS treatment, RGS16 mRNA was transiently up-regulated 3.7-fold, followed by transient protein induction (24 h: 2.5-fold; 72 h: 1.5-fold) . Similarly, RGS4 protein was transiently induced (24 h: 3.1-fold; 72 h: 1.5-fold), whereas expression of RGS1 and RGS5 was not altered . Similar to the LPS-treated rat hearts, RGS16 expression was transiently induced by LPS in cultured neonatal rat cardiomyocytes (24 h: 1.6-fold, 72 h: 0.9-fold) . To determine the functional consequences of the RGS protein induction phospholipase C (PLC) activity was analyzed in membranes obtained from solvent and LPS-treated hearts . Basal and endothelin-1-stimulated PLC activity was transiently repressed by LPS with a maximum after 24 h although no apparent changes in PLCbeta1 or endothelin receptor expression could be detected . CONCLUSION: These data suggest that the rapid up-regulation of cardiac RGS4 and RGS16 is associated with a desensitization of endothelin-1 receptor signaling . Up-regulation of these RGS proteins may thus be involved in the early onset of cardiac failure during sepsis.

Biochem J, 2002 Jan 1, 361(Pt 1), 49 - 56
Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria; Harper JA et al.; Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice . Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein . This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein) . UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria . We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria . Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological . However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein . As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1 . Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact . We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

J Agric Food Chem, 2001 Dec, 49(12), 5755 - 60
4-(Methylthio)-3-butenyl isothiocyanate, a principal antimutagen in daikon (Raphanus sativus; Japanese white radish); Nakamura Y et al.; The antimutagenic activity of n-hexane extracts from eight strains of daikon (Raphanus sativus; Japanese white radish) have been examined using the UV-induced mutation assay of Escherichia coli B/r WP2 . A correlation was found between the potency of antimutagenicity and the amount of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) in their n-hexane extracts . Because the pure MTBITC also showed antimutagenicity, MTBITC is presumably the active antimutagen principle in n-hexane extracts of daikon . Among the eight strains of daikon studied, Aokubi, the improved common strain in Japan, contained 71.0 micromol of MTBITC in 100 g of fresh daikon . In contrast, Karami and Momoyama, which are original wild strains, contained much more MTBITC (363.5 and 168.0 micromol/100 g, respectively) . In addition, phenethyl isothiocyanate was found in a lesser amount (5-33 nmol/100 g) in eight strains of daikon, and allyl isothiocyanate and benzyl isothiocyanate were not detectable in any strains (<3 nmol/100 g) . The amount of total isothiocyanate in grated daikon was 7.0 times higher than that in cut daikon measured after 30 min of cooking . Through eating habits, humans might be able to consume substantial amounts of the antimutagen MTBITC from dishes using the grated form of wild strains of daikon . Therefore, it is possible to substantially increase the intake of the antimutagenic ingredient of daikon (i.e., MTBITC) by changing food preferences and preparation procedures (i.e., using the grated form of the wild strains).

J Mol Biol, 2001 Dec 14, 314(5), 1181 - 90
Dynamic association of trigger factor with protein substrates; Maier R et al.; Trigger factor is a ribosome-bound folding helper, which, apparently, combines two functions, chaperoning of nascent proteins and catalyzing prolyl isomerization in their folding . Immediate chaperone binding at the ribosome might interfere with rapid protein folding reactions, and we find that trigger factor indeed retards the in vitro folding of a protein with native prolyl isomers . The kinetic analysis of trigger factor binding to a refolding protein reveals that the adverse effects of trigger factor on conformational folding are minimized by rapid binding and release . The complex between trigger factor and a substrate protein is thus very short-lived, and fast-folding proteins can escape efficiently from an accidental interaction with trigger factor . Protein chains with incorrect prolyl isomers cannot complete folding and therefore can rebind for further rounds of catalysis . Unlike DnaK, trigger factor interacts with substrate proteins in a nucleotide-independent binding reaction, which seems to be optimized for high catalytic activity rather than for chaperone function . The synthetic lethality, observed when the genes for both DnaK and trigger factor are disrupted, might result from an indirect linkage . In the absence of trigger factor, folding is retarded and more aggregates form, which can neither be prevented nor disposed of when DnaK is lacking as well .

J Mol Biol, 2001 Dec 14, 314(5), 1167 - 79
GroEL channels the folding of thioredoxin along one kinetic route; Bhutani N et al.; Many proteins display complex folding kinetics, which represent multiple parallel folding pathways emanating from multiple unfolded forms and converging to the unique native form . The small protein thioredoxin from Escherichia coli is one such protein . The effect of the chaperonin GroEL on modulating the complex energy landscape that separates the unfolded ensemble from the native state of thioredoxin has been studied . It is shown that while the fluorescence change accompanying folding occurs in five kinetic phases in the absence of GroEL, only the two slowest kinetic phases are discernible in the presence of saturating concentrations of GroEL . This result is shown to be consistent with only one out of several available folding routes being operational in the presence of GroEL . It is shown that native protein, which forms via fast as well as slow routes in the absence of GroEL, forms only via a slow route in its presence . The effect of GroEL on the folding of thioredoxin is shown to be the consequence of it binding differentially to the many folding-competent forms . While some of these forms can continue folding when bound to GroEL, others cannot . All molecules are then drawn into the operational folding route by the law of mass action . This observation indicates a new role for GroEL, which is to bias the energy landscape of a folding polypeptide towards fewer available pathways . It is suggested that such channeling might be a mechanism to avoid possible aggregation-prone routes available to a refolding polypeptide in vivo .

