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J Mol Biol, 2002 May 10, 318(4), 1019 - 29
Substrate binding induces domain movements in orotidine 5'-monophosphate decarboxylase; Harris P et al.; Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate (UMP) . We have earlier determined the structure of ODCase from Escherichia coli complexed with the inhibitor 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid (BMP); here we present the 2.5 A structure of the uncomplexed apo enzyme, determined from twinned crystals . A structural analysis and comparison of the two structures of the E . coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12 degrees around a hinge that crosses the active site . Hence, the ODCase dimer, which contains two active sites, may be divided in three domains: a central domain that is fixed, and two lids which independently move 12 degrees upon binding . Corresponding analyses, presented herein, of the two Saccharomyces cerevisiae ODCase structures (with and without BMP) and the Methanobacterium thermoautotrophicum ODCase structures (with and without 6-aza UMP) show very similar, but somewhat smaller domain movements . The domain movements seem to be initiated by the phosphoryl binding to the enzyme and can explain why the binding of the phosphoryl group is essential for the catalytic function . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 975 - 83
Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon; Gyohda A et al.; Specific cleavages within the shufflon-specific recombination site of plasmid R64 were detected by primer extension when a DNA fragment carrying the recombination site was incubated with the shufflon-specific Rci recombinase . Rci-dependent cleavages occurred in the form of a 5' protruding 7 bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon system take place at these positions . As a result, shufflon crossover sites were designated as sfx sequences consisting of a central 7 bp spacer sequence, and left and right 12 bp arms . R64 sfx sequences are unique among various site-specific recombination sites, since only the spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the left arm sequence is not conserved and is not related to the right arm sequence . From nuclease protection analyses, Rci protein was shown to bind to entire R64 and artificial sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second to the sfx left arm in a non-specific manner . The sfx left arm sequences as well as the right arm sequences were shown to determine affinity to Rci and subsequently inversion frequency . Asymmetry of the sfx sequence may be the reason why Rci protein acts only on the inverted sfx sequences . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Apr 19, 318(1), 189 - 97
The crystal structure of a major dust mite allergen Der p 2, and its biological implications; Derewenda U et al.; The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209 . The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand . This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor . The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein .

J Mol Biol, 2002 Apr 19, 318(1), 149 - 59
Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA; Moscardini M et al.; All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome . Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection . The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro . Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process . To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1 . The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT . FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription . The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis .

J Mol Biol, 2002 Apr 19, 318(1), 135 - 47
Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen; Kaufmann M et al.; The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution . The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues . The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains . The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins . The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins . Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli . The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase . When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization . The experimental structure is now the basis for the design of antibodies with even higher stability and affinity .

Biochem Biophys Res Commun, 2002 May 31, 294(1), 191 - 7
Expression, purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor; Hough E et al.; The ATP-sensitive potassium channel is made up of four pore forming Kir6.2 subunits, surrounded by four regulatory sulphonylurea receptor (SUR) subunits . The latter subunit contains two nucleotide-binding folds (NBFs) that confer the ability on the channel to sense changes in the metabolic status ({ATP}/{ADP}) of the cell and couple the changes to the membrane potential of the cell . In an attempt to better understand the mechanisms by which NBFs influence the activity of the channel, we have expressed the NBF domains with C-terminally added epitopes (FLAG to NBF1 and His(6) to NBF2) in Escherichia coli and the rabbit reticulocyte lysate system and examined the ability of these domains to interact with each other and with Kir6.2 . Both NBFs could be expressed to high levels in E . coli and purified to homogeneity from inclusion bodies . Re-folding of the proteins proved to be unsuccessful . However, we were able to obtain small amounts of radio-labelled NBFs in a soluble state . Using co-immunoprecipitation, we demonstrate that the radio-labelled NBF1 and NBF2 interact with each other . Neither of the NBFs bound to Kir6.2 expressed in the presence of canine microsomes.

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1530 - 5
Successful genetic transduction in vivo into synovium by means of electroporation; Ohashi S et al.; This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues . Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes . Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium . The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm . When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times . Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product . Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis . The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1389 - 95
Hsc62, Hsc56, and GrpE, the third Hsp70 chaperone system of Escherichia coli; Yoshimune K et al.; Hsc62 is the third Hsp70 homolog of Escherichia coli, which we found previously . Hsc62 is structurally and biochemically similar to DnaK, but hscC gene encoding Hsc62 did not compensate for the defects in the dnaK-null mutant of E . coli MC4100 strain . We cloned the ybeV gene and purified the gene product named Hsc56, a 55,687-Da protein with a J-domain like sequence . Hsc56 stimulated the ATPase activity of only Hsc62 but not those of the other Hsp70 homologs, DnaK and Hsc66 . Hsc56 contains the -His-Pro-Glu- sequence corresponding to the His-Pro-Asp motif in DnaJ, which is indispensable for DnaJ to interact with DnaK . Conversion of -His-Pro-Glu- to -Ala-Ala-Ala- abolished the ability of Hsc56 to stimulate the ATPase activity of Hsc62 . GrpE, a nucleotide exchange factor for DnaK, also stimulated the ATPase activity of Hsc62 in the presence of Hsc56 . Hsc62-Hsc56-GrpE is probably a new Hsp70 chaperone system of E . coli.

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1377 - 82
Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure; Zolk O et al.; The technique of representational difference analysis of cDNA has been applied to screen for differentially expressed genes in a canine model of pacing-induced heart failure . We identified the canine homolog of the cardiac ankyrin repeat protein (CARP) which has been shown to be involved in the regulation of the transcription of cardiac genes . To confirm the significance for human heart failure, cardiac tissue specimens obtained from non-failing donor hearts and from explanted hearts from patients with end-stage heart failure were investigated . CARP mRNA and protein levels were markedly increased in failing left ventricles . Interestingly, alterations in CARP expression were restricted to ventricular tissue and were not observed in atria . Fractionation experiments revealed that CARP was expressed predominantly in the nuclei consistent with the proposed function of CARP as a modulator of transcription . Together, these findings raise the possibility that augmented ventricular CARP expression may play a role in the pathogenesis of human heart failure.

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1374 - 6
Characterization of the chondroitin sulfates in wild type Caenorhabditis elegans; Beeber C et al.; The purpose of this study was to isolate and characterize the GAGs from the wild type nematode Caenorhabditis elegans in preparation for the characterization of the transgenic form constructed by Link {Proc . Natl . Acad . Sci . USA 92 (1995) 9368} which expresses various forms of beta-peptide (or A4 peptide) . This peptide forms deposits very similar to the ones found in the neuritic plaques and neurofibrillary tangles in Alzheimer disease (AD) . Characterization has been accomplished by degradation with specific enzymes and analysis of the products by TLC and HPLC . The results were compared with earlier works and shown to differ in disaccharide content.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 542 - 8
Affinity improvement of the high-affinity immunoglobulin E receptor by phage display; Iwasaki A et al.; The immunoglobulin E (IgE)-binding site of its high-affinity receptor is localized in the second immunoglobulin-like domain (D2) of the alpha-subunit (Fc epsilon RI alpha) . In this study, the randomized pentapeptides were introduced between Glu(132) and Ile(138) of Fc epsilon RI alpha D2 and displayed on a filamentous phage . After eight rounds of panning, a phage clone having a mutation of Asp(135)Tyr(136)Met(137) in Fc epsilon RI alpha D2 was obtained . The binding affinity of the mutant phages to immobilized IgE was approximately 500 times higher than that of the wild type . The mutant phages competitively inhibited the binding of IgE to the soluble receptor at a 50% inhibition (IC(50)) value of 116 pM . The mutant Fc epsilon RI alpha D2, which had been expressed as a fusion protein with glutathione S-transferase in Escherichia coli, also showed higher IgE-binding capacity than the wild type . The mutant Fc epsilon RI alpha D2 is expected to manifest its improved IgE-binding affinity together with any fusion partner.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 537 - 41
Silent mutations affect in vivo protein folding in Escherichia coli; Cortazzo P et al.; As an approach to investigate the molecular mechanism of in vivo protein folding and the role of translation kinetics on specific folding pathways, we made codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five minor codons with their synonymous major ones . The altered region corresponds to a turn between two short alpha helices . One of the silent mutations of EgFABP1 markedly decreased the solubility of the protein when expressed in Escherichia coli . Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region seems to have special translation kinetic requirements that ensure proper folding of the protein . Our results highlight the importance of codon usage in the in vivo protein folding.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 344 - 8
Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes; Yasuda K et al.; Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination . Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences . In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages . Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs . pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages . Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs . Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 155 - 62
Characterization of the Mycoplasma hominis ftsZ gene and its sequence variability in mycoplasma clinical isolates; Momynaliev KT et al.; We cloned and sequenced Mycoplasma hominis chromosomal fragment containing ftsZ gene . The wild-type expression of the gene was shown at RNA level by reverse transcription followed by PCR amplification . We revealed that M . hominis FtsZ had a comparatively low similarity to proteins of Mycoplasma genitalium and Mycoplasma pneumoniae . After full ftsZ gene sequencing for 14 clinical isolates of M . hominis, single-nucleotide substitutions were found in 21 positions, 6 of them being common for almost all isolates . This ftsZ gene polymorphism may be used for subtyping of M . hominis in clinical samples . Expression of the M . hominis ftsZ gene in different Escherichia coli strains was also demonstrated, and M . hominis FtsZ protein was purified from E . coli cells transformed with recombinant expression plasmid . Complementation between the M . hominis FtsZ and E . coli FtsZ could be shown . The comparison of FtsZ protein structures may also be used for investigation of bacterial phylogenetic relationships.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 145 - 9
Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response; Beigneux AP et al.; Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection . Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively . Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver . These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment . LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h . LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486 . In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA . These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.

Biochem Biophys Res Commun, 2002 May 3, 293(2), 747 - 52
Chloroplast SecE: evidence for spontaneous insertion into the thylakoid membrane; Steiner JM et al.; SecE, an essential component of the bacterial SecAYEG translocase, mediates protein translocation across the cytoplasmic membrane . In the thylakoid membranes of chloroplasts an SecE homologue, cpSecE, has recently been identified . In this report we show that insertion of cpSecE does not require stromal extract, indicating that signal recognition particle is not involved . Removal of nucleoside triphosphates has apparently no effect on the integration, again ruling out an involvement of SRP or its partner protein, FtsY . The use of well-known inhibitors of the Sec- and Tat pathways, sodium azide and nigericin, respectively, also had no influence on membrane insertion . The data presented here point towards cpSecE as another passenger of a wholly spontaneous import/insertion pathway in the thylakoids of chloroplasts .

Biochem Biophys Res Commun, 2002 May 3, 293(2), 675 - 9
Expression and functional analysis of an inhibitor of apoptosis protein from Trichoplusia ni; Liao WT et al.; An inhibitor of the apoptosis protein (IAP) family gene from Trichoplusia ni, Tn-IAP1v, a variant of lepidopteran Tn-IAP1, was cloned by RT-PCR . There are six single nucleotide polymorphisms between the two Tn-IAP1 variants, resulting in three predicted single amino acid polymorphisms . With the GST fusion expression system, soluble recombinant Tn-IAP1v was highly expressed in Escherichia coli and then purified by affinity chromatography . Caspase inhibition assays indicated that recombinant Tn-IAP1v could specifically inhibit human caspase-9 in vitro instead of caspase-3, -7, and -8, which was further confirmed by the observation that recombinant Tn-IAP1v can directly bind caspase-9 in the protein pull-down assay . These results suggested that Tn-IAP1v might serve as an initiator caspase inhibitor in vivo in the conserved mitochondria apoptotic pathway .

Biochem Biophys Res Commun, 2002 May 17, 293(4), 1301 - 8
Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase; Hsiang CY; The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function . The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase . To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity . PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion . Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity . PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates . The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed . Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro . (c) 2002 Elsevier Science (USA).

Arch Biochem Biophys, 2002 May 1, 401(1), 81 - 90
Implication by site-directed mutagenesis of Arg314 and Tyr316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase; Lee P et al.; Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr316 is completely conserved and is equivalent to the E . coli Tyr345, which interacts with the 2'-phosphate of NADP in the crystal structure {Hurley et al., Biochemistry 30 (1991) 8671-8678} . Lys321 is also completely conserved in the five isocitrate dehydrogenases . Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg314 . While Arg323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation . The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis . Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method . Wild-type and mutant enzymes were expressed in E . coli and purified to homogeneity . All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme . Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements . The kinetic parameters (V(max) and K(M) values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys321 and Arg323 are not involved in enzyme function . R314Q exhibits a 10-fold increase in K(M) for NADP as compared to that of wild-type, while they have comparable V(max) values . These results suggest that Arg314 contributes to the affinity between the enzyme and NADP . The hydroxyl group of Tyr316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type . Y316L shows a 4-fold increase in K(M) for NADP and a decrease in V(max) as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis . The K(i) for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg314 and Tyr316 may be located near the 2'-phosphate of enzyme-bound NADP . (c) 2002 Elsevier Science (USA).

Arch Biochem Biophys, 2002 May 15, 401(2), 164 - 72
Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean; Vogel A et al.; Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence . In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells . kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T . Klabunde et al., Eur . J . Biochem . 226 (1994) 369-375) . N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum . A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker . Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP . Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP . The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents . (c) 2002 Elsevier Science (USA).

Arch Biochem Biophys, 2002 May 15, 401(2), 155 - 63
Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase; Poyner RR et al.; Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala . In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site . With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously {J.M . Brewer, C.V . Glover, M.J . Holland, L . Lebioda, Biochim . Biophys . Acta 1383 (2) (1998) 351-355} . Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining . Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate . Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities . The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA . The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH) . The structure was solved and refined at a resolution of 2.1 A . The structure confirms the conjecture that the active-site flap is opened in the mutant protein . PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex . Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein . His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center . A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site . (c) 2002 Elsevier Science (USA).

Arch Biochem Biophys, 2002 May 15, 401(2), 125 - 33
Canine sulfotransferase SULT1A1: molecular cloning, expression, and characterization; Tsoi C et al.; Sulfotransferases (SULTs) are involved in detoxification and activation of various endogenous and exogenous compounds including important drugs and hormones . SULT1A, the phenol-SULT subfamily, is the most prominent subfamily in xenobiotic metabolism and has been found in several species, e.g., human, rat, and mouse . We have cloned a phenol-sulfating phenol SULT from dog (cSULT1A1) and expressed it in Escherichia coli for characterization . cSULT1A1 showed 85.8, 82.7, 76.3, and 73.6% identities to human P-PST, human M-PST, rat PST-1, and mouse STp1, respectively . It consists of 295 amino acids, which is in agreement with the human ortholog and sulfate substrates typical for the SULT1A family, i.e., p-nitrophenol (PNP), alpha-naphthol, and dopamine . The K(m) for PNP was found to be within the nanomolar range . It also sulfates minoxidil and beta-estradiol but not dehydroepiandrosterone . Western blot analysis indicated that this newly cloned enzyme was found to be ubiquitously expressed in canine tissues with highest expression in male and female liver . (c) 2002 Elsevier Science (USA).

Anal Biochem, 2002 Jun 15, 305(2), 236 - 41
Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran; Ishida A et al.; A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described . These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission . However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants . When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution . The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx . The method was applied to the determination of ATP in Escherichia coli extracts . When a 5% solution of TCA was used for the extraction of ATP from E . coli cells, the detection limit corresponded to 250 cells ml(-1) of E . coli .

Anal Biochem, 2002 Jun 15, 305(2), 214 - 26
Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1; Daniels DA et al.; G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy . Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography . Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers . Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants . Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays . Eight aptamers demonstrated specificity for the neurotensin receptor . One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity . P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells . P19 represents the first example of a GPCR-specific RNA aptamer .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 93 - 98
Preparation and Use of a Monoclonal Antibodyagainst Luffin b; Yan M et al.; Luffin b is one of the most toxic single chain plant ribosome inactivating proteins . It has been successfully used to prepare an immunotoxin against human melanoma cells . Two strains of hybridomas (1E5 and 2E1) were screened out using cell fusion technique which steadily secreted monoclonal antibodies against luffin b . These antibodies specifically reacted with luffin b . The affinity constants of 1E5 and 2E1 monoclonal antibodies were determined to be 1.1x10(9) mol(-1).L and 7.5x10(8) mol(-)1.L, by RIA,respectively . An immunoaffinity gel composed with the 1E5 monoclonal antibody and Sepharose 4B was prepared . The luffin b was successfully purified by one step immunoaffinity chromatography from the crude extract of Luffa cylindrica seeds . An immunoconjugate 1E5-HRP was also prepared and it was successfully used in Western blotting for detection of recombinant luffin b from E.coli total proteins.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 82 - 86
Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts; Yang W et al.; The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD and 45 kD subunits . The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the genomic DNA degradation into nucleosomal fragments during apoptosis . As an inhibitor for DFF40, the 45 kD subunit (DFF45) complexes with DFF40, inhibiting DNase activity until certain apoptosis signals are received . In cells undergoing apoptosis, the cleavage of DFF45 by activated caspase-3 frees DFF40from the complex and initiates the apoptosis-specific DNA fragmentation . In this report, the coding region of human DFF45 gene was amplified from the total RNA of HeLa cells by RT-PCR . The resulting 1 kb DNA fragment was cloned into the bacterial expression vector pET-28a(+) with a 6xhistidine tag fused to the N-terminus of DFF45, generating plasmid pET28a-DFF45, which was then used to transform E.coli BL21(DE3) . Induced by IPTG, the recombinant DFF45 was expressed efficiently with a yield of 56.6% of total bacterial proteins . The product was purified to homogeneity through a nickel affinity column, followed by heat treatment, and approximately 4--6 mg of DFF was purified from 100 ml culture . Purified recombinant human DFF45, added into the apoptotic cell-free system of Xenopus egg extracts, could effectively inhibit both the digestion of lambdaDNA and the degradation of chromosomal DNA into nucleosomal fragments in the nuclei of chicken red blood cells . Our results demonstrated the existence of an apoptosis-specific endonuclease in this cell-free system, the activity of which could be inhibited by recombinant human DFF45.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 71 - 76
Reconstruction and Analysis of A Human Small Molecular Antibody to Tumor Necrosis Factor Alpha; Chen P et al.; A Fab antibody gene was constructed on the basis of the reconstruction of the linker of a human anti-TNF-alpha ScFv gene . The two ScFvs before and after reconstruction were cloned into expression vector pBV220 . About 30 kD recombinant proteins were expressed by induction and they constituted 6.5% and 13.8% of the total bacterial protein, respectively . A soluble Fab expression vector was constructed and transformed into E.coli HB2151.After induction by IPTG, a new protein band about 50 kD appeared on SDS-PAGE . The expressed ScFv and Fab were purified from E.coli lysates, and further experiments showed that 1) the expression amount of reconstructed ScFv was increased distinctly 2) ScFv and Fab could bind rhTNF-alpha . The ScFv containing GGGGS had an affinity constant of 6.70x10(4)(mol/L)(-1), and the ScFv containing (GGGGS) (3) had an affinity constant of 7.27x10(5) (mol/L) (-1).The affinity constant of Fab was 7.61x10(5) (mol/L) (-1) . The Fab and reconstructed ScFv was indifferent in affinity activity 3) ScFv and Fab neutralized the cytotoxicity of rhTNF-alpha . The neutralizing ability of Fab was the same as the reconstructed ScFv, but lower than a mouse anti-TNF-alpha mAb . These data may be helpful for using human anti-TNF-alpha small molecular Ab in antagonizing the activity of TNF-alpha in therapy.