J Mol Biol, 2001 Dec 14, 314(5), 1087 - 95
Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution; Gopal B et al.; Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population . This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery . NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes . Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains . They are organised as two distinct entities . The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains . The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain . The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination . They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli .

J Mol Biol, 2001 Dec 14, 314(5), 1077 - 85
Archaeal RadA protein binds DNA as both helical filaments and octameric rings; Yang S et al.; The Escherichia coli RecA protein has been a model for understanding homologous eukaryotic recombination proteins such as Rad51 . The active form of both RecA and Rad51 appear to be helical filaments polymerized on DNA, in which an unusual helical structure is induced in the DNA . Surprisingly, the human meiosis-specific homolog of RecA, Dmc1, has thus far only been observed to bind DNA as an octameric ring . Sequence analysis and biochemical studies have shown that archaeal RadA proteins are more closely related to Rad51 and Dmc1 than the bacterial RecA proteins . We find that the Sulfolobus solfataricus RadA protein binds DNA in the absence of nucleotide cofactor as an octameric ring and in the presence of ATP as a helical filament . Since it is likely that RadA is closely related to a common ancestral protein of both Rad51 and Dmc1, the two DNA-binding forms of RadA may provide insight into the divergence that has taken place between Rad51 and Dmc1 .

J Mol Biol, 2001 Dec 14, 314(5), 1067 - 75
Modulation of Lrp action in Escherichia coli by leucine: effects on non-specific binding of Lrp to DNA; Chen S et al.; Lrp is a global regulator of metabolism in Escherichia coli that helps cells respond to changes in environmental conditions . The action of Lrp as a transcriptional activator or repressor is sometimes affected by whether the medium contains exogenous leucine . The abundance of Lrp in cells is relatively high (about 15 microM in monomer), and given the relatively high Lrp binding affinity in vitro for specific binding sites (nanomolar apparent dissociation constants), the expectation is that all binding sites will be saturated with Lrp in vivo . Here we consider the fraction of the total Lrp in cells that is free and the fraction that is bound to DNA . Using minicell-producing strains, we measured the distribution of Lrp between cytoplasm and nucleoid in cells grown under different nutritional conditions and in cells in different phases of growth . In E . coli cells grown in minimal medium to mid-log phase, the ratio of free to DNA-bound Lrp was about 0.67 . This ratio decreased about threefold when the cells were grown in minimal medium supplemented with leucine . Our results also confirmed the previous finding that growth rate regulates lrp expression by as much as three to fourfold . Growth rate-regulated lrp expression, along with changes in the extent of non-specific binding, influences the level of free Lrp in vivo over a 16-fold range . We propose that the net effect of these processes is to regulate the relative concentrations of free Lrp hexadecamer and leucine-bound octamer, leading to promoter selection in response to environmental conditions .

Anal Biochem, 2002 Jan 1, 300(1), 77 - 86
Quantitative analysis of tryptophan analogue incorporation in recombinant proteins; Senear DF et al.; Three different methods to quantitate tryptophan (Trp) analogue incorporation into recombinant proteins are described: first, spectroscopic analysis based on a linear combination of the absorption spectra of the aromatic residues in the denatured Trp-containing or analogue-substituted protein; second, chromatographic separation of analogue-substituted and Trp-containing proteins by HPLC; and third, mass spectrum analysis of the mixture of analogue-substituted and Trp-containing proteins . An accurate estimate of analogue incorporation in single-Trp proteins can be obtained directly by either analysis of the absorption spectrum or HPLC chromatography . While analysis of the absorption spectrum or HPLC chromatogram can provide an assessment of the average level of analogue incorporation for proteins that contain two or more Trp residues, mass spectroscopy analysis of peptides generated by protease digestion and separated by HPLC provides a general method for a complete quantitative description of the distribution of analogue incorporation . The more complex analysis by mass spectroscopy becomes important for multi-Trp proteins because the distribution of analogue versus Trp-containing polypeptide chains may not be the same as that predicted on the basis of average level of analogue incorporation . (c)2002 Elsevier Science.

Science, 2001 Dec 14, 294(5550), 2353 - 7
Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF; de Groot BL et al.; "Real time" molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented . We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels . Both proteins act as two-stage filters: Conserved fingerprint {asparagine-proline-alanine (NPA)} motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter . Hydrophobic regions near the NPA motifs are rate-limiting water barriers . In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity . In GlpF, a glycerol-mediated "induced fit" gating motion is proposed to generate selectivity for glycerol over water.

Plant Physiol, 2001 Dec, 127(4), 1667 - 75
Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus; Chisholm ST et al.; Restriction of long-distance movement of tobacco etch virus (TEV) in Arabidopsis ecotype Col-0 plants requires the function of at least three genes: RTM1 (restricted TEV movement 1), RTM2, and RTM3 . The mechanism of TEV movement restriction remains poorly understood, although it does not involve a hypersensitive response or systemic acquired resistance . A functional characterization of RTM1 and RTM2 was done . The RTM1 protein was found to be soluble with the potential to form self-interacting complexes . The regulatory regions of both the RTM1 and RTM2 genes were analyzed using reporter constructs . The regulatory sequences from both genes directed expression of beta-glucuronidase exclusively in phloem-associated cells . Translational fusion proteins containing the green fluorescent protein and RTM1 or RTM2 localized to sieve elements when expressed from their native regulatory sequences . Thus, components of the RTM system may function within phloem, and sieve elements in particular, to restrict TEV long-distance movement.

EMBO J, 2001 Dec 17, 20(24), 7313 - 22
The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA; Bork JM et al.; The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps . The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding . In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs . RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly . RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments . An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps . This function is countered by the RecF/RecO competition for association with the RecR protein.

EMBO J, 2001 Dec 17, 20(24), 7284 - 93
A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H; Mansell JB et al.; Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences . Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency . The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present . The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA . Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate . The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.

EMBO J, 2001 Dec 17, 20(24), 7160 - 7
A simple mechanism for co-dependence on two activators at an Escherichia coli promoter; Wade JT et al.; The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP . In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence . MelR binds to four sites, centred at positions -42.5, -62.5, -100.5 and -120.5 relative to the melAB transcription start point . When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions -62.5 and -100.5 . This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation . We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter . At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase alpha subunit . Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase . Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome.

Am J Physiol Regul Integr Comp Physiol, 2002 Jan, 282(1), R311 - 6
Fever responses of Zucker rats with and without fatty mutation of the leptin receptor; Ivanov AI et al.; Leptin is thought to be involved in febrigenic signaling from the periphery to the brain . Zucker obese rats have a so-called fatty mutation in the leptin receptor gene and express a dysfunctional protein . Studies comparing the fever responses of fatty (fa/fa) rats and of their lean (Fa/Fa and Fa/fa) counterparts yield contradictory results . To resolve these contradictions, we evaluated the effect of fatty mutation on infectious and stress-associated fevers at thermoneutrality (29 degrees C) and in a cool environment (20 degrees C) . Zucker fa/fa and Fa/? rats were infused with Escherichia coli lipopolysaccharide (LPS; 10 microg/kg) through a jugular catheter (infectious fever) or with saline through the catheter (control) or received a painful intramuscular injection of saline (stress fever) . At thermoneutrality, the colonic temperature (T(c)) responses of fatty rats to all stimuli tested were no different from the responses of lean rats . In a cool environment, T(c) responses of fatty rats to all stimuli were ~0.5 degrees C lower than those of lean rats . The observed attenuation of LPS-induced and stress-associated fevers in Zucker fatty rats in the cold agrees with the literature data showing that brown adipose tissue (the major heat production effector) is morphologically and functionally defective in these rats . The normal febrile responses of fatty Zucker rats to pyrogenic stimuli at thermoneutrality indicate that fatty mutation does not interrupt febrigenic signaling from the periphery to the brain.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Jan, 131(1), 37 - 46
Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA; Cheung HY et al.; The cDNA encoding the goldfish (Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector . The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni(2+) column . The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography . Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum . The IgG fraction of the antiserum was isolated on an immobilized Protein A-agarose column . The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses . The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA . The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.

Biosens Bioelectron, 2002 Jan, 17(1-2), 119 - 31
High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants; Tsotsou GE et al.; A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described . The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions . The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE) . The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate . Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3 . The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates . Three derivatives were identified as substrates . The method gave a significant different signal for two isomeric furazan derivatives . All results found on the cell lysate were verified and confirmed with the purified enzyme . This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

J Virol Methods, 2002 Feb, 100(1-2), 121 - 31
A rapid and easy method for production and selection of recombinant adenovirus genomes; Renaut L et al.; Adenoviruses are used widely as vectors for gene therapy . Due to the large size of their genome there is a low frequency of unique restriction sites and many techniques have been described to construct recombinant viruses . Whatever the considered technique, the Escherichia coli strain BJ5183 is used to obtain recombinant adenovirus genomes in a plasmid, or to construct defective viral backbones which will be used to produce infectious viral particles by homologous recombination in HEK293 cells . Unfortunately BJ5183 bacteria do not produce a sufficient amount of plasmid DNA to allow for restriction analysis . Plasmids have to be transferred into another strain to detect the expected construction . It is reported now that the common E . coli strain, Top10F' can be used for the construction of recombinant adenovirus genomes . A plasmid carrying a kanamycin resistance gene and containing the two ends of the adenovirus genome was used . It permits modification by classical molecular biology techniques or homologous recombination at both ends of the genome . The remainder of the genome is introduced by homologous recombination in Top10F' . Several homologous recombination steps were successfully performed without the steps of extraction and introduction of plasmid DNA in another strain to check the plasmids obtained.

Biochem J, 2002 Jan 1, 361(Pt 1), 143 - 51
Isolation and characterization of a Drosophila homologue of mitogen-activated protein kinase phosphatase-3 which has a high substrate specificity towards extracellular-signal-regulated kinase; Kim SH et al.; A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library . DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8 kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3 . Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity . In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells . DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain . In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro . DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development . Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes.

Biochem J, 2002 Jan 1, 361(Pt 1), 119 - 23
Monomers of the catalytic domain of human neuropathy target esterase are active in the presence of phospholipid; Atkins J et al.; NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727-1216) of neuropathy target esterase . NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions . Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis . The pattern of digestion by proteinase K of NEST-phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium . Significant quantities of NEST were liberated from NEST-phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values . Treatment of NEST-phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers . Similarly, the inactivation of esterase activity in NEST-phospholipid complexes by radiation indicated that NEST monomers are catalytically active . The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.