Mol Cell Biol, 2002 Jul, 22(13), 4677 - 89
Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p; Kabani M et al.; We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M . Kabani, J.-M . Beckerich, and C . Gaillardin, Mol . Cell . Biol . 20:6923-6934, 2000) . We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release . Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p . Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays . Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity . The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates . However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved . In support of this hypothesis, Fes1p was found to be associated with ribosomes.

J Biol Chem, 2002 Aug 30, 277(35), 31441 - 7 Epub 2002 Jun 06.
Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity; Yu J et al.; Bile salt export pump (BSEP) is a major bile acid transporter in the liver . Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage . BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis . In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells . We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR) . Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types . This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR . These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR . In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression . Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells . In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm . In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation . These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR . Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.

Adv Drug Deliv Rev, 2002 Jun 17, 54(4), 587 - 606
Polyethylene glycol-superoxide dismutase, a conjugate in search of exploitation; Veronese FM et al.; Without a doubt PEG-SOD has been the enzyme most studied in PEGylation . One can say that it represents the preferred model to assess chemistries for PEG activation, analytical procedures suitable for conjugate characterization, the influence of PEG size in conjugate removal from circulation and elimination of immunogenicity and antigenicity, and the effect of route of administration . The effect of PEG conjugation was studied in vitro and in vivo models in comparison with the free enzyme and the following conclusions may be drawn: (1) At the blood vessel level, PEG-SOD has been shown to provide a greater resistance to oxidant stress, to improve endothelium relaxation and inhibit lipid oxidation . (2) In the heart, PEG-SOD proved to be at least as effective as native SOD in treatment of reperfusion-induced arrhythmias and myocardial ischemia . (3) In the lung, PEG-SOD appeared to be able to reduce oxygen toxicity and E . coli-induced lung injury, but not in the treatment of lung physiopathology associated with endotoxin-induced acute respiratory failure and in the reduction of asbestos-induced cell damage . (4) On cerebral ischemia/reperfusion injuries the effect of PEG-SOD was uncertain, also due to the difficulty of cerebral cell penetration . (5) In kidney and liver ischemia both enzyme forms were found to ameliorate reperfusion damage . In view of so much positive research on PEG-SOD, it is surprising that no approved application in human therapy has been established and approved.

J Biotechnol, 2002 Jul 17, 97(1), 51 - 8
Heterologous production of two unusual acyclic carotenoids, 1,1'-dihydroxy-3,4-didehydrolycopene and 1-hydroxy-3,4,3',4'-tetradehydrolycopene by combination of the crtC and crtD genes from Rhodobacter and Rubrivivax; Steiger S et al.; Acyclic hydroxy carotenoids were produced from lycopene and 3,4-didehydrolycopene in Escherichia coli by combining different carotenogenic genes including the carotene hydratase gene crtC and the carotene 3,4-desaturase gene crtD . The genes originated either from Rhodobacter species or Rubrivivax gelatinosus . It was shown that the product of crtD from Rubrivivax unlike the one from Rhodobacter is able to convert 1-HO-3,4-didehydrolycopene to 1-HO-3,4,3',4'-tetradehydrolycopene (=3,4,3',4'-tetradehydro-1,2-dihydro-psi,psi-caroten-1-ol) . Thus, only when the desaturase from Rubrivivax is expressed can this novel carotenoid be obtained . In the presence of crtC from Rubrivivax, another carotenoid 1,1'-(HO)(2)-3,4-didehydrolycopene (=3,4-didehydrolycopene-1,2,1',2'-tetrahydro-psi,psi-caroten-1,1'-diol) not found in a non-transgenic organism before is formed in E . coli . Its accumulation under these conditions and its absence when crtC from Rubrivivax is replaced by the corresponding gene from Rhodobacter is discussed . The function of the different crtC and crtD genes in the pathway leading to the individual carotenoids is outlined . Since 1,1'-(HO)(2)-3,4-didehydrolycopene could not be produced in substantial amounts and 1-HO-3,4,3',4'-tetradehydrolycopene has not been described before, their structural characteristics were determined for the definite assignment of their identity . This included spectral properties, determination of relative molecular mass as well as the number of hydroxy groups by mass spectroscopy and NMR spectroscopy for 1,1'-(HO)(2)-3,4-didehydrolycopene.

Diagn Microbiol Infect Dis, 2002 May, 43(1), 7 - 12
Detection of EspB using reversed passive latex agglutination: application to determination of enteropathogenic Escherichia coli; Lu Y et al.; We developed a new practical method to identify enteropathogenic Escherichia coli (EPEC) by detecting the pathogenic factor, EspB . E . coli were cultured in Dulbecco's Modification of Eagle's Medium (DMEM), and EspB was detected in the culture supernatant by reversed passive latex agglutination (RPLA) . All 63 E . coli strains harboring the eaeA gene encoding intimin were positive for RPLA, and all 25 strains without the eaeA gene were negative . Among these 63 eaeA-positive strains, 38 Shiga toxin-producing E . coli (STEC) produced Shiga toxin (Stx) under the same culture conditions (DMEM) . Subtypes of EspB alpha, beta and gamma were antigenically cross-reactive to each other as determined by RPLA and Western blotting . A kit for Stx detection (RPLA) is commercially available and therefore this RPLA for detection of EspB could be a practical method to define EPEC in both clinical laboratories and the field.

FEMS Microbiol Lett, 2002 May 21, 211(1), 57 - 64
Characterization of regions of the cyanobacterial tRNA(pro) gene that affect the expression of a beta-glucuronidase reporter gene; Chungjatupornchai W et al.; The E3 strong promoter-active fragment harbors the tRNA(pro) (GGG) gene upstream of the promoterless beta-glucuronidase (GUS) reporter gene in plasmid pKG-E3 . The 74-bp tRNA(pro) coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes . Results in this study showed that the promoter region of tRNA(pro) gene located upstream of its coding sequence and harbored the putative -10 (TACATT) and -35 (TTGGCA) regions which conformed to the Escherichia coli sigma(70) promoter . Differentiation of the 5' end of tRNA(pro)-GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions -3, -4, and -6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA(pro) coding sequence . The presence of block A decreased GUS activity about three-fold, whereas block B and the 3' end of tRNA(pro) gene completely abolished GUS expression . However, the presence of full-length tRNA(pro) gene did not affect the GUS expression . Downstream of the tRNA(pro) coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted DeltaG value of -21.7 kcal x mol(-1) . The absence of this stem-loop structure downstream of the tRNA(pro) coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.

FEMS Microbiol Lett, 2002 May 21, 211(1), 37 - 41
Glutamate dehydrogenase of Halobacterium salinarum: evidence that the gene sequence currently assigned to the NADP+-dependent enzyme is in fact that of the NAD+-dependent glutamate dehydrogenase; Hayden BM et al.; A GDH gene from Halobacterium salinarum has been cloned and sequenced and the publication assigns the sequence to the NADP+-glutamate dehydrogenase of this organism . We have expressed this gene in Escherichia coli and find that it encodes an NAD+-dependent glutamate dehydrogenase without activity towards NADP+ . Further, peptide sequence from the two corresponding proteins supports the view that the deposited sequence is indeed that of the NAD+-dependent glutamate dehydrogenase . Sequence from the NAD+-dependent protein matches the published gene sequence, whereas sequence from the NADP+ glutamate dehydrogenase does not.

Vet Microbiol, 2002 Jul 9, 87(3), 213 - 20
Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams; Zygmunt MS et al.; To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA) . The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E . coli cells in a soluble form . This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography . After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein . The degree of purity appeared satisfactory so that it could be directly used in I-ELISA . Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B . ovis hot saline (HS) extract as antigen, the high number of sera from B . ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B . ovis infection in rams.

Reproduction, 2002 Jun, 123(6), 847 - 57
Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model; Srivastava N et al.; Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins . The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis . In addition to rec-dZP2, E . coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system . Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone . Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT . Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility . In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility . The block in fertility was associated with anti-dZP3 antibody titres . Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida . These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.

Curr Pharm Des, 2002, 8(15), 1349 - 61
Nitroreductase-based GDEPT; Denny WA; Nitroreductases that metabolise aromatic nitro groups to hydroxylamines are attractive as enzymes for GDEPT because of the very large electronic change that this metabolism generates, providing an efficient switch that can be exploited to generate potent cytotoxins . While nitroreductase enzymes are widespread, nearly all the work using these in GDEPT has been with the nfsB gene product of Escherichia coli, an oxygen-insensitive flavin mononucleotide nitroreductase (NTR) . Four classes of prodrugs for NTR have been described; dinitroaziridinylbenzamides, dinitrobenzamide mustards, 4-nitrobenzylcarbamates and nitroindolines . While some quinones are excellent substrates for NTR, none have been identified as potential GDEPT prodrugs . The most widely studied prodrug used for GDEPT in conjunction with NTR is the dinitroaziridinylbenzamide CB 1954 . This shows high selectivity (>1000-fold) in cell lines transfected with NTR, has potent and long-lasting inhibition of NTR-transfected tumours in mice, and is in Phase I trial in conjunction with virally-delivered NTR enzyme . The related mustard SN 23862 has similar selectivity and superior bystander effects in animal models . Nitrobenzyl carbamates of a variety of cytotoxic amines (including aniline mustards, enediynes, duocarmycin analogues, pyrrolobenzodiazepines and the antitumour antibiotics doxorubicin, actinomycin D and mitomcyin C) are metabolised efficiently by NTR to the hydroxylamines, that fragment to release the amines . Nitroindoline derivatives of duocarmycins also show moderate selectivity for NTR-transfected cell lines in culture.

Biotechnol Prog, 2002 May-Jun, 18(3), 672 - 4
Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers; Khlebnikov A et al.; The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose-inducible promoters P(tac) and P(trc) by the natural inducer lactose and the synthetic inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) was investigated . Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall . In E . coli strains lacking a functional LacY, IPTG is required for induction of P(tac) and P(trc) . In E . coli strains carrying a functional LacY, induction of P(trc) and P(tac) with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used . In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY(+) strains.

J Theor Biol, 2002 Mar 21, 215(2), 239 - 51
Dependence of control coefficient distribution on the boundaries of a metabolic system: a generalized analysis of the effects of additional input and output reactions to a linear pathway; de Atauri P et al.; Both experimental and theoretical studies of metabolism are likely to relate to a segment that has been isolated for analytical purposes . In practice, it will be embedded in the whole of cellular metabolism . Thus, it is necessary to consider how conclusions about the control of an isolated pathway may be modified in this wider context where the input and output metabolites are considered as variables of cellular metabolism . Here, we analyse the effect of expanding a linear metabolic pathway by adding an extra input or an extra output . In particular, we analyse the effect of the elasticities of the extra steps on control coefficients . We derive matrix algebraic relationships for obtaining flux and concentration control coefficients from expressions depending on these extra elasticities and on parameters (elasticities and control coefficients) of the original pathway . These equations can be shown in certain cases to be generalized versions of earlier rescaling relationships and to be related to top-down analysis, but also apply where the new variable metabolite of the expanded pathway is an effector of more than one step of the original pathway . We use our relationships to analyse the dependence or independence of control coefficients upon these extra elasticities for the published analyses of the pathway of mammalian serine biosynthesis (Fell & Snell, 1988) and Escherischia coli threonine biosynthesis (Chassagnole et al., 2001) . The same analysis can be applied to determine whether the transport reactions of substrates and products of a pathway in and out of a cell need to be included in estimations of the control coefficients of the enzymes .

J Theor Biol, 2002 Mar 21, 215(2), 151 - 67
Analysis of protein homeostatic regulatory mechanisms in perturbed environments at steady state; Sewell C et al.; Nine different protein homeostatic regulatory mechanisms were analysed for their ability to maintain a generic protein P within a specified range of a set-point steady-state concentration while perturbed by external processes that altered the rates at which P was produced and/or consumed . Steady state regulatory effectiveness was defined by the area within a rectangular region of "perturbation space", where axes correspond to rates of positive and negative perturbations . The size of this region differed in accordance with the regulatory elements composing the homeostatic mechanism . Such elements included basic negative feedback control of transcription (in which P, at some high concentration relative to its set-point value, binds to the gene G that encodes it, thereby inhibiting transcription), multiple sequential binding of a feedback effector (two P's bind sequentially to G), and dimerization of a feedback effector (a P(2) dimer binds to G) . Two homeostatic mechanisms included a cascade structure, one with and one without translational feedback control . Another mechanism included feedback control of P degradation . Finally, two mechanisms illustrated the limits of regulatory systems . One lacked all regulatory elements (and included only an invariant rate of P synthesis and degradation) while the other assumed perfect (Boolean) regulation, in which transcription is completely inhibited at {P}>{P}(sp) and is fully active at {P}<{P}(sp) . All of the systems evaluated are known, but the analytical expressions developed here allow quantitative comparisons between them . These expressions were evaluated at values typical of the average protein in Escherichia coli . A method for building regulatory networks by linking semi-independent regulatory modules is discussed .

J Mol Biol, 2002 May 24, 319(1), 183 - 9
On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase; Bustos-Jaimes I et al.; The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187) . This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161 . This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites . Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state . These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition . In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala . Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme . The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9) . This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand . Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty . These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.

J Mol Biol, 2002 May 24, 319(1), 161 - 71
Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family; Thorell S et al.; Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases . The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%) . The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4 . It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements . The enzyme forms a decamer of identical subunits with point group symmetry 52 . Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer . A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit . In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation . The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme . The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture . The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

J Mol Biol, 2002 May 24, 319(1), 107 - 27
The order of strand exchanges in Cre-LoxP recombination and its basis suggested by the crystal structure of a Cre-LoxP Holliday junction complex; Martin SS et al.; Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences . In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges . However, the particular sequence of events has been in question . From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm . Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage . We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side . In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses . A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order . Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine . These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation . Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.

J Mol Biol, 2002 May 31, 319(2), 341 - 9
Both temperature and medium composition regulate RNase E processing efficiency of the rpsO mRNA coding for ribosomal protein S15 of Escherichia coli; Le Derout J et al.; Cleavage by RNase E is believed to be the rate-limiting step in the degradation of many RNAs . These cleavages are modulated by 5' end-phosphorylation, folding and translation of the mRNA in question . Here, we present data suggesting that these cleavages are also regulated by environmental conditions . We report that rpsO mRNA, 15 minutes after a shift to 44 degrees C, is stabilized in cells grown in minimal medium . This stabilization is correlated with a reduction in the efficiency of the RNase E cleavage which initiates its decay . We also observe the appearance of RNA fragments previously detected following RNase E inactivation and a defect in the adaptation of RNase E concentration . These observations, coupled to the fact that RNase E overproduction slightly reduces the accumulation of the rpsO mRNA, suggest that this stabilization is caused in part by a limitation in RNase E concentration . An increase in the steady-state level of rpsT mRNA is also observed following a shift to 44 degrees C in minimal medium; however, processing of the 9 S rRNA precursor is not affected under these conditions . We thus propose that RNase E concentration changes in the cell in response to environmental conditions and that these changes can selectively affect the processing and the stability of individual mRNAs . Our data also indicate that the efficiency of cleavage of the rpsO mRNA by RNase E is modified by other factor(s) which remain to be identified .

J Mol Biol, 2002 May 31, 319(2), 329 - 39
Limitation of ribosomal protein L11 availability in vivo affects translation termination; Van Dyke N et al.; Historically referred to as "the GTPase center", the L11 binding region (L11BR) of Escherichia coli 23 S rRNA is a highly conserved structure that has been implicated in several essential functions during protein synthesis . Here, in vivo expression of an RNA fragment containing that structure was found to affect translation termination in a codon-specific manner . The cause of these effects appeared to be titration of ribosomal protein L11, since normal phenotypes could be restored by simultaneous overproduction of wild-type L11 but not mutant L11 . Subsequently, altered termination phenotypes were produced when the availability of L11 was limited by overexpression of RNA antisense to L11 mRNA and, finally, by inactivation of the chromosomal L11 gene, and they too were reversible by simultaneous expression of cloned L11 . Our results indicate that in the intact cell the L11BR is an integral functional unit important for translation termination and that the presence of L11 in ribosomes is required for UAG-dependent termination and is somewhat inhibitory of UGA-dependent termination .

J Mol Biol, 2002 Apr 26, 318(2), 519 - 31
Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein; Mateu MG; The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy . The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U) . The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability . This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer . In a second transition, the intermediate cooperatively unfolds into denatured monomer (U) . The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala . A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer . All these residues are involved also in the association process, despite being located far away from the dimerization interface . It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Apr 26, 318(2), 361 - 71
The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism; Noble CG et al.; We have examined the role of the DNA gyrase B protein in cleavage and religation of DNA using site-directed mutagenesis . Three aspartate residues and a glutamate residue: E424, D498, D500 and D502, thought to co-ordinate a magnesium ion, were mutated to alanine; in addition, the glutamate residue and one aspartate residue were mutated to glutamine and asparagine, respectively . We have shown that these residues are important for the cleavage-religation reaction and are likely to be involved in magnesium ion co-ordination . On separate mutation of two of these aspartate residues to cysteine or histidine, the metal ion preference for the DNA relaxation activity of gyrase changed from magnesium to manganese (II) . We present evidence to support the idea that cleavage of each DNA strand involves two or more metal ions, and suggest a scheme for the DNA cleavage chemistry of DNA gyrase involving two metal ions . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Apr 26, 318(2), 351 - 9
Identification of four GyrA residues involved in the DNA breakage-reunion reaction of DNA gyrase; Hockings SC et al.; DNA supercoiling by DNA gyrase involves the cleavage of a DNA helix, the passage of another helix through the break, and the religation of the first helix . The cleavage-religation reaction involves the formation of a 5'-phosphotyrosine intermediate with the GyrA subunit of the gyrase (A(2)B(2)) complex . We report the characterization of mutations near the active-site tyrosine residue in GyrA predicted to affect the cleavage-religation reaction of gyrase . We find that mutations at Arg32, Arg47, His78 and His80 inhibit DNA supercoiling and other reactions of gyrase . These effects are caused by the involvement of these residues in the DNA cleavage reaction; religation is largely unaffected by these mutations . We show that these residues cooperate with the active-site tyrosine residue on the opposite subunit of the GyrA dimer during the cleavage-religation reaction . (c) 2002 Elsevier Science Ltd.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 205 - 209
Cloning and Expression of Luffin-b cDNA from the Seeds of Luffa cylindrica; Yan M et al.; Luffin-b with Mr . 28 kD, isolated from the seeds of Luffa cylindrica,is one of the most toxic single chain plant ribosome inactivating proteins . The cDNA sequence of luffin-b was already reported by Kataoka in 1992 . In this work, the luffin-b gene(lufB) coding sequence was cloned from the fresh seeds of Luffa cylindrica by RT-PCR, and the coding sequence of the gene was shown to be identical with that determined by Kataoka . The lufB expression plasmid was constructed by inserting the lufB cDNA fragment into vector pET24a( ), and the pET24a( )- lufB vector was expressed in E.coli by 0.5 mmol/L IPTG induction . The recombinant product, which mainly existed in inclusion bodies, was identified by SDS-PAGE and Western blotting . The recombinant luffin-b, which was renatured by dialysis with step by step decreasing concentration of urea, showed high activity of ribosome inactivation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(3), 303 - 308
Cloning, Expression and Antibody Production of the Disintegrin Domain of Human Fertilin beta; Ding BB et al.; A cDNA for the disintegrin domain (hf279) was isolated by PCR from human testis cDNAs . DNA sequencing indicated that hf279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin beta . An expression plasmid, pGEX hf279, was constructed by inserting hf279 cDNA into plasmid pGEX-4T-2 containing gst gene . The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21 induced with IPTG . SDS-PAGE analysis revealed that the GST-HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins . The fusion protein was purified by glutathione S-transferase (GST) Sepharose 4B column (purity 90%) and digested by thrombin to obtain the purified HF93 peptide (purity 80%) . Polyclonal antibodies were obtained from the serum of miceimmunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS-PAGE . ELISA and Western blot analysis showed its specificity to HF93 . Therefore this antibody can be used in further studies on the function of HF93.