Protein Sci, 2002 Jan, 11(1), 147 - 57
Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA; Kapur A et al.; Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication . We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS) . ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source . The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful . Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined . These were in agreement with the dissociation constants determined in solution studies . A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of <or=2 x 10(-9) M for Tus where A173 was unchanged . This is the first example in which ESI-MS has been used to compare binding affinities of a DNA-binding protein with mutant proteins for specific DNA recognition sequences . It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.

Protein Sci, 2002 Jan, 11(1), 58 - 64
Characterization of the W321F mutant of Mycobacterium tuberculosis catalase-peroxidase KatG; Yu S et al.; A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis . The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry . Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested . No evidence for Compound II was detected during the spontaneous or substrate-accelerated decay of Compound I . The binding of isoniazid, a first-line anti-tuberculosis pro-drug activated by catalase-peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild-type enzyme . An EPR signal assigned to a protein-based radical tentatively assigned as tyrosyl radical in wild-type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide . These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase . Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants.

Protein Eng, 2001 Nov, 14(11), 943 - 8
Sucrose transport through maltoporin mutants of Escherichia coli; Van Gelder P et al.; Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively . To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB . Three LamB mutants were studied . (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction . The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel . (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A) . Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best . The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure . Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.

Protein Eng, 2001 Nov, 14(11), 929 - 37
Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression; Sroga GE et al.; Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied . Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity . Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide . After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE . In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A . Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes . Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.

Protein Eng, 2001 Nov, 14(11), 903 - 9
The mouse guanylate kinase double mutant E72Q/D103N is a functional adenylate kinase; Stolworthy TS et al.; Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an important enzyme in nucleotide metabolic pathways . Because of its essential intracellular role, guanylate kinase is a target for a number of cancer chemotherapeutic agents such as 6-thioguanine and 8-azaguanine and is involved in antiviral drug activation . Guanylate kinase shares a similarity in function and structure to other nucleoside monophosphate kinases especially with that of the well-studied adenylate kinase . Amino acid substitutions were made within the GMP binding site of mouse guanylate kinase to alter the polarity of the side chains that interact with GMP as a means of evaluating the role that these residues play on substrate interaction . One of these mutants, E72Q/D103N, was shown by functional complementation and enzyme assays to embody both guanylate kinase activity and a novel adenylate kinase activity.

Protein Eng, 2001 Nov, 14(11), 897 - 901
Four-helix bundle topology re-engineered: monomeric Rop protein variants with different loop arrangements; Kresse HP et al.; We converted the small homodimeric four-helix bundle repressor of primer protein (Rop) into a monomeric four-helix bundle by introduction of connecting loops . Both left- and right-handed four-helix bundles were produced . The left-handed bundles were more stable and were used to introduce biologically interesting peptides in one of the loops.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14883 - 8 Epub 2001 Dec 11.
DNA supercoiling allows enhancer action over a large distance; Liu Y et al.; Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance . The mechanism of enhancer action over large distance is unknown . Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro . The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp) . DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14853 - 8 Epub 2001 Dec 11.
Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase; Yamada K et al.; Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the major methyl donor for the conversion of homocysteine to methionine . Two common polymorphisms of the human enzyme have been identified: 677C>T, which leads to the substitution of Ala-222 by valine, and 1298A>C, which leads to the replacement of Glu-429 by alanine; the former polymorphism is the most frequent genetic cause of mild hyperhomocysteinemia, a risk factor for cardiovascular disease . By using a baculovirus expression system, recombinant human MTHFR has been expressed at high levels and purified to homogeneity in quantities suitable for biochemical characterization . The Glu429Ala protein has biochemical properties that are indistinguishable from the wild-type enzyme . The Ala222Val MTHFR, however, has an enhanced propensity to dissociate into monomers and to lose its FAD cofactor on dilution; the resulting loss of activity is slowed in the presence of methyltetrahydrofolate or adenosylmethionine . This biochemical phenotype is in good agreement with predictions made on the basis of studies comparing wild-type Escherichia coli MTHFR with a mutant, Ala177Val, homologous to the Ala222Val mutant human enzyme {Guenther, B . D., et al . (1999) Nat . Struct . Biol . 6, 359-365}.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14895 - 900 Epub 2001 Dec 11.
IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins; Schwartz CJ et al.; IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA . IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors . In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon . In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter . Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a {2Fe-2S}(1+) cluster . The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA . The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15257 - 9 Epub 2001 Dec 11.
Genetic control of manno(fructo)kinase activity in Escherichia coli; Sproul AA et al.; Mutants of Escherichia coli unable to use fructose by means of the phosphoenolpyruvate/glycose phosphotransferase system mutate further to permit growth on that ketose by derepression of a manno(fructo)kinase (Mak(+) phenotype) present in only trace amounts in the parent organisms (Mak-o phenotype) . The mak gene was located at min 8.8 on the E . coli linkage map as an ORF designated yajF, of hitherto unknown function; it specifies a deduced polypeptide of 344 aa . The derepression of Mak activity was associated with a single base change at position 71 (codon 24) of the gene, where GCC (alanine) in Mak-o has been changed to GAC (aspartate) in Mak(+) . By cloning selected portions of the total 1,032-bp mak gene into a plasmid that also carried a temperature-sensitive promoter, we showed that the mutation resided in a 117-bp region that does not specify sequences necessary for Mak activity but was located 46 bp upstream of a 915-bp portion that does . Mak(+) and Mak-o strains differ greatly in the heat stability of the enzyme: at 61 degrees C, mak-o cloned into a mak-o recipient loses 50% of its activity in approximately 6 min, whereas it takes over 30 min to achieve a similar reduction in the activity of mak(+) cloned into a mak-o strain . However, the Mak activity of the cloned fragment specifying the enzyme without the regulatory region lost activity with a half-life of 29 min irrespective of whether it was derived from a mak(+) or a mak-o donor, which indicates that the A24D mutation contributes to the high enzyme activity of Mak(+) mutants by serving to protect Mak from denaturation.