J Virol, 2002 Jul, 76(13), 6518 - 31
Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system; Christensen J et al.; We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand . NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex . In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis . In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding . Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding . However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork . Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template . The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed . NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses . NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding . No NS1 interactions were observed with E . coli SSB or other proteins included as controls.

J Biol Chem, 2002 Aug 16, 277(33), 29654 - 61 Epub 2002 Jun 05.
The physiological role of RNase T can be explained by its unusual substrate specificity; Zuo Y et al.; Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3' maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity . The enzyme was found to be a single-strand-specific exoribonuclease that acts in the 3' to 5' direction in a non-processive manner . However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair . The presence of a free 3'-hydroxyl group is required for the enzyme to initiate digestion . Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues . Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues . A single 3'-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme . In vivo, the substrates of RNase T are similar in that they all contain a double-stranded stem followed by a single-stranded 3' overhang; yet, the action of RNase T on these substrates differs . The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.

Genes Dev, 2002 Jun 1, 16(11), 1371 - 82
Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is required for efficient nuclear localization of Esp1/separase; Agarwal R et al.; Sister chromatid separation at the metaphase-to-anaphase transition is induced by the proteolytic cleavage of one of the cohesin complex subunits . This process is mediated by a conserved protease called separase . Separase is associated with its inhibitor, securin, until the time of anaphase initiation, when securin is degraded in an anaphase-promoting complex/cyclosome (APC/C)-dependent manner . In budding yeast securin/Pds1 not only inhibits separase/Esp1, but also promotes its nuclear localization . The molecular mechanism and regulation of this nuclear targeting are presently unknown . Here we show that Pds1 is a substrate of the cyclin-dependent kinase Cdc28 . Phosphorylation of Pds1 by Cdc28 is important for efficient binding of Pds1 to Esp1 and for promoting the nuclear localization of Esp1 . Our results uncover a previously unknown mechanism for regulating the Pds1-Esp1 interaction and shed light on a novel role for Cdc28 in promoting the metaphase-to-anaphase transition in budding yeast.

Bioinformatics, 2002 May, 18(5), 715 - 24
Evaluation of computational metabolic-pathway predictions for Helicobacter pylori; Paley SM et al.; MOTIVATION: We seek to determine the accuracy of computational methods for predicting metabolic pathways in sequenced genomes, and to understand the contributions of both the prediction algorithms, and the reference pathway databases used by those algorithms, to the prediction accuracy . RESULTS: The comparisons we performed were as follows . (1) We compared two predictions of the pathway complements of Helicobacter pylori that were computed by an early version of our pathway-prediction algorithm: prediction A used the EcoCyc E . coli pathway DB as the reference database (DB) for prediction, and prediction B used the MetaCyc pathway DB (a superset of EcoCyc) as the reference pathway DB . The MetaCyc-based prediction contained 75% more pathway predictions, but we believe a significant number of those predictions were false positives . (2) We compared two predictions of the pathway complement of H . pylori that used MetaCyc as the reference pathway DB, but that used different algorithms: the original PathoLogic algorithm, and an enhanced version of the algorithm designed to eliminate false-positive pathway predictions . The improved algorithm predicted 30\% fewer metabolic pathways than the original algorithm; all of the eliminated pathways are believed to be false-positive predictions . (3) We compared the 98 pathways predicted by the enhanced algorithm with the results of a manual analysis of the pathways of H . pylori . Results: 40 of the computationally predicted pathways were consistent with the manual analysis, 13 pathways are considered false-positive predictions, and four pathways had partially overlapping topologies . Twenty-six predicted pathways were not mentioned in the manual analysis; we believe these are correct predictions by PathoLogic that were not found by the manual analysis . Five pathways from the manual analysis were not found computationally . Agreement between the computational and manual predictions was good overall, with the computational analysis inferring many pathways that the manual analysis did not identify . Ultimately the manual analysis is also partially speculative, and therefore is not an absolute measure of correctness . The algorithm is designed to err on the side of more false positives to bring more potential pathways to the user's attention . The resulting H . pylori pathway DB is freely available at AVAILABILITY: The Pathway Tools software is freely available to academic users, and is available to commercial users for a fee . Contact pkarp@ai.sri.com for information on obtaining the software.

Mol Cell, 2002 May, 9(5), 981 - 91
Insights into specific DNA recognition during the assembly of a viral genome packaging machine; de Beer T et al.; Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses . In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA . The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy . Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold . Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif . Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence . The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.

Biochem J, 2002 Jun 15, 364(Pt 3), 849 - 55
Hydrolysable ATP is a requirement for the correct interaction of molecular chaperonins cpn60 and cpn10; Walters C et al.; Over recent years the binding ability of the molecular chaperone cpn60 (GroEL14) and its co-chaperone cpn10 (GroES7) has been reported to occur under an assortment of specific conditions from the use of non-hydrolysable ATP analogues (namely adenosine 5'-{gamma-thio}triphosphate) to requiring hydrolysable ATP for any interaction to occur . We have investigated this further using the molecular hydrodynamic methods (hydrodynamic bead modelling, sedimentation-velocity analytical ultracentrifugation and dynamic light-scattering), allowing the process to be followed under physiologically relevant dilute solution conditions, combined with absorption spectrophotometry to determine GroES7-GroEL14 interaction through the rate inhibition of the cpn60's ATPase activity by GroES7 . The results found here indicate that the presence of hydrolysable ATP is required to facilitate correct GroES7 interaction with GroEL14 in solution.

Biochem J, 2002 Jun 15, 364(Pt 3), 825 - 31
Identification, cloning and expression of the mouse N-acetylglutamate synthase gene; Caldovic L et al.; In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle . NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria . This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity . Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity . Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive . We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene . NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis . The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus . The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy . His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E . coli, and purified to apparent homogeneity by using a nickel-affinity column . The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein . The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx . 2-6-fold.

Biochem J, 2002 Jun 15, 364(Pt 3), 795 - 805
Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene; Brown AP et al.; Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens . The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels . The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering) . Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards . LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and <or=12.5 pg/microg of membrane protein in developing embryos . The amount of LAT2 reaches a peak at 305 pg/microg of membrane protein 25 daf and is not expressed 20 daf or before . This is the first study to quantify these membrane-bound proteins in a plant tissue . The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds . Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different . Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E . coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot . This is probably due to differences in substrate utilization.

Biochem J, 2002 Jun 15, 364(Pt 3), 679 - 86
Characterization of recombinant glutathionylspermidine synthetase/amidase from Crithidia fasciculata; Oza SL et al.; Trypanothione {N1,N8-bis(glutathionyl)spermidine} is a unique metabolite found only in trypanosomatids, where it subsumes many of the functions of GSH in other organisms . In Crithidia fasciculata, two distinct ATP-dependent ligases, glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), are involved in the synthesis of trypanothione from GSH and spermidine . Both enzymes have been cloned previously, but expression in Escherichia coli produced insoluble and inactive protein . Here we report on the successful expression of soluble (His)6-tagged C . fasciculata GspS in E . coli . Following purification using nickel-chelating affinity chromatography, the tag sequence was removed and the enzyme purified to homogeneity by anion-exchange chromatography . The kinetic parameters of the recombinant enzyme have been determined using a coupled enzyme assay and also by HPLC analysis of end-product formation . Under optimal conditions (0.1 M K+-Hepes, pH 7.3) GspS has synthetase activity with apparent K(m) values for GSH, spermidine and MgATP of 242, 59 and 114 microM respectively, and a k(cat) of 15.5 s(-1) . Glutathionylspermidine is formed as end product and the enzyme lacks TryS activity . Like E . coli GspS, the recombinant enzyme also possesses amidase activity (EC 3.5.1.78), hydrolysing glutathionylspermidine to GSH and spermidine with a k(cat) of 0.38 s(-1) and a K(m) of 500 microM . GspS can also hydrolyse trypanothione at about 1.5% of the rate with glutathionylspermidine . A single amino acid mutation (Cys-79-->Ala) is shown to ablate the amidase activity without affecting the synthetase activity.

Biochem J, 2002 Jun 15, 364(Pt 3), 629 - 34
The structure of procaspase 6 is similar to that of active mature caspase 6; Kang BH et al.; To investigate the structural characteristics and activation mechanism of the precursor caspase, genes encoding the inactive pro-form and the active mature form of caspase 6 were expressed in Escherichia coli and the proteins of both forms were purified to homogeneity . The structure of each protein was characterized by chemical cross-linking, size-exclusion chromatography, CD and fluorescence spectroscopies . The pro-form caspase 6 exhibits a dimeric structure and its overall secondary structure was found to be similar to that of the mature caspase 6 . Upon the maturation of procaspase 6, the maximum fluorescence wavelength lambda(max) was red-shifted from 330 to 337 nm and the fluorescence intensity of lambda(max) was increased . This fluorescence spectral change indicates that the environment of a tryptophan residue in the substrate-binding site can be changed to a more polar one when the procaspase 6 is processed . Taken together, our results strongly demonstrate that precursor caspase 6 exists as a dimer and its overall structure is similar to that of the active caspase 6 . Our results also suggest that the local conformational change at the substrate-binding site, with no drastic change in the overall structure, seems to enable precursor caspase 6 to become the active mature enzyme.

Parasitol Res, 2002 May, 88(5), 421 - 6
S-adenosylmethionine decarboxylase from Leishmania infantum promastigotes: molecular cloning and differential expression; Taladriz S et al.; S-Adenosylmethionine decarboxylase (AdoMetDC), an enzyme involved in the synthesis of polyamines as well as in the cell methylation processes, has been considered in trypanosomes as a specific drug target . We have cloned by RT-PCR a DNA fragment of 1,364 bp which contains the open reading frame and the 5' end fragment of the AdoMetDC encoding gene from the parasite protozoon Leishmania infantum . The 1,197 bp ORF encodes for a 392 amino acid residue polypeptide . The sequence comparison with AdMetDC from different species showed a high level of homology, around 80% . with the American and African trypanosomes and a certain distance from the polypeptides of higher eukaryotes . AdoMetDC has been cloned in a pQE32 vector and overexpressed in a M15 Escherichia coli strain . The gene expression shows variations between the distinct phases of the parasite, being higher in the most infective one . This fact may be related to the multiple defense mechanism of the protozoon against the macrophage action.

Sci Total Environ, 2002 Apr 22, 289(1-3), 123 - 32
An analysis of the combined effects of organic toxicants; Chen CY et al.; This paper presents a basic database for the joint actions of 44 binary mixtures of various organic toxicants on Escherichia coli . The multiple toxicity behaviors observed from the E . coli organisms were analyzed and compared with previous works based on the Microtox tests . The two kinds of tests produced quite different responses, in terms of the joint action mode and the sum of toxic units, to various organic mixtures . However, detailed analyses with the considerations of the chemical's mechanisms of toxicity and the slope of toxicant's dose-response curve have revealed several general criteria for the prediction of combined effects of organic toxicants . First, for both reactive and non-reactive toxicants, either additive or less than additive (antagonistic) joint actions will be observed for chemicals of the same mechanism of toxicity . Second, the mixture of reactive toxicants with different mechanisms is the only category of organic mixtures associated with frequent observations of synergism . Third, greater-than-additive (synergistic) effects are inherently associated with toxicants having flat dose-response curves . Less than additive effects are, however, mainly related to a chemical's display steep dose-response curves . Model analyses indicate that the observed synergistic effects are due to response addition or response multiplication joint actions . Hence, most of the synergistic joint actions are non-interactive in nature and are governed by the dose-response relationships of individual toxicants.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7992 - 7 Epub 2002 Jun 04.
Ligand binding and protein dynamics in neuroglobin; Kriegl JM et al.; Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins . It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown . Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions . To bind exogenous ligands like O(2) and CO, the His E7 endogenous ligand is displaced from the sixth coordination . By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3-353 K) . Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO . Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers . The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K . Flash photolysis near physiological temperature (275-353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 micros); (ii) extremely fast bimolecular exogenous ligand binding (10 micros < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 micros < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks) . All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.

Hepatol Res, 2002 Jun, 23(2), 90 - 97
Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions; Uchiyama Y et al.; The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy . We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli . With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer . The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 degrees C and a pH of 7.5 . The addition of 2.5 mM Mn(2+) increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg(2+) . At higher concentrations of Mn(2+), GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted . This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp.

Trends Genet, 2002 May, 18(5), 239 - 40
Evolutionary genomics: molecular evolution at the genomic scale; Naruya S; The Symposium on Evolutionary Genomics was held in Atami, Japan, from 4 to 6 November 2001.

Immunology, 2002 Jun, 106(2), 200 - 11
Accumulation of a potent gammadelta T-cell stimulator after deletion of the lytB gene in Escherichia coli; Eberl M et al.; Activation of human Vgamma9/Vdelta2 T cells by many pathogens depends on the presence of small phosphorylated non-peptide compounds derived from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis . We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vgamma9/Vdelta2 T-cell activator accumulates by a factor of approximately 150 compared to wild-type E . coli . The compound responsible for the strong immunogenicity of this E . coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway . Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB-deficient E . coli mutant led to upregulation of T-cell activation markers on the surface of Vgamma9/Vdelta2 T cells as well as proliferation and expansion of Vgamma9/Vdelta2 T cells . This response was dependent on costimulatory growth factors, such as interleukin (IL)-2, IL-15 and IL-21 . Significant levels of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were secreted in the presence of IL-2 and IL-15, but not in the presence of IL-21, demonstrating that proliferating phosphoantigen-reactive Vgamma9/Vdelta2 T cells do not necessarily produce proinflammatory cytokines.

Plant J, 2002 Jun, 30(5), 581 - 91
Downregulation of a chloroplast-targeted beta-amylase leads to a starch-excess phenotype in leaves; Scheidig A et al.; A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation . One clone isolated had a sequence very similar to a recently described chloroplast-targeted beta-amylase of Arabidopsis . Expression of the gene in E . coli showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-amylase was imported and processed into pea chloroplasts . To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques . Analysis of plants reduced in the presence of this beta-amylase isoform showed that their leaves had a starch-excess phenotype, indicating a defect in starch degradation . In addition, it was shown that the antisense plants degraded only 8-30% of their total starch, in comparison with 50% in the wild type, over the dark period . This is the first time that a physiological role for a beta-amylase in plants has been demonstrated.

Eur J Biochem, 2002 Jun, 269(11), 2801 - 9
Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor; Tsetlin VI et al.; Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites . The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs . The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein {Fischer, M., Corringer, P., Schott, K., Bacher, A . & Changeux, J . (2001) Proc . Natl Acad . Sci . USA 98, 3567-3570} and glutathione S-transferase (GST) {Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C . & Tsetlin, V . (2001) J . Biol . Chem . 276, 15810-15815} . However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers . In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates . The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions . This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm) . Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS . CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions . The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.

Eur J Biochem, 2002 Jun, 269(11), 2662 - 71
Two independent, light-sensing two-component systems in a filamentous cyanobacterium; Jorissen HJ et al.; Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp . PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp . PCC7601 . While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine . Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction . In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts . For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3' end and expressed in Escherichia coli . The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P(r) and P(fr) forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1 . A red shift in the absorption maxima of the CphB/PCB complex (lambda(max) = 685 and 735 nm for P(r) and P(fr), respectively) is indicative for a noncovalent incorporation of the chromophore (lambda(max) of P(r), P(fr) of CphA: 663, 700 nm) . A CphB mutant generated at the chromophore-binding position (Leu246-->Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB . The kinetics of the light-induced P(fr) formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P(fr) formation . The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator . In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.

Mol Microbiol, 2002 May, 44(4), 1095 - 1107
Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules; Kenny B et al.; Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens . A/E pathogens encode a type III secretion system to transfer effector proteins into host cells . The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria . In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection . Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction . We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells . The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins . The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity . Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection . Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

World J Gastroenterol, 2002 Jun, 8(3), 551 - 4
Expression of CD14 protein and its gene in liver sinusoidal endothelial cells during endotoxemia; Gong JP et al.; AIM: To observe expression of CD14 protein and CD14 gene in rat liver sinusoidal endothelial cells (LSECs) during endotoxemia, and the role of CD14 protein in the activation of lipopolysaccharide (LPS)-induced LSECs . METHODS: Wistar rat endotoxemia model was established first by injection of a dose of LPS (5mg/kg, Escherichia coli O111:B4 ) via the tail vein, then sacrificed after 0 h,3h,6h, 12h, and 24h, respectively . LSECs were isolated from normal and LPS-injected rats by an in situ collagenase perfusion technique . The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody, then stained with goat anti rabbit IgG conjugated fluorescein isothiocyanate(FITC) and flow cytometric analysis (FCM) was performed . The percentage and mean fluorescence intensity (MFI) of CD14-positive cells were taken as the indexes . LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis . The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody, then stimulated with different concentrations of LPS, and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and Interleukin (IL)-6 with ELISA . RESULTS: In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats . CD14 positive cells in rats with endotoxemia were 54.32%, 65.83%, 85.64%, and 45.65% at 3h, 6h, 12h, and 24h respectively, there was significant difference when compared to normal group of animals (4.45%)(P<0.01) . The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats . In LPS group, the levels of TNF-alpha and IL-6 were 54+/-6 ng.L(-1), 85+/-9 ng.L(-1), 206+/-22 ng.L(-1), 350+/-41 ng.L(-1), 366+/-42 ng.L(-1) and 103+/-11 ng.L(-1), 187+/-20 ng.L(-1), 244+/-26 ng.L(-1), 290+/-31 ng.L(-1), and 299+/-34 ng.L(-1), respectively at different concentration points . In anti-CD14 group, the levels of TNF-alpha and IL-6 were 56+/-5 ng.L(-1), 67+/-8 ng.L(-1), 85+/-10 ng.L(-1), 113+/-12 ng.L(-1), 199+/-22 ng.L(-1) and 104+/-12 ng.L(-1), 125+/-12 ng.L(-1), 165+/-19 ng.L(-1), 185+/-21 ng.L(-1), and 222+/-23 ng.L(-1), respectively at different concentration points . There was significant difference between the two groups (P<0.01) . CONCLUSION: LSECs can synthesize CD14 protein and express CD14 gene during endotoxemia . CD14 protein plays an important role in the activation of LPS-induced LSECs . This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.

World J Gastroenterol, 2002 Jun, 8(3), 499 - 504
Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein; Zhu LX et al.; AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV . The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing . METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products . RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively . Deglycosylation analysis showed that this difference was mainly due to different glycosylation . Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94 . Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences . CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2 . As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER . Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Braz J Med Biol Res, 2002 Jun, 35(6), 651 - 61
Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense; Ishida ML et al.; Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans . These A . brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized . The present study suggests that ORF1 and ORF2 correspond to the A . brasilense ntrY and ntrX genes, respectively . The amino acid sequences of A . brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively . Analysis of lacZ transcriptional fusions by the beta-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A . brasilense . The ntrYX operon complemented a nifR3ntrBC deletion mutant of A . brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs . Our data support the existence of another two-component regulatory system in A . brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Neuroimmunomodulation, 2001, 9(6), 340 - 51
Plasma interleukin-1beta, prolactin, ACTH and corticosterone responses to endotoxin after damage of the anterior hypothalamic area; Phelps CP et al.; This report concerns the use of an animal model described by us {J Submicrosc Cytol Pathol 1995;27:83-89} to investigate neural and endocrine sites for endotoxin (ENDO, E . coli 055:B5, 200 microg/100 g body weight in saline intravenously) effects on immunomodulatory hormone and cytokine release . Plasma interleukin-1beta (IL-1beta), prolactin (PRL), ACTH and corticosterone responses to ENDO after neurotoxic damage of neurons residing in the anterior hypothalamic area (AHA) were studied in freely behaving male rats . Excitotoxic cell damage in the AHA was produced by bilaterally injecting N-methyl-DL-aspartate (NMA) in artificial cerebrospinal fluid (aCSF) into this brain site . Injections of comparable volumes of aCSF alone served as controls for brain damage associated with the treatment . In both experimental brain manipulations before ENDO challenge the rise in plasma IL-1beta concentrations in response to ENDO was reduced by 2-fold at 1 h and 3- to 5-fold at 3 h when compared to controls . Nevertheless, experimental and control brain manipulations did not modulate the expected rise in corticosterone concentrations after ENDO exposure which rose 5-fold above the baseline level in all animals . However, AHA manipulation did reduce plasma ACTH and prolactin concentrations differentially . Introduction of either NMA or the control injection of aCSF alone into AHA reduced plasma ACTH concentrations by 2-fold at 0.5 and 1 h after ENDO . However, there was a greater reduction in the rise of plasma PRL concentrations after ENDO found in NMA-treated groups versus rats receiving control aCSF . These results demonstrate that variable-size hypothalamic damage (a larger lesion produced in AHA by NMA treatment vs . a smaller lesion control after aCSF) can result in a differential blunting of PRL, IL-1beta and ACTH release into blood in the face of robust, unmodulated corticosterone increases . In summary, these findings revealed a consistent predominant influence of ENDO on adrenal release of corticosterone as a concomitant to differential IL-1beta, ACTH and PRL release after AHA cell loss . In conclusion, these results constitute further evidence for hypothalamic orchestration of a balance between immunotropic and immunosuppressive neuroendocrine-immune events during acute bacterial infection of mammals .

J Cell Sci, 2002 Jun 15, 115(Pt 12), 2529 - 39
GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange; Kang X et al.; GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli . TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5 . It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region . Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal . Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies . The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine . In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface . VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.

J Biol Chem, 2002 Sep 13, 277(37), 34087 - 100 Epub 2002 Jun 03.
Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination; Napierala M et al.; Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences . However, the frequency of recombination between the CTG.CAG tracts has not been studied . Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli . The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12% . Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain . Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length . The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences . The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication . This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences . Thus, long CTG.CAG tracts are hot spots for genetic recombination.

J Biol Chem, 2002 Sep 6, 277(36), 33092 - 8 Epub 2002 Jun 03.
The crystal structure of metal-free human EF-hand protein S100A3 at 1.7-A resolution; Fritz G et al.; S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins . It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm) . This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3 . The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control . We determined the crystal structure of the protein at 1.7 A . The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding . The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion . Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.

J Biol Chem, 2002 Aug 23, 277(34), 30524 - 34 Epub 2002 Jun 03.
Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase; Abdel-Banat BM et al.; The gene of chitinase in the silkworm, Bombyx mori, generates four mRNA products by alternative splicing . Nucleotide sequences of the entire gene for chitinase and respective cDNAs demonstrate that the pre-mRNA undergoes alternative splicing at both the 5' and 3' regions . At the 5' region, the pre-mRNA experienced differential splicing through two alternative 5'-intron consensus splicing sites . These products differ in the last amino acid of the signal peptide and the first amino acid of the mature N-terminal sequences: one with Cys(20)-Ala(21) and the other with Ser(20)-Asp(21) . The product with Cys(20)-Ala(21) residues is one amino acid larger than the other with Ser(20)-Asp(21) . At the 3' region the pre-mRNA of the chitinase gene undergoes alternative splicing in three different fashions . It is spliced either through retaining or excluding the upstream 121-bp direct repeat found at the 3' region of the coding sequences or through retaining or excluding of an insertion of 9 bp in a combinatorial manner . Retention or exclusion of the upstream 121-bp direct repeat results in a protein with a deduced amino acid sequence similar in size to the one retaining both direct repeats . However, exclusion of the insert of the 9 bp from the mRNA results in a protein with 22 extra amino acids . All of the mRNA products appear to be generated from a single gene as demonstrated by testing the 3' region of the genomic DNA and variant chitinase mRNA products . B . mori chitinase expression in the fifth instar larvae epidermal tissues appears to be developmentally regulated, but the phenomenon of alternative splicing of the pre-mRNA is not stage-dependent . Furthermore, the four mRNA products showed chitinase activity when expressed in Escherichia coli, which demonstrates the role of the alternative splicing process in generating multiple isoforms of the silkworm's chitinase.

J Biol Chem, 2002 Aug 30, 277(35), 31287 - 90 Epub 2002 Jun 03.
Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N; Gumbiowski K et al.; ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1) . The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)) . The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2) . In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation . However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons . Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme . This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.

Mech Ageing Dev, 2002 Apr 30, 123(8), 907 - 15
Large genome rearrangements as a primary cause of aging; Vijg J et al.; In his introductory chapter of the Mutation Research special issue on 'Genetic Instability and Aging', the late Bernard Strehler provided some historical perspectives on the long-standing hypothesis that aging is primarily caused by changes in the genome of somatic cells (Strehler, 1995, Mutat . Res . 338 (1995) 3) . Based on his own findings of a loss of ribosomal RNA gene copies in postmitotic tissues of dogs as well as humans during aging, his main conclusion was that deletional mutations are more likely than point mutations to be a main causal factor in aging . To directly assess the levels of different types of spontaneous mutations in organs and tissues during aging, we have used a mouse model harboring a chromosomally integrated cluster of lacZ-containing plasmids that can be recovered and analyzed in Escherichia coli . Our results indicate the accumulation of mutations in some but not all organs of the mouse with significant differences in mutational spectra . In addition to point mutations, genome rearrangements involving up to 66 Mb of genomic DNA appeared to be a major component of the mutational spectra . Physical characterization of the breakpoints of such rearrangements indicated their possible origin by erroneous, non-homologous DNA double-strand break repair . Based on their increased occurrence during aging in some tissues and their often very large size, we have designed a model for an aging tissue in terms of a cellular mosaic with a gradual increase in genome rearrangements that leads to functional senescence, neoplastic transformation or death of individual cells by disrupting nuclear architecture and patterns of gene regulation.

Biochim Biophys Acta, 2002 Jun 3, 1597(2), 311 - 9
Cloning, expression and mutagenesis of a subunit contact of rabbit muscle-specific (betabeta) enolase; Kornblatt MJ et al.; The cDNA for rabbit muscle-specific (betabeta) enolase was cloned, sequenced and expressed in Escherichia coli . This betabeta-enolase differs at eight positions from that sequenced by Chin (17) . Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts . Recombinant wild-type and mutant enolase were purified from E . coli and compared to enolase isolated from rabbit muscle . Molecular weights were determined by mass spectrometry . All three betabeta-enolases had similar kinetics, and UV and circular dichroism (CD) spectra . The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate . We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation . DeltaG(o) for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant . In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.

Biochim Biophys Acta, 2002 Jun 3, 1597(2), 252 - 9
MALDI-TOF mass spectrometry characterization of recombinant hydrophobic mutants containing the GCN4 basic region/leucine zipper motif; Bird GH et al.; We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize hydrophobic, alanine-rich mutants of the basic region/leucine zipper (bZIP) protein GCN4 . These bacterially expressed proteins were generated to probe how small, alpha-helical proteins bind specific DNA sites . Enzymatic digestion mapping combined with MALDI-TOF MS characterization of protein fragments allowed us to resolve mass discrepancies between the expected and observed molecular mass measurements . Changes in mass were attributed to posttranslational modifications (PTMs) by proteolytic cleavage of the initiating methionine residue, carbamylation at the amino terminus, oxidation of histidine side chains, and oxidative addition of beta-mercaptoethanol (BME) at the cysteine side chain . Proteins can undergo a wide variety of co-translational modifications and PTMs during growth, isolation, and purification . Such changes in mass can only be detected by a high-resolution technique such as MALDI, which in conjunction with enzymatic digestion mapping, becomes a powerful methodology for characterization of protein structure.

FEBS Lett, 2002 Jun 5, 520(1-3), 88 - 92
Biochemical properties of the human REV1 protein; Masuda Y et al.; It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway . We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C . Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.

FEBS Lett, 2002 Jun 5, 520(1-3), 77 - 80
The effect of an agglutogen on virus infection: biotinylated filamentous phages and avidin as a model; Nakamura M et al.; To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages) . Microscopic observation of mixtures of BIO-phages and avidin-fluorescein conjugates revealed many aggregates . Even at low phage concentrations, avidin induced inhibition of infection significantly . Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin . The inhibition by avidin was at > or = 2 microg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli . On the other hand, antibody inhibited the infection at > or = 0.1 microg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations . These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin . Agglutogens may be novel alternative antiviral drugs.

FEBS Lett, 2002 Jun 5, 520(1-3), 25 - 9
Detection of a very rapid first phase in complex formation of DnaK and peptide substrate; Koller MF et al.; Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides . This first phase is completed within the dead time (1-2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence . It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step . These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.

FEMS Microbiol Lett, 2002 May 7, 210(2), 257 - 61
Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli; Strachan G et al.; Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin . In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin . Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.

Neuroscience, 2002, 112(2), 447 - 54
Sensorimotor functions in transgenic mice expressing the neurofilament/heavy-LacZ fusion protein on two genetic backgrounds; Dubois M et al.; NFH-LacZ transgenic mice are characterized by expression of a non-endogenous fusion protein between a truncated form of mouse NFH (neurofilament of heavy molecular weight) and the complete Escherichia coli beta-galactosidase protein . These transgenic mice were compared to their respective controls on two background strains (C3H and FVB) in several sensorimotor tests . NFH-LacZ mice were deficient in tests requiring balance and equilibrium in a manner generally independent of genetic background . In particular, NFH-LacZ mice fell more quickly than controls from two stationary beams and had fewer rears in an open-field . The transgenic mice were also impaired during the initial trials of sensorimotor learning on the rotorod.We conclude that despite the absence of overt signs of sensorimotor weakness in their home cage, the disruption of the NFH gene, causing neurofilament accumulations in the cell body and diminished axonal calibers of motoneurons, is sufficient to cause motor deficits that resemble the early stages of amyotrophic lateral sclerosis.

Biochemistry, 2002 Jun 11, 41(23), 7508 - 18
Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion; Gu F et al.; Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion . It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases . A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations . In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite . When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products . The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry . 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level . HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity . The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA . With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations . In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III . These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems . Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta . Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E . coli polymerase I.

Biochemistry, 2002 Jun 11, 41(23), 7435 - 42
Nitric oxide and peroxynitrite activate the iron regulatory protein-1 of J774A.1 macrophages by direct disassembly of the Fe-S cluster of cytoplasmic aconitase; Cairo G et al.; Posttranscriptional regulation of iron homeostasis involves, among other factors, a reversible conversion of the Fe-S enzyme cytoplasmic aconitase to a mRNA-binding iron regulatory protein (IRP-1) that lacks an Fe-S cluster . Previous studies have shown that aconitase/IRP-1 may be a target of *NO or peroxynitrite (ONOO(-)), formed after reaction of *NO with superoxide anion (O(2)(*-)); however, the mechanisms and consequences of such interactions have remained uncertain . In this study, recombinant aconitase/IRP-1 was exposed to SIN-1, whose thermal decomposition releases *NO and O(2)(*-) . Results showed that SIN-1 was able to induce concomitant inactivation of aconitase and activation of IRP-1, attributable to cluster disassembly induced by ONOO(-) . SIN-1 was used also in lysates of J774A.1 mouse macrophages grown under control conditions, or subjected to iron loading or starvation by treatment with hemin or desferrioxamine, respectively . Three lines of evidence confirmed that ONOO(-) activated IRP-1 by removing iron from the Fe-S cluster of cytoplasmic aconitase . First, IRP-1 activation was accompanied by iron release and loss of aconitase activity . Second, aconitase activity was recovered by reassembling Fe-S clusters with cysteine and ferrous ammonium sulfate . Third, iron release and IRP-1 activation were observed in lysates from control or iron-loaded macrophages, containing increasing levels of Fe-S clusters, but not in lysates from iron-starved macrophages, in which aconitase had already undergone cluster disassembly and switched to IRP-1 . *NO was less efficient than ONOO(-) in attacking the Fe-S cluster of cytoplasmic aconitase; in fact, SIN-1-dependent iron release and IRP-1 activation were diminished by superoxide dismutase, which scavenged O(2)(*-) before it reacted with *NO to form ONOO(-) . Under comparable conditions, however, both *NO and ONOO(-) inactivated an IRP-2 unable to assemble an Fe-S cluster . These results indicate that *NO and ONOO(-) may activate IRP-1 by attacking the Fe-S cluster of cytoplasmic aconitase, while also inactivating the cluster-deficient IRP-2 . Such divergent actions offer clues to explain links between iron homeostasis and reactive nitrogen species in macrophages involved in inflammation or other pathophysiologic conditions.

Biochemistry, 2002 Jun 11, 41(23), 7400 - 6
The Mycobacterium leprae hsp65 displays proteolytic activity . Mutagenesis studies indicate that the M . leprae hsp65 proteolytic activity is catalytically related to the HslVU protease; Portaro FC et al.; The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides . The M . leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins . When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed . The amino acid sequence alignment of the M . leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)) . Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss . These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M . leprae hsp65 . The possible pathophysiological implications of the proteolytic activity of the M . leprae hsp65 are now under investigation in our laboratory.

Biochemistry, 2002 Jun 11, 41(23), 7366 - 72
An isotope-edited FT-IR study of a symporter, the lactose permease; Patzlaff JS et al.; The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulation of lactose . In this report, the effect of the electrochemical gradient on the permease has been studied . Bacteriorhodopsin was co-reconstituted with the lactose permease to provide a light-triggered electrochemical gradient . Reaction-induced Fourier transform infrared spectra were acquired, and bacteriorhodopsin contributions were subtracted . In previous work, positive bands in the 1765-1730 cm(-1) region of the reaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in the permease {Patzlaff, J . S., Brooker, R . J., and Barry, B . A . (2000) J . Biol . Chem . 275, 28695-28700} . In this study, we have globally labeled the permease with (13)C or (15)N . Isotopic labeling demonstrates that features in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amide II vibrational modes . In addition, isotope labeling leads to a tentative assignment of spectral features to lysine, arginine, histidine, glutamine, and/or asparagine in the permease . These results indicate that the electrochemical gradient causes changes in the environment or protonation state of carboxylic acid residues in the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containing amino acid side chains . Evidence for a change in secondary structure, corresponding to an interconversion of secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptide vibrational modes, is also presented . These experiments demonstrate the usefulness of reaction-induced spectroscopy in the study of transmembrane transport.

Biochemistry, 2002 Jun 11, 41(23), 7267 - 74
Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C; Lindhout DA et al.; Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction . The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin . This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy . It also contains the unique cardiac PKC phosphorylation site at residue T142 . To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D {(1)H, (15)N}-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)) . We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study . NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively . This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+) . These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.

Microgravity Sci Technol, 2001, 13(1), 35 - 8
Ion channel are sensitive to gravity changes; Goldermann M et al.; The effects of gravity on alamethicin doped planar lipid bilayers and on reconstituted porins of Escherichia coli outer membrane, respectively, have been investigated in this paper . The aim of the study was to find out whether and how gravity influences the highly stratified system: membrane-ion channel, in order to provide a novel approach to the explanation of gravity effects on living systems . This is necessary, as even single cells can react to gravity changes without having perceptive organelles . The mechanism of this detection is not clear yet . One possibility might be the detection of gravity by the membrane itself, or by the interaction of integral membrane proteins with gravity . Here we show for the first time that gravity directly influences the integral open state probability of native ion channels (porins) incorporated into planar lipid bilayers . Under hypergravity, especially the open state probability of porins is increased, whereas it is decreased in the microgravity case . The dependency is sigmoidal with the steepest region at 1 to 1.3 g . In the light of these experiments, a general effect of gravity on ion channels and membranes seems to be reasonable, possibly providing an explanation for several impacts of gravity on living systems.

Nat Struct Biol, 2002 Jul, 9(7), 553 - 8
Protein building blocks preserved by recombination; Voigt CA et al.; Borrowing concepts from the schema theory of genetic algorithms, we have developed a computational algorithm to identify the fragments of proteins, or schemas, that can be recombined without disturbing the integrity of the three-dimensional structure . When recombination leaves these schemas undisturbed, the hybrid proteins are more likely to be folded and functional . Crossovers found by screening libraries of several randomly shuffled proteins for functional hybrids strongly correlate with those predicted by this approach . Experimental results from the construction of hybrids of two beta-lactamases that share 40% amino acid identity demonstrate a threshold in the amount of schema disruption that the hybrid protein can tolerate . To the extent that introns function to promote recombination within proteins, natural selection would serve to bias their locations to schema boundaries.