J Biol Chem, 2002 Feb 22, 277(8), 5982 - 7 Epub 2001 Dec 12.
Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage; Ren YG et al.; Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail . PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues . Here, we show by site-directed mutagenesis that these residues of human PARN, i.e . Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex . We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN . Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites . Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine . This suggests that these residues coordinate divalent metal ions . We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.

J Biol Chem, 2002 Feb 15, 277(7), 5163 - 7 Epub 2001 Dec 12.
Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA; Levit MN et al.; Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue . It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria . Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm) . These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP . Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA . Previous in vivo functions of E . coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions . The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.

J Biol Chem, 2002 Mar 8, 277(10), 7790 - 8 Epub 2001 Dec 12.
In vivo protein cyclization promoted by a circularly permuted Synechocystis sp . PCC6803 DnaB mini-intein; Williams NK et al.; A synthetic Synechocystis sp . PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli . The system was used to cyclize the NH(2)-terminal domain of E . coli DnaB, the structure of which had been determined previously by NMR spectroscopy . Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield . The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher . Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile . The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form . Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

J Biol Chem, 2002 Feb 22, 277(8), 6005 - 11 Epub 2001 Dec 12.
The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature; Xia B et al.; The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs . This region forms a stable stem-loop structure followed by an AU-rich sequence . Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50% . The AU-rich track, however, slightly destabilizes the mRNA . The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important . Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression . Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes . Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.

J Biol Chem, 2002 Feb 22, 277(8), 5970 - 6 Epub 2001 Dec 12.
IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli; Hou Z et al.; A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site . The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A . Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme . More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element) . Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away . The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.

J Biol Chem, 2002 Mar 1, 277(9), 6929 - 33 Epub 2001 Dec 12.
The mutation K30D disrupts the only salt bridge at the subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis and changes the mechanism of cooperativity; Ceci P et al.; The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89 . Upon mutation of Lys-30 to Asp, the interface is destabilized markedly . Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K(1,2D) = 8 x 10(4) M(-1)) and for the CO-bound (K(1,2L) = 1 x 10(3) m(-1)) and oxygenated (K(1,2L) = 70 m(-1)) derivatives . For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure . In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration . At low protein concentrations (e.g . 1.2 x 10(-5) m heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases . At protein concentrations where both derivatives are dimeric (e.g . 3.3 x 10(-3) m heme), both cooperativity and oxygen affinity decrease . Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.

J Biol Chem, 2002 Feb 22, 277(8), 6333 - 43 Epub 2001 Dec 12.
Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling; Ligos JM et al.; PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control . Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates . One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species . The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo . Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues . Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs . After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein . GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo . In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein . These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.

J Biol Chem, 2002 Mar 1, 277(9), 6806 - 12 Epub 2001 Dec 12.
Spinesin/TMPRSS5, a novel transmembrane serine protease, cloned from human spinal cord; Yamaguchi N et al.; A cDNA encoding a novel serine protease, which we designated spinesin, has been cloned from human spinal cord . The longest open reading frame was 457 amino acids . A homology search revealed that the human spinesin gene was located at chromosome 11q23 and contained 13 exons, the gene structure being similar to that of TMPRSS3 whose gene is also located on 11q23 . Spinesin has a simple type II transmembrane structure, consisting of, from the N terminus, a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger receptor-like domain, and a serine protease domain . Unlike TMPRSS3, it carries no low density lipoprotein receptor domain in the stem region . The extracellular region carries five N-glycosylation sites . The sequence of the protease domain carried the essential triad His, Asp, and Ser and showed some similarity to that of TMPRSS2, hepsin, HAT, MT-SP1, TMPRSS3, and corin, sharing 45.5, 41.9, 41.3, 40.3, 39.1, and 38.5% identity, respectively . The putative mature protease domain preceded by H(6)DDDDK was produced in Escherichia coli, purified, and successfully activated by immobilized enterokinase . Its optimal pH was about 10 . It cleaved synthetic substrates for trypsin, which is inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride but not by antipain or leupeptin . Northern blot analysis against mRNA from human tissues including liver, lung, placenta, and heart demonstrated a specific expression of spinesin mRNA in the brain . Immunohistochemically, spinesin was predominantly expressed in neurons, in their axons, and at the synapses of motoneurons in the spinal cord . In addition, some oligodendrocytes were clearly stained . These results indicate that spinesin is transported to the synapses through the axons after its synthesis in the cytoplasm and may play important roles at the synapses . Further analyses are required to clarify its roles at the synapses and in oligodendrocytes.