Pharmacogenetics, 2002 Jun, 12(4), 299 - 306
A novel mutant allele of the CYP2A6 gene (CYP2A6*11 ) found in a cancer patient who showed poor metabolic phenotype towards tegafur; Daigo S et al.; In a clinical study, a newly developed anticancer drug, TS-1 capsule, which contained tegafur (FT) and 5-chloro-2,4-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase, was orally administered to five gastric cancer patients (patients 1-5) . The total area under the plasma FT concentration-time curve in patient 1 was four-fold higher than in other patients . Since cytochrome P450 2A6 (CYP2A6) has been reported to metabolize FT to yield 5-fluorouracil (5-FU), it was postulated that the poor metabolic phenotype of patient 1 was caused by mutations of the CYP2A6 gene . Thus, alleles for the CYP2A6 genes derived from patient 1 were completely sequenced . It was found that one allele was CYP2A6*4C, which was a whole deleted allele for the human CYP2A6 gene . The other allele was a novel mutant allele (CYP2A6*11) in which thymine at nucleotide 670 was changed to cytosine . The nucleotide change caused an amino acid change from serine at residue 224 to proline . To examine whether or not the amino acid change affected CYP2A6 activity, we expressed an intact or mutant CYP2A6 together with NADPH-P450 oxidoreductase in Escherichia coli, and compared the capacity of the wild and mutant enzymes to metabolize FT to 5-FU . The Vmax value for FT metabolism by the mutant CYP2A6 was approximately one-half of the value of the intact CYP2A6, although the Km values were nearly the same . From these results, we conclude that the poor metabolic phenotype of patient 1 was caused by the existence of the two mutant alleles, CYP2A6*4C and the new variant CYP2A6*11.

J Biol Chem, 2002 Aug 16, 277(33), 29537 - 49 Epub 2002 May 31.
The Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis in response to lysine availability; Brinkman AB et al.; Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea . Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsive regulatory protein) . Whereas Lrp-like proteins in bacteria are involved in regulation of amino acid metabolism, their physiological role in archaea is unknown . Although several archaeal Lrp-like proteins have been characterized recently, no target genes apart from their own coding genes have been discovered yet, and no ligands for these regulators have been identified so far . In this study, we show that the Lrp-like protein LysM from Sulfolobus solfataricus is involved in the regulation of lysine and possibly also arginine biosynthesis, encoded by the lys gene cluster . Exogenous lysine is the regulatory signal for lys gene expression and specifically serves as a ligand for LysM by altering its DNA binding affinity . LysM binds directly upstream of the TFB-responsive element of the intrinsically weak lysW promoter, and DNA binding is favored in the absence of lysine, when lysWXJK transcription is maximal . The combined in vivo and in vitro data are most compatible with a model in which the bacterial-like LysM activates the eukarya-like transcriptional machinery . As with transcriptional activation by Escherichia coli Lrp, activation by LysM is apparently dependent on a co-activator, which remains to be identified.

Theriogenology, 2002 Feb, 57(3), 1161 - 77
Lochial secretions of Escherichia coli- or Arcanobacterium pyogenes-infected bovine uteri modulate the phenotype and the functional capacity of neutrophilic granulocytes; Zerbe H et al.; It has been suggested that in cases of puerperal endometritis of cattle infected with Escherichia coli and Arcanobacterium pyogenes, the neutrophils are compromised in their defense capacity or downregulated functionally . In addition to direct bacterial effects, contents of lochial secretions and secreted products of locally activated polymorphonuclear neutrophilic granulocytes (PMNs) may also account for changes in function of freshly immigrating neutrophils . In this study, lochial secretions were obtained from healthy cows and from cows infected by E . coli or A . pyogenes . Separated uterine PMN of infected cows displayed an altered phenotype and function which correlated with the degree of bacterial contamination . Concurrently tested circulating PMN showed no such changes . Infected lochial secretions sterilized by filtration also changed the phenotype of blood PMN . Lochial secretions of healthy cows displayed only minor effects . The effects on PMN function in infected cows varied: ingestion was less affected, whereas generation of reactive oxygen species (ROS) was severely depressed . Concurrently tested purified bacterial products (solubles and fragments) of E . coli and A . pyogenes did not induce the phenotypical and functional changes observed in blood PMN . Since infected lochia also contained high numbers of immigrated and probably activated PMN, the influence of supernatants from phorbol myristate acetate-activated PMN were tested on freshly isolated blood PMN . Such supernatants also increased the expression of certain surface molecules and inhibited the ROS generation . Thus, reduced function and altered phenotypes of PMN which immigrate into the uteri of cows with bacterial endometritis is due not only to interactions with bacteria or bacterial products, but is also to the uterine milieu.

Southeast Asian J Trop Med Public Health, 2001, 32 Suppl 2, 98 - 104
Recent advances in serodiagnosis for cysticercosis; Sako Y et al.; Neurocysticercosis (NCC) caused by infection with the larval stage of Taenia solium is an important cause of neurological disease worldwide . Up to the present, many studies on characterizing species-specific antigens of T . solium have been done and several high quality antigens for serodiagnosis are available . Hence the research on serodiagnosis has been shifted to the next phase, stable production of diagnostic antigens using molecular techniques . In order to establish an enzyme-linked immunosorbent assay (ELISA) using recombinant proteins, we carried out molecular cloning and identified four diagnostic antigen candidates (Ag1, Ag1V1, Ag2, and Ag2V1) . Recombinant proteins, except Ag2V1, were successfully expressed using an Escherichia coli expression system . Immunoblot analysis using NCC patient sera detected recombinant proteins . But as reactivity to rAg1 was too weak, Ag1 was not suitable for the immunodiagnosis antigen . Therefore Ag1V1 and Ag2 were chosen for ELISA antigens and Ag1V1/Ag2 chimeric protein was expressed . Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA . Serum samples from patients with other parasitic infections did not recognized Ag1V1/Ag2 chimeric protein . Ag1V1/Ag2 chimeric protein obtained in this study is of value for differential immunodiagnosis.

Southeast Asian J Trop Med Public Health, 2001, 32 Suppl 2, 159 - 64
Diagnosis of intestinal amebiasis using salivary IgA antibody detection; Punthuprapasa P et al.; Attempts were made to use soluble antigen extract of strain HK-9 of Entamoeba histolytica to detect salivary IgA antibodies in intestinal amebiasis patients by using ELISA . Total salivary samples of 109 individuals were divided into four groups . Group I comprised 32 patients whose stools were positive only for E . histolytica cysts and/or trophozoites . Group II comprised 12 individuals whose stools were positive for E . histolytica and other intestinal parasites . Group III comprised 36 individuals whose stools were negative for E . histolytica but contained other intestinal parasites such as E . coli, E . nana, Blastocystis hominis, Trichomonas hominis, Giardia lamblia, Opisthorchis viverrini, and hookworm . Group IV comprised 29 healthy individuals whose stools were free from any intestinal parasitic infections . Based on the mean optical density, OD + 2SD of the results from 29 parasitologically negative healthy individuals, the cut-off OD value for salivary IgA antibodies was 1.265 . Therefore, the assays were positive in 14 out of 32 (43.75%) of group I and 2 out of 12 (16.6%) of group II . The assays were positive in 16 out of 36 (44.44%) for group III whereas 2 out of 29 (6.90%) for group IV were positive . The overall sensitivity and specificity of the assays were 36% and 72%, respectively . The false positive rate was 28% and the false negative rate was 64% . The predictive values of positive and negative results were 47% and 63%, respectively . The diagnostic accuracy of ELISA for the presence of salivary IgA antibodies was 58%.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(4), 457 - 462
Identification and Funtional Characterization of Three Postsynaptic Short-chain Neurotoxins from Hydrophiinae, Lapemis hardwickii Gray; Zhong XF et al.; Three cDNA clones, sn12, sn36 and sn160, encoding isoforms of postsynaptic short-chain neurotoxins, were cloned by screening a cDNA library of the venom from Hydrophiinae, Lapemis hardwickii Gray . The sequences of three cDNA clones encoded proteins consisting of 60 amino acid residues . There was only one amino acid substitution among the three isoforms SN12, SN36 and SN160 at the position 46 of mature proteins, and they were Pro(46), His(46) and Arg(46), respectively . The three molecules were expressed in Escherichia coli and the recombinant proteins were characterized . Different LD(50) were obtained, namely 0.0956 mg/kg, 0.3467 mg/kg and 0.2192 mg/kg, when the SN12, SN36 and SN160 were injected into Kunming mice(i.p.) . In analgesic effect assayed by the acetic acid-induced writhing method, SN12 and SN160 showed similar analgesic effect, but SN36 had effects significantly different with the other two . Our studies suggested that the amino acid residues on position 46 could affect the combination between the postsynaptic short-chain neurotoxins and the nicotinic acetylchoine receptor, since different amino acid substitution resulted in different biological activities.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(4), 379 - 385
Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins; Wei J et al.; Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed . GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG . The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE . 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B . Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide . Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established . The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients . Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients . Detection system established in the study is promising for clinical application.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(4), 368 - 372
Asp(126), Asp(130) and Asp(134) are Necessary for Human IL-18 to Elicit IFN-gamma Production from PBMC; Fu Y et al.; To identify the amino acid residues which are critical to interleukin 18 (IL-18) function, three highly-conserved amino acids (Asp(126), Asp(130) and Asp(134)) were mutated to Asn, Lys and Lys . The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, renatured by stepwise dilution and purified by Sephadex G-75 chromatography . The purity of the recombinant proteins was over 95% and Western blot showed that the mutant rhIL-18 had the same immunogenicity as that of wild type rhIL-18 . The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma(IFN-gamma) production from human peripheral blood mononuclear cells(PBMC) . The results showed that the three mutants induced significantly less amount of IFN-gamma from PBMC(32%, 8% and 10% of wild type for hIL-18D(126)N, hIL-18D(130)K and hIL-18Df(134)K, respectively) indicating that the three highly conserved amino acids are necessary for human IL-18 function.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(5), 531 - 536
Cloning and Expression of SpltMNPV Sl136 Gene and Functions of the Expressed Product; Zhang P et al.; By computer-assisted analysis, it was revealed that ORF136 gene product in SpltMNPV genome had the basic properties of membrane protein . A putative signal peptide was present at the N-terminal and a transmembrane region near the C-terminal of SL136 protein . In the N-terminal half region, there was a coiled-coil domain, which is a typical feature of a number of viral fusion proteins . After PCR amplification, a recombinant plasmid pBVSl136 and a recombinant AcMNPV containing Sl 136 were constructed, in order to express Sl136 gene in E.coli and insect Hi5 cells, respectively . The SDS-PAGE results showed that both expression levels were high . Cell membrane fusion was induced in the Sl-zsu-1 cells, which had been transfected with Sl136 gene alone, by lowering pH of the medium . These results suggested that SL136 protein may be an envelope fusion protein.

Int Immunol, 2002 Jun, 14(6), 647 - 58
Modulation of B lymphocyte signalling by the B subunit of Escherichia coli heat-labile enterotoxin; Bone H et al.; The non-toxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent mucosal adjuvant and immunomodulator capable of blocking autoimmune disease . These effects are linked with its ability to modulate lymphocyte populations--a feature that is dependent on binding to ubiquitously expressed cell surface receptors . Here, we demonstrate that EtxB can trigger up-regulated expression of class II MHC and CD25 on purified populations of B lymphocytes, suggesting that EtxB can directly activate biochemical signalling pathways in these cells . The nature of the intracellular signalling events was investigated . B cells cultured with EtxB, but not a non-receptor binding mutant protein, EtxB(G33D), caused the activation of the extracellular signal-regulated kinase (Erk) forms of mitogen-activated protein (MAP) kinase in a process that was dependent on MAPK/Erk kinase (MEK), phosphoinositide 3-kinase (PI3-kinase) and protein kinase C (PKC), as determined by the use of specific inhibitors . PI3-kinase was critical not only in the activation of MAP kinase but also in the up-regulation of both class II and CD25 . However, MEK inhibition only partially abrogated the EtxB-mediated up-regulation of MHC class II expression and did not affect CD25 expression--findings suggesting that additional pathways downstream of PI3-kinase are involved . A role for PKC in these processes was suggested by the finding that inhibitors of PKC completely blocked EtxB-mediated CD25 up-regulation . Thus, we have shown that receptor binding by EtxB triggers multiple signalling pathways in B cells that regulate the expression of key cell surface molecules.

Appl Environ Microbiol, 2002 Jun, 68(6), 3138 - 40
Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase; Woo HJ et al.; Synechocystis sp . strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli . The E . coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase . Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E . coli.

Appl Environ Microbiol, 2002 Jun, 68(6), 2794 - 801
Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy; Vigeant MA et al.; The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek) . Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased . In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy . We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces . However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions . We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells . We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained . A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.

J Pharm Biomed Anal, 2002 Jun 1, 28(5), 953 - 63
An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161; Wan M et al.; Staphylokinase, a bacterially-derived protein which functions as a plasminogen activator, has potential utility as a human therapeutic for thrombotic disorders . A recombinant version of this protein, SY161, contains 13 amino acid substitutions designed to decrease immunogenicity, and has been covalently modified by crosslinking a 5 kDa polyethyleneglycol (PEG) group to the N-terminal region to prolong the drug circulating half-life . The recombinant PEG-modified SY161 staphylokinase is currently in phase II clinical trials as a treatment for acute myocardial infarction . We have developed a sensitive product specific host cell protein (HCP) assay in the ELISA format to monitor in process host-derived contaminant clearance and final drug product purity . The assay is based upon use of goat polyclonal antibodies raised against E . coli host strain cell proteins from a null cell line, extracted by the same manufacturing process used to produce SY161 . The identification and clearance of HCP contaminants was confirmed during drug product production using SDS-PAGE and Western blotting utilizing the same polyclonal HCP antibodies . The assay is specific for E . coli host cell strain proteins with a useful detection range from 1 to 100 ng/ml, and is not affected by product level . The level of residual HCPs in the clinical product produced by our manufacturing process was determined to be less than 1 ng/ml at a product concentration of 1 mg/ml.

Bioorg Med Chem Lett, 2002 Jun 17, 12(12), 1691 - 4
Development of a genetic selection for catalytic antibodies; Gildersleeve J et al.; The design and evaluation of a new genetic selection system for evolving catalytic antibodies with aldolase activity are described . Through a series of model selections, we have identified selection conditions where expression of a catalytically active antibody confers a growth advantage to Escherichia coli . In addition, we provide evidence that the growth advantage is a direct result of catalytic activity.

J Biochem (Tokyo), 2002 Jun, 131(6), 855 - 9
Acidic residues on the N-terminus of proinsulin C-Peptide are important for the folding of insulin precursor; Chen LM et al.; To investigate the role of C-peptide in the folding of insulin precursor, a series of C-peptide mutant proinsulin genes were constructed, overexpressed in Escherichia coli and the proteins purified . Correct disulfide linkages of these proteins were confirmed by both tryptic peptide mapping and insulin receptor binding analyses . In vitro refolding experiments were performed with the purified proteins and showed that mutations on the glycine-rich middle segment of C-peptide, GGGPGAG, and deletion of the C-terminal pentapeptide, EGSLQ, as well as mutations on the two pairs of dibasic residues at the two ends of C-peptide did not significantly affect the refolding yields . However, both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N-terminus of the C-peptide resulted in serious aggregation during refolding . The results indicate that the highly conserved acidic N-terminal part of C-peptide is very important for insulin precursor folding, and that C-peptide may have some intramolecular chaperone-like function in the folding of insulin precursor.

J Biochem (Tokyo), 2002 Jun, 131(6), 839 - 47
Another cut for lysine tRNA: application of the hyperprocessing reaction reveals another stabilization strategy in metazoan lysine tRNAs; Tanaka T et al.; Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions . We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing . We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure . There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted . Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible . The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P . The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions . We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme . The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications . A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs . Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).

Biochem J, 2002 Aug 15, 366(Pt 1), 323 - 32
Identification and characterization of GSTT3, a third murine Theta class glutathione transferase; Coggan M et al.; A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database . The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes . mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis . Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes . A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate.

Immunogenetics, 2002 May, 54(2), 67 - 73 Epub 2002 Mar 16.
Possible association of non-binding of HSP70 to HLA-DRB1 peptide sequences and protection from rheumatoid arthritis; Maier JT et al.; The beta-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70-74 . This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation . It is known that the Escherichia coli M(r)70,000 heat shock protein DnaK can bind peptides from the shared epitope . Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK . Similar binding specificities could be found for the constitutively expressed mammalian M(r)70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72 . However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70 . Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70-74 fragments and protection from RA.

Cell Tissue Res, 2002 May, 308(2), 287 - 97 Epub 2002 Apr 13.
Immunolocalization of chitin synthase in the tobacco hornworm; Zimoch L et al.; To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase . Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells . In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut . Moreover, in the anterior midgut they were localized in the apical region of columnar cells . Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome . To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein . In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa . Immunohistochemistry showed intense labeling in midgut brush border membranes . Immunofluorescence was restricted to the apical ends of microvilli . Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules . This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1071 - 3 Epub 2002 May 29.
Crystallization and preliminary X-ray crystallographic analysis of enoyl-acyl carrier protein reductase from Helicobacter pylori; Lee HH et al.; Enoyl-acyl carrier protein reductase (ENR) catalyzes the NADH-dependent stereospecific reduction of alpha,beta-unsaturated fatty acids bound to the acyl-carrier protein . ENR from Helicobacter pylori has been overexpressed in Escherichia coli and has been crystallized in the presence of its cofactor NADH and the inhibitor triclosan (or its analogue diclosan) at 296 K using polyethylene glycol (PEG) 400 as a precipitant . For the triclosan (or diclosan) complex, diffraction data to 2.5 (or 2.3) A resolution have been collected using synchrotron X-rays . The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 73.35, b = 94.91, c = 75.38 A, beta = 106.21 degrees for the triclosan complex (or a = 73.25, b = 95.07, c = 75.02 A, beta = 106.53 degrees for the diclosan complex) . The asymmetric unit contains one homotetramer, with a corresponding V(M) of 2.10 A(3) Da(-1) and a solvent content of 41% by volume.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1068 - 70 Epub 2002 May 29.
Crystallization and preliminary X-ray diffraction analysis of brefeldin A-ADP ribosylated substrate (BARS); Nardini M et al.; Brefeldin A-ADP ribosylated substrate (BARS) is a newly discovered enzyme involved in membrane fission, catalyzing the formation of phosphatidic acid by transfer of an acyl group from acyl-CoA to lysophosphatidic acid . A truncated form of BARS, lacking the C-terminal segment expected to interact with the Golgi membrane, has been expressed in soluble form in Escherichia coli, purified and crystallized . BARS crystals diffract up to 2.5 A resolution using synchrotron radiation and belong to space group P6(2)22/P6(4)22, with unit-cell parameters a = b = 89.2, c = 162.6 A, alpha = beta = 90, gamma = 120 degrees and one molecule (39.5 kDa) per asymmetric unit . SeMet-substituted BARS has been crystallized under growth conditions very similar to those of the native protein.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1066 - 7 Epub 2002 May 29.
Crystallization of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli; Krzywda S et al.; FtsH is a membrane-anchored ATP-dependent protease that degrades misfolded or misassembled membrane proteins as well as a subset of cytoplasmic regulatory proteins . It belongs to the family of AAA(+) ATPases with roles in diverse cellular processes . The ATPase domain of FtsH from Escherichia coli has been crystallized from ammonium sulfate solutions and crystals diffracting to 1.5 A resolution have been obtained.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1051 - 3 Epub 2002 May 29.
Crystallization and preliminary crystallographic studies on the chromo shadow domain (CSD) of mouse heterochromatin protein M31; Gao Y et al.; Members of the heterochromatin protein 1 (HP1) class of non-histone chromosomal proteins are components of heterochromatin and are involved in the epigenetic regulation of the genome . HP1 proteins are modular and consist of two sequence-related domains called the chromodomain (CD) and the chromo shadow domain (CSD) . In order to investigate the role of the murine HP1-like protein M31 in heterochromatin formation and gene silencing, recombinant CSD was overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as precipitant . Diffraction data to 2.9 A were collected from a native crystal belonging to space group C222(1), with unit-cell parameters a = 60.0, b = 95.6, c = 91.7 A, alpha = beta = gamma = 90 degrees.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1030 - 1 Epub 2002 May 29.
Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 nucleotide-binding domain 2 (NBD2); Li J et al.; Escherichia coli Hsp100 ClpB has been identified recently as playing critical roles in multi-chaperone systems . ClpB binds and disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides . ClpB contains two nucleotide-binding domains (NBD1 and NBD2) in its primary sequence . Walker A and Walker B motifs exist in both nucleotide-binding domains . Therefore, ClpB belongs to the large ATPase family known as ATPase associated with various cellular activities (AAA) . The mechanisms by which NBD1 and NBD2 function to support the ClpB molecular-chaperone activity are currently unknown . To investigate how NBD2 participates in ClpB function to disaggregate denatured proteins, ClpB NBD2 has been cloned and crystallized . The ClpB NBD2 crystals diffract X-rays to 2.5 A using synchrotron X-ray sources . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 99.57, b = 149.34, c = 164.69 A.

J Magn Reson, 2002 Apr, 155(2), 236 - 43
Tailored HCCH-TOCSY experiment for resonance assignment in the proximity of a paramagnetic center; Piccioli M et al.; The presence of a paramagnetic center may disturb both coherent and incoherent communication between nuclear spins that are affected, to some extent, by the hyperfine interaction . This is a limiting factor to an extensive use of paramagnetic probes in NMR spectroscopy to enhance partial alignment and to exploit cross correlation effects and pseudocontact shifts . We propose here an HCCH-TOCSY experiment tailored to identify spin systems involving resonances that are partly or completely affected by hyperfine interaction . The efficiency of polarization transfer steps when fast relaxing nuclei are involved is discussed . The sequence is tested for the protein Calbindin D(9k), in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion as well as for the oxidized form of cytochrome b(562) . (c) 2002 Elsevier Science (USA).

Mol Plant Microbe Interact, 2002 May, 15(5), 456 - 62
Roles for riboflavin in the Sinorhizobium-alfalfa association; Yang G et al.; Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S . meliloti . Riboflavin-synthesis genes in S . meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes . The ribBA and ribC loci complemented corresponding E . coli rib mutants . S . meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin . Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain . No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N . These results support the importance of extracellular riboflavin for alfalfa root colonization by S . meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.

Clin Nephrol, 2002 May, 57(5), 398 - 401
Disseminated strongyloidiasis in nephrotic syndrome; Morimoto J et al.; Strongyloides stercoralis is endemic in the southwestern islands Amami and Ryukyu in Japan . Systemic strongyloidiasis occurs in immunocompromised hosts . We report here on a 60-year-old patient with minimal-change nephrotic syndrome (MCNS) without eosinophilia or HTLV-I infection . She was treated with corticosteroid for MCNS and died of disseminated strongyloidiasis . The patient developed systemic purpura, ileus, respiratory distress, malabsorption, pancytopenia, pulmonary hemorrhage and sepsis due to Escherichia coli before death . Massive infestation with Strongyloides stercoralis was disclosed by autopsy, and the larvae was considered as a pathomechanism or exacerbating agent of nephrotic syndrome in endemic areas.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 925 - 7
Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase; Yamamoto H et al.; The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation . Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 910 - 2
Expression of Nicotiana glutinosa ribonucleases in Escherichia coli; Hino M et al.; We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively . In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity . RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I) . Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU . Furthermore, kinetic parameters for RNase NW (Km, 0.778 mM and kcat, 1938 min(-1)) and RNase NGR3 (Km, 0.548 mM and kcat, 408 min(-1)) were calculated using GpU as a substrate.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 770 - 6
Kinetic analysis of the effect of (-)-epigallocatechin gallate on the DNA scission induced by Fe(II); Ohashi Y et al.; The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (-)-epigallocatechin gallate (EGCg) were kinetically analyzed . The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission . Coordinated EGCg accelerated these reactions . Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl- ions and EGCg added . Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one . This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.

Biosci Biotechnol Biochem, 2002 Apr, 66(4), 754 - 61
Cloning of cold-active alkaline phosphatase gene of a psychrophile, Shewanella sp., and expression of the recombinant enzyme; Murakawa T et al.; A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp . is a cold-active enzyme that has high catalytic activity at low temperature {Ishida et al . (1998) Biosci . Biotechnol . Biochem., 62, 2246-2250} . Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques . The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures . The recombinant enzyme expressed in E . coli was purified to homogeneity with the molecular mass of 41 kDa . The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.

Chem Pharm Bull (Tokyo), 2002 May, 50(5), 578 - 82
Selective inhibition of Fe- versus Cu/Zn-superoxide dismutases by 2,3-dihydroxybenzoic acid derivatives; Soulere L et al.; A series of catechol derivatives were synthesised and tested for their ability to inactivate the iron-containing superoxide dismutase (Fe-SOD) from Escherichia coli and the bovine erythrocytes Cu/Zn-SOD . Incubation of catechols with Fe- or Cu/Zn SODs resulted in a time-dependent loss of enzyme activity with highly selective inhibition for the iron-dependent enzyme . Catechol-induced inactivation of SODs was correlated with the auto-oxidation of the catechol compounds to their corresponding ortho-quinone derivatives, which was found to be non-dependent on the presence of enzymes . Mass electrospray experiments on catechol-incubated Fe-SOD provided evidence for the irreversible nature of the inhibition process, yielding to a complex mixture of modified proteins.

Life Sci Space Res, 1965, 3, 185 - 205
Satellite biological experiments--major results and problems; Sisakyan NM et al.; The data on the results of biological experiments carried out on Vostok 5 and Vostok 6 are presented . Space flight factors are shown to cause in hereditary structures of some biological objects (seeds of higher plants, lysogenic bacteria, Tradescantia microspores, etc.) distortions of a small but statistically significant value . Changes in physiological functions of certain objects (seeds of higher plants, etc.) have been also detected . These data are in good agreement with the results of flight experiments carried out in 1960-1962 . Prospects of research of the biological effect of cosmic radiation and weightlessness are considered with respect to flight experiments.

Biochem Soc Trans, 2002 Apr, 30(2), 150 - 5
The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes; Carpousis AJ; mRNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript . The RNase e of Escherichia coli is a single-strand-specific endo-nuclease involved in the processing of rRNA and the degradation of mRNA . A nucleolytic multi-enzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E . Two other components are a 3' exoribonuclease (polynucleotide phosphorylase, PNPase) and a DEAD-box RNA helicase (RNA helicase B, RhlB) . RNase E is a large multidomain protein with N-terminal ribonucleolytic activity, an RNA-binding domain and a C-terminal "scaffold" that binds PNPase, enolase and RhlB . RhlB by itself has little activity but is strongly stimiulated by its interaction with RNase E . RhlB in vitro can facilitate the degradation of structured RNA by PNPase . Since the discovery of the RNA degradosome in E . coli, related complexes have been described in other organisms.

Microb Ecol, 2000 Dec, 40(4), 336 - 344
Humic Materials Offer Photoprotective Effect to Escherichia coli Exposed to Damaging Luminous Radiation; Muela A et al.; The behavior of Escherichia coli immersed in aqueous systems amended with humic acids, under PAR, UV-A, UV-B, and simulated solar radiation was examined . Culturability, ability to elongate, functioning of the electron transport systems, and glucose uptake were assessed . Humic substances in the range from 1 to 50 mg L-1 protected cells from photoinactivation . Decrease in culturability and cellular activities was significantly (p <0.05) less in the presence of humic material . However, humic acids were not used as nutrients . Neither irradiated nor nonirradiated humic solutions (50 mg L-1) supported the growth of 105 cells ml-1 . However, humic acids dissolved in 0.9% NaC1 efficiently absorbed light over wavelengths from 270 to 500 nm . Also, a photoprotective effect against simulated sunlight was observed when humic acids were not in contact with but rather enveloped the cellular suspensions in double-wall microcosms . The protection afforded by humic acids against luminous radiation likely derives from their ability to absorb these radiations and hence reduces the amount of energy reaching the cells.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 707 - 712
Cloning and Expression of Chinese Duck Interferon-gamma Gene; Long JE et al.; The efficacy of cytokine therapy has been demonstrated in several viral diseases . Interferon-gamma is a cytokine that has potent antiviral property and immunomodulatory activity . To investigate the role of IFN-gamma in viral clearance during natural infection and to define the antiviral mechanism, DHBV-infected ducks was used as an animal model . To clone, express, and develop the method of quantifying DuIFN-gamma gene transcription and expression, DuIFN -gamma cDNA was amplified by RT-PCR from PHA stimulated duck PBMC . Recombinant plasmid expressing DuIFN-gamma was used to transfect COS-7, and the cell culture supernatant was analyzed by CPE inhibitory assay and MTT methods to determine the antiviral titer of IFN-gamma . The GST-DuIFN-gamma fusion protein was expressed in E.coli and purified using the GST sepharose 4B . Results indicated that the supernatant collected from COS-7 cells transfected with DuIFN-gamma cDNA was able to prevent duck fibroblasts from VSV induced CPE in a dose dependent manner . An anti-DuIFN-gamma antibody neutralized this antiviral activity.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 703 - 706
Fusion Expression and Purification of OSBP PH Domain and Preliminary Analysis of Its Second Structure; Shen L et al.; Oxysterol binding protein (OSBP) is a regulator of oxysteroid metabolism . To investigate the function and the structure-function relationship of OSBP, the recombinant vector OSBP PH-pRSET-A was transformed into E.coli JM109(DE3), and the strain highly expressing soluble 6His-OSBP PH domain in minimal medium were obtained . The fusion protein was purified by Ni(2 )-NTA agarose beads . The secondary structure of the purified 6His-OSBP PH domain fusion protein was analysed by circular dichronism . The results indicated the PH domain was composed of alpha-helix 7.2%, beta-pleated sheets 71.1% and radom coil 21.7%.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 647 - 652
Expression, Purification and Functional Identification of Extracellular Part of Discoidin Domain Receptor 2; Wang JC et al.; Discoidin domain receptor 2 (DDR2) is a new type of receptor tyrosine kinases, and was thought to be involved in the metastasis of some tumors . Its ligand is fibrillar collagen . The activation of DDR2 induced by collagen mediates the over-expression of matrix metalloproteinase 1 (MMP-1) in cells . A specific inhibitor of DDR2 was necessary for the study of DDR2 function . Theoretically, a soluble receptor could possibly be used as specific inhibitor for the native receptor on cell membrane . In this report, a fragment (DB) of extracellular part of DDR2 was cloned and expressed for the use as potential inhibitor . This DB fragment corresponded to the polypeptide from the 23rd amino acid residue to the 293rd amino acid residue of DDR2 . The fragment was amplified by RT-PCR from human lung cancer tissue, and the product was cloned into pMD18-T vector . After identification by sequence analysis, the fragment was sub-cloned into pGEX-4T-1 vector . Fusion protein of GST-DB was expressed in JM109 E.coli cells as expected and the soluble part accounted for about 13% of the total fusion protein . The soluble fusion protein was then purified with glutathione affinity resin, and GST-DB with purity of 86.1% was obtained . Competitive combination inhibitory test showed that the purified GST-DB inhibited the interaction between collagen II and DDR2 on the surface of RA synovial fibroblasts . Zymography analysis showed that the level of MMP-1 of both NIH 3T3 cell and RA synovial fibroblasts with collagen II-stimulation decreased after adding GST-DB fusion protein . The results indicated that the fusion protein GST-DB could inhibit the function of DDR2 on cells, and DDR2 might mediate collagen II-induced over-expression of MMP-1 in these cells.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 621 - 628
A Negative Element Located in the Upstream Flanking Region of the Gene Encoding Arginyl-tRNA Synthetase (argS) from Escherichia coli; Liu MF et al.; The gene, argS, encoding the arginyl-tRNA synthetase (ArgRS) from Escherichia coli ( E.coli ) was overexpressed 1 000 fold in the transformant when E . coli TG1 was transformed with the recombinant plasmid containing argS and pUC18 . In order to investigate the regulation of expression of E . coli argS, a series of deletion mutations was constructed . The results of SDS-PAGE showed that deletions of the whole 5' flanking region (argSdelta1) or the region in front of Shine-Dalgarno Sequence (argSdelta2) or the -10 region of promoter (argSdelta3), caused no overexpression of argS . If argS was deleted from 3' end of the flanking region (-189 nt) to the upstream of -10 region of promoter (argSdelta4), the -35 region (argSdelta5), -52 nt (argSdelta6), -70 nt (argSdelta7) and -122 nt (argSdelta8), respectively, the mutant gene was overexpressed to a level similar to that of argS bringing the full length 5' flanking region . However, in the expression of argSdelta4, argSdelta5, argSdelta6, some of ArgRS formed an inclusion body . By determination of RNA dot hybridization, the amount of mRNA produced in the transcription of argSdelta4, argSdelta5 and argSdelta6 was about 2--3 times than that of the wild type argS, argS delta7 and argS delta8 . This indicated that the deletion of a 19 nt sequence (AATAGTGAAAACGGCAATA) located between -52 nt and -70 nt of the gene increased the transcription of argS . The 19 nt sequence is a negative region that represses transcription of argS . Deletion of the negative element may result in a faster production of ArgRS and the accumulation of some unfolding protein intermediates aggregating to form the inclusion body . The result by analysis of gel retardation shows that a factor binds to the negative element . Arginine induced specifically the transcription of argS and its effect correlated with the above negative element.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(6), 591 - 599
The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling; Fei H et al.; Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins . Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli . Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L) . Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor . (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude . (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition . (4) A hydrophobic residue is preferential at P(3) site . Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 8066 - 71 Epub 2002 May 28.
Proton-motive force stimulates the proteolytic activity of FtsH, a membrane-bound ATP-dependent protease in Escherichia coli; Akiyama Y; FtsH is a membrane-bound, ATP-dependent metalloprotease in Escherichia coli that degrades some integral membrane proteins and cytoplasmic proteins . In this study, we show that FtsH-dependent degradation of both membrane-bound and soluble proteins is retarded when cells are treated with carbonyl cyanide-3-chlorophenylhydrazone or 2,4-dinitrophenol uncouplers, which dissipate the proton-motive force . In vitro casein degradation by membrane-integrated FtsH was stimulated by succinate, a respiratory substrate; this stimulation was counteracted by cyanide-3-chlorophenylhydrazone . Potassium thiocyanate, which specifically collapses Deltapsi, partially canceled the effect of succinate, but ammonium sulfate, which collapses DeltapH, showed little effect . These results indicate that the proton-motive force, in particular the Deltapsi component, plays a role in efficient degradation of substrates by FtsH in its native state . FtsH variants with altered transmembrane regions did not receive proton-motive force stimulation, suggesting that the proton-motive force activates FtsH, directly or indirectly, through the transmembrane region.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 8348 - 53 Epub 2002 May 28.
Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: a soluble, secreted CD30 homologue; Panus JF et al.; Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus . Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene, vCD30, encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus . The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51-59% identity with highly conserved extracellular segments of both mouse and human CD30 . A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions . Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses . Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases.

Protein Eng, 2002 May, 15(5), 437 - 41
Structural restoration of inactive recombinant fish growth hormones by chemical chaperonin and solvent restraint approaches; Chang CC et al.; Recombinant proteins may undergo conformational distortion, leading to aggregation and loss of function, when they are expressed in heterologous systems . The structural and functional restoration of such inactive proteins is highly desirable . We have over-expressed recombinant growth hormones from the fish ayu (Plecoglossus altivelis) and yellow grouper (Epinephelus awoara) by a pET expression system . Both recombinant proteins accumulate as insoluble form in Escherichia coli . We refolded these inactive proteins into the active form using a stepwise refolding process with a dilute denaturing agent as a steric blocker and chemical chaperonin . Optical characterization showed that stable folding intermediates with a helical conformation can be detected in the molten globule state . Moreover, the function of restored recombinant growth hormones was demonstrated by its ability to stimulate proliferation in zebrafish liver cells.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2538 - 45
Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase; Tardif KD et al.; Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine . To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA(Val) . Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3'-terminus of tRNA(Val) is recognized differently at the two sites . Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA(Val) at the aminoacylation and editing sites, and by probing tRNA(Val) for editing determinants that are distinct from those required for aminoacylation . Mutational analysis of Escherichia coli tRNA(Val) and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS . This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2492 - 500
The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system; Verhoeven EE et al.; Nucleotide excision repair in Escherichia coli involves formation of the UvrB-DNA complex and subsequent DNA incisions on either site of the damage by UvrC . In this paper, we studied the incision of substrates with different damages in varying sequence contexts . We show that there is not always a correlation between the incision efficiency and the stability of the UvrB-DNA complex . Both stable and unstable UvrB-DNA complexes can be efficiently incised . However some lesions that give rise to stable UvrB-DNA complexes do result in a very low incision . We present evidence that this poor incision is due to sterical hindrance of the damage itself . In its C-terminal region UvrC contains two helix-hairpin-helix (HhH) motifs . Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)2 domain . This (HhH)2 domain was previously shown to be important for the 5' incision on a substrate containing a (cis-Pt).GG adduct, but not for 3' incision . Here we show that, mainly depending on the sequence context of the lesion, the (HhH)2 domain can be important for 3' and/or 5' incision . We propose that the (HhH)2 domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2407 - 16
eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments; Moore GL et al.; We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization . Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective . We find that the free energy of annealing between the recombining DNA sequences is a much better descriptor of the extent of crossover formation than sequence identity . Three different diversity targets are investigated for the DNA shuffling protocol to showcase the utility of the eCodonOpt framework: (i) maximizing the average number of crossovers per recombined sequence; (ii) minimizing bias in family DNA shuffling so that each of the parental sequence pair contributes a similar number of crossovers to the library; and (iii) maximizing the relative frequency of crossovers in specific structural regions . Each one of these design challenges is formulated as a constrained optimization problem that utilizes 0-1 binary variables as on/off switches to model the selection of different codon choices for each residue position . Computational results suggest that many-fold improvements in the crossover frequency, location and specificity are possible, providing valuable insights for the engineering of directed evolution protocols.

Nucleic Acids Res, 2002 Jun 1, 30(11), 2390 - 7
23S rRNA assisted folding of cytoplasmic malate dehydrogenase is distinctly different from its self-folding; Sanyal SC et al.; The role of the 50S particle of Escherichia coli ribosome and its 23S rRNA in the refolding and subunit association of dimeric porcine heart cytoplasmic malate dehydrogenase (s-MDH) has been investigated . The self-reconstitution of s-MDH is governed by two parallel pathways representing the folding of the inactive monomeric and the dimeric intermediates . However, in the presence of these folding modulators, only one first order kinetics was observed . To understand whether this involved the folding of the monomers or the dimers, subunit association of s-MDH was studied using fluorescein-5-isothiocyanate-rhodamine-isothiocyanate (FITC-RITC) fluorescence energy transfer and chemical cross-linking with gluteraldehyde . The observation suggests that during refolding the interaction of the unstructured monomers of s-MDH with these ribosomal folding modulators leads to very fast formation of structured monomers that immediately dimerise . These inactive dimers then fold to the native ones, which is the rate limiting step in 23S or 50S assisted refolding of s-MDH . Furthermore, the sequential action of the two fragments of domain V of 23S rRNA has been investigated in order to elucidate the mechanism . The central loop of domain V of 23S rRNA (RNA1) traps the monomeric intermediates, and when they are released by the upper stem-loop region of the domain V of 23S rRNA (RNA2) they are already structured enough to form dimeric intermediates which are directed towards the proper folding pathway.