J Biol Chem, 2002 Feb 15, 277(7), 5017 - 23 Epub 2001 Dec 11.
Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated; Paglia MJ et al.; Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin . We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35) . Phosphorylation of Pgamma at either residue caused a minor decrease--not the previously reported increase--in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers . Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits . The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R . H . (2001) J . Biol . Chem . 276, 27527-27534) . In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced . Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work . In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only approximately 10% of the endogenous Pgamma was phosphorylated . This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme . We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6 . However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g . mitogen-activated protein kinase; Wan, K . F., Sambi, B . S., Frame, M., Tate, R., and Pyne, N . J . (2001) J . Biol . Chem . 276, 37802-37808) in photoreceptor cells.

J Biol Chem, 2002 Mar 29, 277(13), 10861 - 8 Epub 2001 Dec 10.
Characterization of Escherichia coli null mutants for glutaredoxin 2; Vlamis-Gardikas A et al.; Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase . To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations . Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties . Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides . Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1 . grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase . Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway . The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.

J Biol Chem, 2002 Mar 8, 277(10), 8626 - 34 Epub 2001 Dec 10.
The crystal structure of indoleglycerol-phosphate synthase from Thermotoga maritima . Kinetic stabilization by salt bridges; Knochel T et al.; The crystal structure of the thermostable indoleglycerol-phosphate synthase from Thermotoga maritima (tIGPS) was determined at 2.5 A resolution . It was compared with the structures of the thermostable sIGPS from Sulfolobus solfataricus and of the thermolabile eIGPS from Escherichia coli . The main chains of the three (beta alpha)(8)-barrel proteins superimpose closely, and the packing of side chains in the beta-barrel cores, as well as the architecture of surface loops, is very similar . Both thermostable proteins have, however, 17 strong salt bridges, compared with only 10 in eIGPS . The number of additional salt bridges in tIGPS and sIGPS correlates well with their reduced rate of irreversible thermal inactivation at 90 degrees C . Only 3 of 17 salt bridges in tIGPS and sIGPS are topologically conserved . The major difference between the two proteins is the preference for interhelical salt bridges in sIGPS and intrahelical ones in tIGPS . The different implementation of salt bridges in the closely related proteins suggests that the stabilizing effect of salt bridges depends rather on the sum of their individual contributions than on their location . This observation is consistent with a protein unfolding mechanism where the simultaneous breakdown of all salt bridges is the rate-determining step.

J Biol Chem, 2002 Mar 1, 277(9), 7477 - 82 Epub 2001 Dec 10.
Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine . Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine; Huang Y et al.; D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase) . Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors . Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase . The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues . The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+) . However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD . The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes . The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues . The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+) . By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction . DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor . On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.

J Biol Chem, 2002 Feb 15, 277(7), 4704 - 12 Epub 2001 Dec 10.
The single transmembrane domains of ErbB receptors self-associate in cell membranes; Mendrola JM et al.; Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor . However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers . In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo . Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM . A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer . We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor . Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization . Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.

J Biol Chem, 2002 Mar 1, 277(9), 6934 - 42 Epub 2001 Dec 07.
Mechanism-based inhibition of enzyme I of the Escherichia coli phosphotransferase system . Cysteine 502 is an essential residue; Garcia-Alles LF et al.; Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system . The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP . The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations . Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm . E-Cl-PEP was not an inhibitor . Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI . Z-Cl-PEP is a suicide inhibitor . About 10-50 turnovers sufficed to inactivate EI completely . Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations . Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm . The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI . E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP . This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species . Cys-502 of EI was identified by mass spectrometry as the reacting residue . The C502A EI mutant showed less than 0.06% wild-type activity . Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.

J Biol Chem, 2002 Feb 15, 277(7), 5378 - 84 Epub 2001 Dec 07.
Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis; Reuter K et al.; We have solved the 2.5-A crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis . The structure reveals that the enzyme is present as a homodimer . Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain . The connective domain is responsible for dimerization and harbors most of the active site . The strictly conserved acidic residues Asp(150), Glu(152), Glu(231), and Glu(234) are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity . The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal . A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers . A possible involvement of this loop in an induced fit during substrate binding is discussed.

J Biol Chem, 2002 Feb 1, 277(5), 3733 - 42 Epub 2001 Dec 07.
Intricate interactions within the ccd plasmid addiction system; Dao-Thi MH et al.; The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid . It codes for a stable toxin (CcdB) and a less stable antidote (CcdA) . Both are expressed at low levels during normal cell growth . Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase . The interactions between CcdB, CcdA, and its promoter DNA were analyzed . In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry . CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one . CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates . The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA*CcdB*DNA complex with a CcdA:CcdB ratio of one . This (CcdA(2)CcdB(2))(n) complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.

J Bacteriol, 2002 Jan, 184(1), 233 - 40
Tn5 transposase with an altered specificity for transposon ends; Naumann TA et al.; Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon . The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences . Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE) . In Escherichia coli, IE is methylated by Dam methylase (IE(ME)) . This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons . Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE(ME)-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE(ME) defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons . This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.