EMBO Rep, 2002 Jun, 3(6), 532 - 6 Epub 2002 May 24.
A dual role for the FtsK protein in Escherichia coli chromosome segregation; Capiaux H et al.; FtsK is a multifunctional protein that acts in Escherichia coli cell division and chromosome segregation . Its C-terminal domain is required for XerCD-mediated recombination between dif sites that resolve chromosome dimers formed by recombination between sister chromosomes . We report the construction and analysis of a set of strains carrying different Xer recombination sites in place of dif, some of which recombine in an FtsK-independent manner . The results show that FtsK-independent Xer recombination does not support chromosome dimer resolution . Furthermore, resolution of dimers by the Cre/loxP system also requires FtsK . These findings reveal a second role for FtsK during chromosome dimer resolution in addition to XerCD activation . We propose that FtsK acts to position the dif regions, thus allowing a productive synapse between dif sites.

J Biol Chem, 2002 Aug 9, 277(32), 28564 - 71 Epub 2002 May 28.
Structural independence of the two EF-hand domains of caltractin; Veeraraghavan S et al.; Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans . The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions . In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli . Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy . Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays . Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain . Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways . However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range . Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.

J Biol Chem, 2002 Aug 16, 277(33), 29369 - 76 Epub 2002 May 28.
Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids . Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport; Weimar JD et al.; Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC ) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA . In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids . The E . coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif ((213)YTGGTTGVAKGA(224) and (356)GYGLTE(361)) placing it in the superfamily of adenylate-forming enzymes . A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport . This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD(G216A), fadD(T217A), fadD(G219A), and fadD(K222A)) and 2) those with 10% or less wild-type activity (fadD(Y213A), fadD(T214A), and fadD(E361A)) . Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type . The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP . The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids . These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

Am J Respir Cell Mol Biol, 2002 Jun, 26(6), 650 - 8
Extrinsic coagulation blockade attenuates lung injury and proinflammatory cytokine release after intratracheal lipopolysaccharide; Miller DL et al.; Initiation of coagulation by tissue factor (TF) is a potentially powerful regulator of local inflammatory responses . We hypothesized that blockade of TF-factor VIIa (FVIIa) complex would decrease lung inflammation and proinflammatory cytokine release after tracheal instillation of Escherichia coli lipopolysaccharide (LPS 0111:B4) . At the time of injury, rats received one dose of site-inactivated FVIIa (FFR-FVIIa) or saline intravenously . At 0, 6,12, 24, and 48 h after injury, lungs were examined for histologic changes and bronchoalveolar lavage (BAL) was performed to assess protein, lactate dehydrogenase (LDH) activity, cell counts, and cytokine levels . LPS-injured rats treated with FFR-FVIIa showed decreased intra-alveolar inflammation and fibrin deposition by light microscopy compared with untreated rats . This was accompanied by decreased protein leakage (P < 0.0001), LDH activity (P < 0.0001), and local elaboration of interleukin (IL)-1beta, IL-6, and IL-10 (all P < 0.0001), but not tumor necrosis factor (TNF)-alpha . Protection was associated with reduction of TF mRNA expression in whole lung, but not with changes in nuclear translocation of nuclear factor (NF)-kappaB . FFR-FVIIa given 6 h after LPS afforded equivalent lung protection . Therefore, blockade of TF-FVIIa complex protects the lung from injury by LPS in part by reducing local expression of proinflammatory cytokines and may offer promise for therapy of acute lung injury.

Gene, 2002 Apr 17, 288(1-2), 85 - 94
The use of synthetic genes for the expression of ciliate proteins in heterologous systems; Lin Y et al.; The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens . These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development . Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward . To overcome this problem, we utilized 'assembly polymerase chain reaction' to manufacture synthetic versions of two genes (designated IAG52A{G5/CC} and IAG52B{G5/CC}) encoding approximately 52/55 kDa i-antigens from parasite strain G5 . This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes . To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A{G5/CC} into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting . When cloned downstream of appropriate promoters, IAG52A{G5/CC} was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses . Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems . Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems.

Gene, 2002 Apr 17, 288(1-2), 1 - 8
Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 3' side; Stenstrom CM et al.; The downstream region (DR) located immediately after the initiation codon acts as a translational enhancer and depending on its sequence gene expression can vary considerably . In order to determine the influence of the DR on the apparent translation initiation, we have analyzed several naturally occurring DRs (a stretch of five codons) in a lacZ reporter gene . The efficiency of expression, associated with these DRs did not show any correlation to the expression levels connected with the natural genes . Changes of the iso-codon composition in the DR, thus maintaining the amino acid sequence in the gene product, gave significant variations in gene expression . Thus, the messenger RNA base sequence, and not the encoded amino acid sequence, in the early coding region is the determinant for the apparent efficiency of translation initiation and/or early elongation.

Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 118 - 28
Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis; Umbach AL et al.; The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP . Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate . The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli . Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate . Uncharged substitutions (Gln, Leu) produced an inactive enzyme . These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis . Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site . Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site . In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.

Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 66 - 74
HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria; Schmitz O et al.; NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety . This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase . HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli . The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated . The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp . PCC 6803 and Synechococcus sp . PCC 6301 . The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively . H(2)-evolution of the purified enzyme of S . sp . PCC 6803 is highest at 60 degrees C and pH 6.3 . Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S . sp . PCC 6301 . SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE . The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).

Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 22 - 8
Electron transfer at the low-spin heme b of cytochrome bo(3) induces an environmental change of the catalytic enhancer glutamic acid-286; Prutsch A et al.; Intramolecular proton transfer of heme-copper oxidases is performed via the K- and the transmembrane D-channels . A carboxyl group conserved in a subgroup of heme-copper oxidases, located within the D-channel close to the binuclear center (=glutamic acid-286 in cytochrome bo(3) from Escherichia coli) is essential for proton pumping . Upon electron transfer to the fully oxidized (FO) enzyme, this amino acid has been shown to undergo a cyanide-independent environmental change . The redox-induced environmental transition of glutamic acid-286 is preserved in the site-directed mutant Y288F, which has lost its Cu(B) binding capacity . Furthermore, the mixed-valence (MV) redox state of cytochrome bo(3) (in which Cu(B) and high-spin heme are reduced, whereas the low-spin heme stays oxidized) was prepared by anaerobic exposure of the protein to carbon monoxide . This complex was converted (i) to the FO state by reaction with the caged dioxygen donor mu-peroxo) (mu-hydroxo) bis {bis (bipyridyl) cobalt (III)} and (ii) to the fully reduced (FR) state via caged electron donors; the environmental change of glutamic acid-286 could be observed only upon reduction . Taken together, these results from two different lines of evidence clearly show that the redox transition of the low-spin heme b center alone triggers the change in the chemical environment of this acidic side chain . It is suggested that glutamic acid-286 is a kinetic enhancer of proton translocation, which is energetically favoured in mesophilic oxidases.

Biophys Chem, 2002 May 2, 96(2-3), 305 - 18
In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor; Bazarsuren A et al.; The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E . coli and isolated as inclusion bodies . Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material . A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration . Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry . The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin . Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.

Biophys Chem, 2002 May 2, 96(2-3), 243 - 57
Refolding and structural characterization of the human p53 tumor suppressor protein; Bell S et al.; The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level . We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies . To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding . The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces . The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody . Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.

Biophys Chem, 2002 May 2, 96(2-3), 163 - 71
Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability; Siebold C et al.; Cyclized subunits of the E . coli glucose transporter were produced in vivo by intein mediated trans-splicing . IIA(Glc) is a beta-sandwich protein, IICB(Glc) spans the membrane eight times . Genes encoding the circularly permuted precursors U(Cdelta)-IIA(Glc)-U(Ndelta) and U(Cdelta)-IICB(Glc)-U(Ndelta) were assembled from DNA fragments encoding the 3' and 5' segments of the recA intein of M . tuberculosis and crr and ptsG of E . coli, respectively . A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins . The cyclized proteins complemented growth of glucose auxotrophic strains . Purified, cyclized IIA(Glc) and IICB(Glc) had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity . They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin . Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding (75 vs . 70 degrees C; 1.52 vs . 1.05 M).

Biophys Chem, 2002 May 2, 96(2-3), 153 - 61
Formation of 70S ribosomes: large activation energy is required for the adaptation of exclusively the small ribosomal subunit; Blaha G et al.; Association of ribosomal subunits is an essential reaction during the initiation phase of protein synthesis . Optimal conditions for 70S formation in vitro were determined to 20 mM Mg2+ and 30 mM K+ . Under these conditions, the association reaction proceeds with first order kinetics, suggesting a conformational change to be the rate-limiting step . 70S formation separates into two sub-reactions, the adaptation of the ribosomal subunits to the association conditions and the association step itself . The activation energy of the process was determined to 78 kJ/mol and revealed to be required exclusively for the adaptation of the small subunit, rather than the large subunit or the association step . The presence of mRNA {poly(U)} together with cognate AcPhe-tRNA, accelerates the association rate significantly, forming a well-defined 70S peak in sucrose gradient profiles . mRNA alone provokes an equivalent acceleration, however, the resulting 70S couple impresses as an ill-defined, broad peak, probably indicating the readiness of the ribosome for tRNA binding, upon which the ribosome flips into a defined state.

Vaccine, 2002 Jun 21, 20(21-22), 2764 - 71
Activation of antigen-presenting cells by immunostimulatory plant DNA: a natural resource for potential adjuvant; Wang Y et al.; Genomic DNA sequences (bacteria, insect, nematodes and molluscs) or synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-DNA/ODN) are regarded as promising candidates for new medical adjuvants for their ability to stimulate the mammalian immune system and enhance immune responses to specific antigens . Here, we first report the immunostimulatory activity of total genomic DNA from two plants, Brassica chinensis L . and Zea may, the CpG methylation status of which is incomplete compared with E . coli DNA . These plant DNA can activate B cells to proliferate . Plant DNA promotes secretion of IL-12, and increases expression of MHC and costimulatory molecules by bone marrow-derived dendritic cells (BMDC) . Plant DNA can also enhance antigen presentation capacity of BMDC and macrophages . When administrated in vivo, plant DNA can inhibit tumor growth in situ or metastasis in tumor-bearing mice . The immunostimulatory activity of plant DNA could be abolished by methylation . Our data showed that plant DNA can activate antigen-presenting cells (APC) including DC, macrophages and B cells, indicating that plant DNA is a new kind of potential adjuvant . Therefore, we conclude that plant DNA is another natural source of CpG-DNA, and that green plants may provide abundant resources for this potential medical adjuvant.

J Interferon Cytokine Res, 2002 Mar, 22(3), 321 - 8
Rat interleukin-18 binding protein: cloning, expression, and characterization; Im SH et al.; Interleukin-18 binding protein (IL-18BP) is a constitutively expressed and secreted protein that lacks a transmembrane domain . IL-18BP binds specifically to mature IL-18 and inhibits its activity . To study the immunomodulating role of IL-18BP in models of autoimmune diseases in rats, we cloned and characterized rat IL-18BP . Rat IL-18BP has 193 amino acid residues and is highly homologous to human and mouse IL-18BP . Recombinant rat IL-18BP binds to rat IL-18, reacts with antibodies to human or mouse IL-18BP, and inhibits IL-18-dependent interferon-gamma (IFN-gamma) production in vitro . Thus, rat IL-18BP can be employed to antagonize the proinflammatory responses induced by endogenous IL-18 in rat models of autoimmune diseases.

Biochem J, 2002 Sep 1, 366(Pt 2), 557 - 64
The AtNFS2 gene from Arabidopsis thaliana encodes a NifS-like plastidial cysteine desulphurase; Leon S et al.; NifS-like proteins are cysteine desulphurases required for the mobilization of sulphur from cysteine . They are present in all organisms, where they are involved in iron-sulphur (Fe-S) cluster biosynthesis . In eukaryotes, these enzymes are present in mitochondria, which are the major site for Fe-S cluster assembly . The genome of the model plant Arabidopsis thaliana contains two putative NifS-like proteins . A cDNA corresponding to one of them was cloned by reverse-transcription PCR, and named AtNFS2 . The corresponding transcript is expressed in many plant tissues . It encodes a protein highly related (75% similarity) to the slr0077-gene product from Synechocystis PCC 6803, and is predicted to be targeted to plastids . Indeed, a chimaeric AtNFS2-GFP fusion protein, containing one-third of AtNFS2 from its N-terminal end, was addressed to chloroplasts . Overproduction in Escherichia coli and purification of recombinant AtNFS2 protein enabled one to demonstrate that it bears a pyridoxal 5'-phosphate-dependent cysteine desulphurase activity in vitro, thus being the first NifS homologue characterized to date in plants . The putative physiological functions of this gene are discussed, including the attractive hypothesis of a possible role in Fe-S cluster assembly in plastids.

Biochemistry, 2002 Jun 4, 41(22), 7116 - 24
Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic; Jiang YL et al.; We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate {Werner, R . M., and Stivers, J . T . (2000) Biochemistry 39, 14054-14064} . Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM) . The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds >30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA) . The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)) . Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)) . These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate . This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.

Biochemistry, 2002 Jun 4, 41(22), 7074 - 81
Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table; Sera T et al.; We have developed a novel and simple method to rationally design artificial zinc-finger proteins (AZPs) targeting diverse DNA sequences using a nondegenerate recognition code table . The table was constructed based on known and potential DNA base-amino acid interactions . The table permits identification of an amino acid for each position (-1, 2, 3, and 6) of the alpha-helical region of the zinc-finger domain (position 1 is the starting amino acid in the alpha-helix) from overlapping 4-bp sequences in a given DNA target . Based on the table, we designed ten 3-finger AZPs, each of which targeted an arbitrarily chosen 10-bp DNA sequence, and characterized the binding properties . In vitro DNA-binding assays showed five of the AZPs tightly and specifically bound to their targets containing more than three guanine bases in the first 9-bp region . In addition, 6-finger AZPs, each of which was produced by combining two functional 3-finger AZPs, bound to their 19-bp targets with the dissociation constant of less than 3 pM . The in vivo functionality of the AZP was tested using Arabidopsis protoplasts . The AZP fused to a transcriptional activation domain efficiently activated expression of a reporter gene linked to a native promoter containing the AZP target site . Our simple AZP design method will provide a powerful approach to manipulation of endogenous gene expression by enabling rapid creation of numerous artificial DNA-binding proteins.

Biochemistry, 2002 Jun 4, 41(22), 7021 - 9
Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access; Fritz TA et al.; Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction . These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate . Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate {Barrett, J . E., Lucero, C . M., and Schultz, P . G . (1999) J . Am . Chem . Soc . 121, 7965-7966.} formed after THF changes its binding from the cofactor pocket to a putative alternate site . To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF . The mutant enzyme has a cavity in the active site continuous with bulk solvent . This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene . The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene . Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

Biochemistry, 2002 Jun 4, 41(22), 6920 - 7
Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD; Goulding CW et al.; Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds . Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond . DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA . The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)) . The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C) . Isolated DsbD(N) is functional in electron transport in vitro . Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands . The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70) . A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.

Biochemistry, 2002 Jun 4, 41(22), 6902 - 10
Truncated hemoglobin from the cyanobacterium Synechococcus sp . PCC 7002: evidence for hexacoordination and covalent adduct formation in the ferric recombinant protein; Scott NL et al.; The glbN gene for the hemoglobin of Synechoccocus sp . PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli . The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein . Mass spectrometric analysis confirmed the identity of the polypeptide . NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp . PCC 6803 {Scott, N . L., and Lecomte, J . T . J . (2000) Protein Sci . 9, 587-597} . The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side . Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix . In this protein, as in that from Synechocystis sp . PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein . Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

J Vet Diagn Invest, 2002 May, 14(3), 260 - 2
Endocarditis associated with Escherichia coli in a sea lion (Zalophus californianus); Kim JH et al.; Endocarditis associated with Escherichia coli was diagnosed in a 2-year-old male California sea lion (Zalophus californianus) . The diagnosis was based on light microscopic examination and bacterial isolation from the valvular lesion . This is the first case of bacterial endocarditis reported in a sea lion.

J Immunoassay Immunochem, 2002, 23(2), 129 - 43
Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA; Rau D et al.; A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli . The expression vector pASK75 was modified by sequentially inserting the E . coli aP coding region and the scFv cloning cassette . Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments . These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector . An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA . Therefore, this system permits the production of scFv-aP conjugates in E . coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays . These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

Anal Chem, 2002 Apr 1, 74(7), 1680 - 6
Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures; Engen JR et al.; The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described . Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein . The isotope pattern was used as a signature for peptides originating from the SIL protein . In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information . Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein . Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate . This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.