J Bacteriol, 2002 Jan, 184(1), 152 - 64
Metabolic flux responses to pyruvate kinase knockout in Escherichia coli; Emmerling M et al.; The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with {U-13C6}glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model . The general response to disruption of both pyruvate kinase isoenzymes in E . coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme . Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions . In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E . coli, with a turnover that is comparable to the specific glucose uptake rate . Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E . coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate . Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

J Bacteriol, 2002 Jan, 184(1), 111 - 8
Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway; Tian H et al.; We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the Escherichia coli cytoplasmic membrane . The screen yielded mutations in each of the known genes encoding components of the E . coli signal recognition particle pathway: ffh, ffs, and ftsY, which encode Ffh, 4.5S RNA, and FtsY, respectively . In addition, the screen yielded mutations in secM, which is involved in regulating levels of the SecA component of the bacterium's protein export pathway . We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ . Among the mutations that resulted in membrane protein insertion defects, only the secM mutations also showed defects in the translocation of proteins into the E . coli periplasm . Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.

J Bacteriol, 2002 Jan, 184(1), 59 - 66
The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production; York GM et al.; Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules . Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis . Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase . Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation . R . eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting . The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected . In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type . This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA . Transfer of the R . eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly . Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway . Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

J Bacteriol, 2002 Jan, 184(1), 51 - 8
Regulation of the nfsA Gene in Escherichia coli by SoxS; Paterson ES et al.; In Escherichia coli, the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by the soxRS regulon . One member of the soxRS regulon, nfsA, encodes the major oxygen-insensitive nitroreductase in Escherichia coli which catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH . In this study we investigate the regulation of nfsA in response to the superoxide generating compound paraquat . The transcription start site (TSS) of nfsA was located upstream of the ybjC gene, a small open reading frame of unknown function located directly upstream of nfsA, suggesting that these two genes form an operon . The activity of the promoter associated with this TSS was confirmed with lacZ fusions and was shown to be inducible by paraquat . Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the -35 promoter sequence in the forward orientation, suggesting that the ybjC-nfsA promoter is a class I SoxS-dependent promoter.

Am J Physiol Lung Cell Mol Physiol, 2002 Jan, 282(1), L50 - 5
Escherichia coli FPG and human OGG1 reduce DNA damage and cytotoxicity by BCNU in human lung cells; He YH et al.; The pulmonary complications of 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) are among the most important dose-limiting factors of BCNU-containing cancer chemotherapeutic regimens . BCNU damages DNA of both cancer cells and normal cells . To increase the resistance of lung cells to BCNU, we employed gene transfer of Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) and human 8-oxoguanine-DNA glycosylase (hOGG1) to A549 cells, a lung epithelial cell line, using a bicistronic retroviral vector, pSF91-RE, that encoded both FPG/hOGG1 and an enhanced green fluorescent protein . The transduced epithelial cells were sorted by flow cytometry, and expression of FPG/hOGG1 protein was determined by the level of FPG/hOGG1 RNA and enzyme activity . The single-cell gel electrophoresis (comet assay) measured DNA damage induced by BCNU . FPG/hOGG1-expressing A549 cells incubated with 40-500 microg/ml BCNU exhibited significantly less DNA damage than vector-transduced cells . In addition, FPG- and/or hOGG1-expressing cells incubated with 10-40 microg/ml BCNU showed at least a 25% increase in cell survival . Gene transfer of FPG/hOGG1 reduced BCNU-induced DNA damage and cytotoxicity of cultured lung cells and may suggest a new mechanism to reduce BCNU pulmonary toxicity.

Bioorg Med Chem, 2002 Feb, 10(2), 325 - 32
Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin; Chui HM et al.; The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported {BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl} . The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine . Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated . Modifications were present at positions 1911, 1915, and 1917 . The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy

FEBS Lett, 2001 Dec 7, 509(2), 317 - 22
Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli; Hintz M et al.; The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli . In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E . coli, but is apparently absent in gcpE-deficient mutants . Here, this compound was purified from E . coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) . HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

FEBS Lett, 2001 Dec 7, 509(2), 298 - 302
Phosphatidylglycerophosphate synthases from Arabidopsis thaliana; Muller F et al.; Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively . Apart from their N-terminal regions, the two proteins exhibit high sequence similarity . Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS . The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus . Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A . thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity.

Mol Cell, 2001 Nov, 8(5), 1053 - 62
Crystal structure of colicin E3: implications for cell entry and ribosome inactivation; Soelaiman S et al.; Colicins kill E . coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it . The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site . The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms . Active site residues are D510, H513, E517, and R545 . IP is buried between T and C . Rather than blocking the active site, IP prevents access of the active site to the ribosome.

Cytokine, 2001 Nov 7, 16(3), 106 - 19
Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation; Leong SR et al.; A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework . Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding . Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo . The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile . PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region . In vitro binding and bioassays showed little or no loss of activity . The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits . Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold . In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody . These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity .

Cytokine, 2001 Nov 7, 16(3), 88 - 92
Molecular cloning, expression and characterization of the Canis familiaris interleukin-4; Wondimu A et al.; Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes . The rapid amplification of cDNA ends (RACE) was used to clone the canine IL-4 gene . It was expressed in mammalian cells and Escherichia coli . Monoclonal antibodies were raised to rcIL-4 for use in an enzyme-linked immunosorbent assay (ELISA) . This will facilitate studies on the role of cIL-4 in inflammatory diseases, particularly rheumatoid arthritis .

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1301 - 6
A novel RNase G mutant that is defective in degradation of adhE mRNA but proficient in the processing of 16S rRNA precursor; Wachi M et al.; Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA . Consequently, defects in RNase G result in elevation of AdhE levels . Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation . We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng . Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430 . DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430 . This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2) . However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants . We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced . These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1268 - 74
Chaperone-like properties of lysophospholipids; Kern R et al.; Lysophospholipids are metabolic intermediates in phospholipid turnover, detergent molecules with membrane-modulating effects, and multifunctional cellular growth factors in eukaryotic cells . In bacterial cells, lysophospholipids are mostly found in the form of lysophosphatidylethanolamine . We show that a heat shock from 30 to 42 degrees C increases four-fold the Escherichia coli pool of lysophosphoethanolamine and that lysophospholipids display chaperone-like properties . Lysophosphatidylethanolamine, like molecular chaperones such as DnaK, promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation . Like chaperones, lysophophatidylethanolamine, lysophosphatidylcholine, lysophosphatidylinositol and lysophosphatidic acid prevent the aggregation of citrate synthase at 42 degrees C . The renaturation and solubilisation of proteins by lysophospholipids occur at micromolar concentrations of these compounds, close to their critical micellar concentration . Furthermore, lysophosphatidylethanolamine is much more efficient than other detergents tested for the renaturation and solubilisation of citrate synthase . In contrast with lysophospholipids, phosphatidylethanolamine and phosphatidylcholine are not able to promote citrate synthase folding nor to prevent its aggregation at 42 degrees C . The chaperone-like properties of lysophospholipids suggest that, in addition to their known functions, they might affect the structure and function of hydrophilic proteins.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1257 - 61
Site-directed mutation on the only universally conserved residue Leu122 of small heat shock protein Hsp16.3; Mao Q et al.; Hsp16.3 from Mycobacterium tuberculosis belongs to the small heat shock protein family and has chaperone-like activity in vitro . The only universally conserved hydrophobic residue Leu122 was substituted by Val and Ala, respectively . The mutations on the Leu122 of Hsp16.3 have resulted in much lower structural stability in vivo and in vitro . Both mutant proteins exhibited much weaker chaperone-like activities than the Hsp16.3 WT under heat shock conditions . Taken together, the highly hydrophobic residue L122 of Hsp16.3 was suggested to play a very important role in maintaining not only the structural stability but also the chaperone-like activity.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1118 - 24
Glycine at the 65th position plays an essential role in ATP-dependent protein folding by Archael group II chaperonin; Iizuka R et al.; In the previous study, we have found that G65C and I125T double mutant of alpha chaperonin homo-oligomer from a hyperthermophilic archaeum, Thermococcus sp . strain KS-1, lacks ATP-dependent protein refolding activity despite showing ATPase activity and the ability to bind the denatured proteins . In this study, we have characterized several mutant Thermococcus chaperonin homo-oligomers with the amino acid substitutions of Gly-65 or Ile-125 . The results showed that amino acid residue at 65th position should be a small amino acid such as glycine or alanine for the ATP-dependent refolding activity . The alpha chaperonin homo-oligomers with amino acid substitution of Gly-65 by amino acids whose side chains are larger than the methyl group did not have ATP-dependent protein refolding activity, but exhibited an increase of the binding affinity for unfolded proteins in the presence of ATP or AMP-PNP . (c)2001 Elsevier Science.

J Infect Dis, 2001 Dec 15, 184(12), 1566 - 71 Epub 2001 Dec 03.
Inflammatory reactions in extraoral tissues in mice after intragingival injection of lipopolysaccharide; Funayama H et al.; Intragingival (ig) injection into mice of lipopolysaccharide (LPS) from Prevotella intermedia or Escherichia coli elevated the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), in the mandible, liver, lung, and spleen, with a time course similar to that seen with intravenous (iv) injection . The effect of i.g . injection was less than that of i.v . injection but similar to that of intraperitoneal (ip) injection . The i.g . injection also increased hepatic serotonin, reflecting platelet accumulation . In galactosamine-treated mice, the minimum ig dose of LPS needed to induce lethal hepatitis was very small (less than that needed by ip injection) . These results support the idea that the LPS produced in oral tissues may be transported easily to extraoral tissues and, in some cases, may cause inflammatory or immune responses . It also may influence the pathogenesis of some systemic diseases.

J Infect Dis, 2001 Dec 15, 184(12), 1556 - 65 Epub 2001 Nov 01.
Clonal and pathotypic analysis of archetypal Escherichia coli cystitis isolate NU14; Johnson JR et al.; Escherichia coli NU14, a cystitis isolate used to study the pathogenesis of cystitis and to develop a FimH (type 1 fimbrial adhesin) vaccine, was assessed for extended virulence genotype, phylogenetic background, and FimH sequence and binding phenotype(s) . NU14 exhibited the same virulence genotype and was derived from the same (meningitis- and cystitis-associated) subclone of E . coli O18:K1:H7 as the archetypal neonatal bacterial meningitis (NBM) isolate RS218 . NU14 also displayed the same Ser62Ala FimH polymorphism as did NBM isolates RS218 and IHE3034-conferring both collagen binding and a distinct monomannose binding capability (which characterizes uropathogenic but not commensal E . coli and dramatically increases adherence to uroepithelial cells) . These findings establish that strain NU14 exhibits numerous urovirulence-associated traits and derives from the single most prevalent clonal group in acute cystitis . They provide further evidence of clonal and pathotypic similarities between cystitis and NBM isolates of E . coli O18:K1:H7.

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