Gene Ther, 2002 Jun, 9(11), 686 - 90
Modification of hepatic genomic DNA using RNA/DNA oligonucleotides; Kren BT et al.; The ideal gene therapy is one that repairs the precise genetic defect without additional modification of the genome . Such a strategy has been developed for correcting single nucleotide mutations by using RNA/DNA oligonucleotides, or chimeraplasts . This approach for in situ repair is based on the delivery of exogenous DNA designed to mediate genomic base conversion, insertion, or deletion, thereby, correcting the genetic mutation . Using in vivo delivery systems to hepatocytes via the asialoglycoprotein receptor, we targeted rat liver DNA and successfully modified the genomic sequence by chimeraplasty . The changes in both the hepatic genes, and their associated phenotypes remained stable for 2 years . In addition, we also examined the potential to alter sequence defects in mitochondrial DNA . Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeraplast-mediated DNA changes . Using an in vitro DNA repair assay of mutagenized plasmids and an Escherichia coli readout system, we showed that extracts from highly purified rat liver mitochondria have the essential enzymatic activity to mediate precise single-nucleotide changes at a frequency similar to liver nuclear extracts . Moreover, single-stranded oligonucleotides carrying a single nucleotide mismatch with the target sequence were capable of promoting gene conversion using either mitochondrial or nuclear extracts . Several approaches now exist for the precise repair of genetic mutations using either single-stranded or RNA/DNA chimeric oligonucleotides.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7496 - 501
ERK2 enters the nucleus by a carrier-independent mechanism; Whitehurst AW et al.; In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus . Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood . To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay . GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner . Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes . The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins . The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors . These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors . In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin . Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7450 - 4
Identification of residues in TIP47 essential for Rab9 binding; Hanna J et al.; TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-dependent and cation-independent mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells . TIP47 recognizes distinct determinants in the cytoplasmic domains of these two receptors, and its ability to bind to the cation-independent MPR is enhanced by the concomitant binding of the Rab9 GTPase . We show here that TIP47 residues 161-169 are essential, but likely not sufficient, for Rab9 binding . Mutation of these residues led to a significant decrease in Rab9 binding, but did not alter the global folding of the protein . The most impaired mutant was indistinguishable from wild-type TIP47 in its circular dichroism spectrum, and mutant proteins that showed decreased Rab9 binding retained full capacity to bind to MPR cytoplasmic domains . Closely related sequences in a related protein, adipophilin, did not confer Rab9 binding capacity to that protein . Partial proteolysis of TIP47 and TIP47 mutant proteins revealed subtle conformational differences, suggesting that residues 161-169 reside in a portion of TIP47 that is important for its conformation . These experiments reveal distinct binding domains for the Rab9 GTPase and MPR cytoplasmic domains in the cargo selection protein TIP47.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7402 - 7
Amyloid aggregates of the HET-s prion protein are infectious; Maddelein ML et al.; The {Het-s} infectious element of the filamentous fungus Podospora anserina is a prion . We have recently reported that recombinant HET-s protein aggregates in vitro into amyloid fibers . In vivo, the protein aggregates specifically in the {Het-s} prion strains . Here, we show that biolistic introduction of aggregated recombinant HET-s protein into fungal cells induces emergence of the {Het-s} prion with a high frequency . Thus, we demonstrate that prion infectivity can be created de novo, in vitro from recombinant protein in this system . Although the amyloid filaments formed from HET-s could transmit {Het-s} efficiently, neither the soluble form of the protein nor amorphous aggregates would do so . In addition, we have found that (i) {Het-s} infectivity correlates with the ability to convert HET-s to amyloids in vitro, (ii) {Het-s} infectivity is resistant to proteinase K digestion, and (iii) HET-s aggregates formed in vivo in {Het-s} strains have the ability to convert the recombinant protein to aggregates . Together, our data designate the HET-s amyloids as the molecular basis of {Het-s} prion propagation.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7390 - 5
Elucidation of the function of lipoprotein-sorting signals that determine membrane localization; Masuda K et al.; Escherichia coli lipoproteins are anchored to the inner or outer membrane depending on the residue at position 2 . Aspartate at this position makes lipoproteins specific to the inner membrane, whereas other residues cause the release of lipoproteins from the inner membrane in a manner dependent on both ATP binding cassette (ABC) transporter LolCDE and molecular chaperone LolA, followed by LolB-dependent localization in the outer membrane . The function of lipoprotein-sorting signals was examined in proteoliposomes reconstituted from LolCDE and lipoproteins . The release of outer membrane-specific lipoproteins was inhibited on reconstitution with other outer membrane-specific, but not inner membrane-specific, lipoproteins . Outer membrane-specific lipoproteins stimulated ATP hydrolysis by LolCDE whereas inner membrane-specific ones did not . LolA was not required for the stimulation of ATP hydrolysis . These results revealed a previously undocumented function of aspartate at position 2, i.e., lipoproteins having this signal avoid being recognized by LolCDE, thereby remaining in the inner membrane.

Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7373 - 7
Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity; Morgan-Kiss RM et al.; Expression systems based on the Escherichia coli arabinose operon P(BAD) promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the lac operon . Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced . Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations {Khlebnikov, A., Risa, O., Skaug, T., Carrier, T . A . & Keasling, J . D . (2000) J . Bacteriol . 182, 7029-7034} . However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in E . coli . We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (araE araFGH) and degradation (araBAD) . This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer . This approach is readily adapted to other sugar-regulated expression systems.

J Biol Chem, 2002 Aug 9, 277(32), 28512 - 20 Epub 2002 May 24.
Distinct roles of the N-terminal-binding domain and the C-terminal-solubilizing domain of alpha-synuclein, a molecular chaperone; Park SM et al.; alpha-Synuclein, an acidic neuronal protein of 140 amino acids, is extremely heat-resistant and is natively unfolded . Recent studies have demonstrated that alpha-synuclein has chaperone activity both in vitro and in vivo, and that this activity is lost upon removing its C-terminal acidic tail . However, the detailed mechanism of the chaperone action of alpha-synuclein remains unknown . In this study, we investigated the molecular mechanism of the chaperone action of alpha-synuclein by analyzing the roles of its N-terminal and C-terminal domains . The N-terminal domain (residues 1-95) was found to bind to substrate proteins to form high molecular weight complexes, whereas the C-terminal acidic tail (residues 96-140) appears to be primarily involved in solubilizing the high molecular weight complexes . Because the substrate-binding domain and the solubilizing domain for chaperone function are well separated in alpha-synuclein, the N-terminal-binding domain can be substituted by other proteins or peptides . Interestingly, the resultant engineered chaperone proteins appeared to display differential efficiency and specificity in terms of the chaperone function, which depended upon the nature of the binding domain . This finding implies that the C-terminal acidic tail of alpha-synuclein can be fused with other proteins or peptides to engineer synthetic chaperones for specific purposes.

EMBO J, 2002 Jun 3, 21(11), 2778 - 87
Structural origins of amino acid selection without editing by cysteinyl-tRNA synthetase; Newberry KJ et al.; Cysteinyl-tRNA synthetase (CysRS) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other tRNA synthetases . To elucidate how CysRS is able to distinguish cysteine from non-cognate amino acids, crystal structures of the Escherichia coli enzyme were determined in apo and cysteine-bound states . The structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the base of the active site cleft, in a trigonal bipyramidal geometry together with four highly conserved protein side chains . Cysteine binding induces movement of the zinc ion towards substrate, as well as flipping of the conserved Trp205 indole ring to pack on the thiol side chain . The imidazole groups of five conserved histidines lie adjacent to the zinc ion, forming a unique arrangement suggestive of functional significance . Thus, amino acid discrimination without editing arises most directly from the favorable zinc-thiolate interaction, which is not possible for non-cognate substrates . Additional selectivity may be generated during the induced-fit conformational changes that help assemble the active site.

EMBO J, 2002 Jun 3, 21(11), 2509 - 16
Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature; Shin S et al.; A large group of hydrolytic enzymes, which contain a conserved stretch of approximately 130 amino acids designated the amidase signature (AS) sequence, constitutes a super family that is distinct from any other known hydrolase family . AS family enzymes are widespread in nature, ranging from bacteria to humans, and exhibit a variety of biological functions . Here we report the first structure of an AS family enzyme provided by the crystal structure of malonamidase E2 from Bradyrhizobium japonicum . The structure, representing a new protein fold, reveals a previously unidentified Ser-cisSer-Lys catalytic machinery that is absolutely conserved throughout the family . This family of enzymes appears to be evolutionarily distinct but has diverged to acquire a wide spectrum of individual substrate specificities, while maintaining a core structure that supports the catalytic function of the unique triad . Based of the structures of the enzyme in two different inhibited states, an unusual action mechanism of the triad is proposed that accounts for the role of the cis conformation in the triad.

Thromb Res, 2002 Feb 15, 105(4), 331 - 7
Expression and characterization of the ScFv fragment of antiplatelet GPIIIa monoclonal antibody SZ-21; An G et al.; Platelet thrombus formation is a major contributor to various cardiovascular diseases caused by vascular occlusion . Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in platelet aggregation and hence the formation of thrombi . In the present study, the genes encoding the light- and heavy-chain variable regions (V(H) and V(L)) of SZ-21, which is a monoclonal antibody (MoAb) against GPIIIa integrin have been cloned by RT-PCR from the SZ-21 hybridoma . The genes of V(H) and V(L) were attached to the oligonucleotide of the linker peptide and single-chain antibody fragment (ScFv) was constructed . The ScFv was ligated into phage display vector pHEN1, and the phagemid pHEN1-21ScFv was constructed . The high-affinity phage display technology was used to retain the SZ-21ScFv binding activity to platelets in great effort . After four rounds of enrichment, the screening clone of SZ-21ScFv gene with good reactivity to platelets was ligated into highly expressed vector pET20b and expressed in Escherichia coli BL21(DE3)PlysS for the fusion protein . Recombinant ScFv fragment was produced mostly in the form of inclusion bodies, with its yield up to 21% of the total amount of bacteria protein . The ScFv fragment with the similar binding activity to platelets as MoAb SZ-21 was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot . ADP-induced platelet aggregation can be inhibited by ScFv fragment in a dose-dependent manner and the maximal inhibition rate was obtained at a concentration of 750 nM . In addition, the ScFv fragment has ability to inhibit the binding of fibrinogen to platelets and react with endothelial cells . In this study, we have successfully produced the SZ-21ScFv, which retained the binding affinity to platelets and antithrombotic ability of their murine counterparts.

J Inorg Biochem, 2002 Jun 7, 90(3-4), 155 - 8
Differential genotoxic effects of antitumor trans-{PtCl(2)(E-iminoether)(2)} and cisplatin in Escherichia coli; Janovska E et al.; In the present work, we measured survival and the platinum on the genome after treatment of repair-proficient or repair-deficient Escherichia coli strains with trans-{PtCl(2)(E-iminoether)(2)} and compared these results with the effects of "classical" cisplatin . We found that toxicity of antitumor trans-{PtCl(2)(E-iminoether)(2)} in repair-deficient trains was much less than that of cisplatin . This markedly reduced toxicity was not a consequence of the reduced uptake or low levels of DNA binding in the bacteria cells but rather appeared to reflect DNA binding mode of this trans-platinum drug different from that of cisplatin.

Biochim Biophys Acta, 2002 May 8, 1589(3), 247 - 60
Myotrophin-kappaB DNA interaction in the initiation process of cardiac hypertrophy; Gupta S et al.; To investigate how cardiac hypertrophy and heart failure develop, we isolated and characterized a candidate initiator, the soluble 12-kDa protein myotrophin, from rat and human hearts . Myotrophin stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes . Recombinant myotrophin from the cloned gene showed structural/functional motifs, including ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and casein kinase II . One repeat, homologous with I kappaB, interacts with rel/NF-kappaB in vitro . We analyzed the interaction of recombinant myotrophin and nuclear extracts prepared from neonatal and adult cardiomyocytes; gel mobility shift assay showed that myotrophin bound to kappaB DNA . To define PKC's role in myotrophin-induced myocyte growth, we incubated neonatal rat myocytes (normal and stretch) with specific inhibitors and found that myotrophin inhibits {3H}leucine incorporation into myocytes and different hypertrophic gene expression in neonatal myocytes . Using confocal microscopy, we observed that a basal level of myotrophin was present in both cytoplasm and nucleus under normal conditions, but under cyclic stretch, myotrophin levels became elevated in the nucleus . Myotrophin gene levels were upregulated when myocytes underwent cyclic stretch or were treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta and also when excised beating hearts were exposed to high pressure . Our data showed that the myotrophin-kappaB interaction was increased with age in spontaneously hypertensive rats (SHRs) only . Our data provide evidence that myotrophin-kappaB DNA interaction may be an important step in initiating cardiac hypertrophy.

Chem Biol, 2002 May, 9(5), 575 - 83
The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase; Beck BJ et al.; The pikromycin (Pik) polyketide synthase (PKS) from Streptomyces venezuelae comprises four multifunctional polypeptides (PikAI, PikAII, PikAIII, and PikAIV) . This PKS can generate 12- and 14-membered ring macrolactones (10-deoxymethynolide and narbonolide, respectively) through the activity of its terminal modules (PikAIII and PikAIV) . We performed a series of experiments involving the functional replacement of PikAIV in mutant strains with homodimeric and heterodimeric PikAIV modules to investigate the details of macrolactone ring size determination . The results suggest a new and surprising mechanism by which the penultimate hexaketide chain elongation intermediate is transferred from PikAIII ACP5 to PikAIV ACP6 before release by the terminal thioesterase domain . Elucidation of this chain transfer mechanism provides important new details about alternative macrolactone ring size formation in modular PKSs and contributes to the potential for rational design of structural diversity by combinatorial biosynthesis.

Brain Res, 2002 Jun 21, 941(1-2), 1 - 10
Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2; Kim EJ et al.; In neurodegenerative disease, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been regarded as beneficial . The NSAID, an inhibitor of cyclooxygenase (COX), has been also suggested as a ligand of the peroxisome proliferator-activated receptor (PPAR) . In cortical neuron-glial co-cultures, we examined the effect of PPAR agonists on lipopolysaccharide(LPS)-induced neuronal death, which has been known to be NO-dependent . LPS induced iNOS expression and the release of nitric oxide in microglia, and COX-2 expression in neurons . PPAR-gamma agonists such as 15d-PGJ(2), ciglitazone and troglitazone prevented LPS-induced neuronal death and abolished LPS-induced NO and PGE(2) release, however PPAR-alpha agonists such as clofibrate and WY14,643 did not produce the same results . PPAR-gamma agonists also reduced LPS-induced iNOS and COX-2 expression, which suggested by interfering with the NF-kappaB signal pathway.

Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 110 - 8
DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751; Kostelidou K et al.; The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well . Both KorA(RK2) (IncP-1 alpha) and KorA(R751) (IncP-1 beta) recognise the 5'-GTTTAGCTAAAC-3' palindrome . Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter . Class I (high affinity) operators for KorA(RK2) are flanked by an A-A-A/T sequence in the upstream half; the T base was shown to greatly influence strong repression . A C-A-G triplet was present in the same region in the R751 O(A) sequences and the G base was accordingly found to be important for strong KorA(R751) repression . An obvious difference between the two KorA proteins is a histidine to serine change at the C-proximal end of the putative recognition helix of the HTH motif (aa 56) . An IncP-1 alpha KorAH56S mutant protein had higher affinity for all operators but had improved more on R751 operators than on RK2 operators . This indicates that KorA of RK2 is not maximised for DNA binding activity and that the aa difference at position 56 may play a role in differentiation between alpha and beta KorA operators.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Jun, 132(2), 423 - 31
Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions; Tafalla C et al.; In the present study, the ability of mussel (Mytilus galloprovincialis) hemocytes to produce nitric oxide (NO) in response to phorbol myristate acetate (PMA) was determined using the Griess reaction . Significant NO production was found in these cells in response to PMA . This stimulation was reversed in the presence of the NO synthase inhibitor, N(G)-methyl-L-arginine (L-NMMA) . Moreover, the effect of the pre-incubation of hemocytes with NO was also determined on phagocytic immune functions of mussel hemocytes using two NO donors, glycerin trinitrate (GTN) and S-nitroso-N-acetyl-penicillamine (SNAP) . In the case of GTN, a visible cytotoxic effect of the compound at the higher doses was observed . Those GTN concentrations that did not have a negative effect on hemocyte viability did not produce sufficient NO to significantly alter the chemiluminescent response to zymosan in all cases, nor the ability of hemocytes to phagocytose bacteria (Escherichia coli) . SNAP, however, did not affect cell viability at either of the concentrations used and produced NO levels up to 13-fold higher than controls after 2 h of incubation . In this case, NO exogenously produced by SNAP significantly inhibited the chemiluminescent response of mussel hemocytes, whereas it did not have a significant effect on the capability of these cells to phagocytose bacteria.

Phytochemistry, 2002 Jun, 60(3), 255 - 61
Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.); Bennett MH et al.; Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A . A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins . Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs . RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons . Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana . Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family . The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.

Hybrid Hybridomics, 2002 Apr, 21(2), 117 - 22
Plasmid immunization primes unique DTH responses to HIV-1MN envelope epitopes as compared to recombinant protein vaccination; Chattergoon MA et al.; Current evidence suggests that the induction of cell-mediated immunity is required for a successful HIV-1 vaccine . Delayed type hypersensitivity (DTH) and cellular cytotoxicity are closely linked elements of the cellular immune response, both are favored by immunizations that result in a T-helper (Th)-1 response . The classical experimental animal for the study of DTH is the guinea pig . Here we report that guinea pigs can readily be sensitized for DTH skin reactions to envelope protein with a plasmid expressing HIV-1(MN) (subtype B) envelope, as well as with the recombinant HIV-1 envelope protein . Further, utilizing peptide probes that in aggregate represent the entire gp120 molecule, common and unique dominant epitopes induced by each method of immunization were identified.

Oral Microbiol Immunol, 2002 Jun, 17(3), 150 - 6
Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28; Slakeski N et al.; We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein . The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases . A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P . gingivalis . Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli . Western blot analyses of purified rOmp28 with rabbit antisera to a P . gingivalis outer membrane preparation, protective rat anti-whole P . gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera . Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P . gingivalis from the United States, Sudan, Romania and Norway . These results suggest that Omp28 is expressed by a wide distribution of P . gingivalis strains.

Anim Genet, 2002 Jun, 33(3), 220 - 3
Characterization of the porcine FABGL gene; Jacobs K et al.; The porcine major histocompatibility complex, also called swine lymphocyte antigen (SLA) complex, is of particular interest not only because of its central role in the immune response, but also because of its influence on many traits such as reproduction, fatness and meat quality . The porcine FABGL (FabG (beta-ketoacyl-{acyl-carrierprotein} reductase, Escherichia coli) like) gene, coding for a 17beta-hydroxysteroid dehydrogenase (17beta-HSD), is a candidate gene for these traits . The complete gene was sequenced and compared with human and mouse FABGL sequences . The deduced amino acid sequence showed 85 and 83% sequence identity to human and mouse sequences, respectively . Polymorphicic BbvI and DdeI restriction sites were found in the porcine FABGL gene . The promoter was compared with the promoter regions of human and mouse FABGL sequence in order to identify putative regulatory elements . The transcription profile of the porcine gene was determined and showed a widespread tissue distribution.

Environ Microbiol, 2002 May, 4(5), 306 - 13
The influence of host dynamics on the clonal composition of Escherichia coli populations; Davis SA et al.; Species, be they plant or animal, vary in their capacity for population growth or decline . Populations of the same species may also differ in their capacity for population change . A series of mathematical models were developed with the aim of determining if host population dynamics could influence the clonal composition of the Escherichia coli community in that host population . The biological assumptions underlying the models are described in some detail . Analytical and numerical approaches were used to investigate the behaviour of these models . The results demonstrate that host dynamics can have a profound influence on the E . coli clonal composition of the host population . This outcome is largely independent of the nature of the assumptions underlying the models . The ways in which the predictions of these models may be tested empirically are discussed, as are the implications of these models for understanding the nature of host-bacterial pathogen dynamics.

J Mol Evol, 2002 Jun, 54(6), 825 - 40
Molecular evolution within the L-malate and L-lactate dehydrogenase super-family; Madern D; The NAD(P)-dependent malate (L-MalDH) and NAD-dependent lactate (L-LDH) form a large super-family that has been characterized in organisms belonging to the three domains of life . In the first part of this study, the group of {LDH-like} L-MalDH, which are malate dehydrogenases resembling lactate dehydrogenase, were analyzed and clearly defined with respect to the other enzymes . In the second part, the phylogenetic relationships of the whole super-family were presented by taking into account the {LDH-like} L-MalDH . The inferred tree unambiguously shows that two ancestral genes duplications, and not one as generally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within the super-family: L-LDH, {LDH-like} L-MalDH, and dimeric L-MalDH . In addition, various cases of functional changes within each group were observed and analyzed . The direction of evolution was found to always be polarized: from enzymes with a high stringency of substrate recognition to enzymes with a broad substrate specificity . A specific phyletic distribution of the L-LDH, {LDH-like} L-MalDH, and dimeric L-MalDH over the Archaeal, Bacterial, and Eukaryal domains was observed . This was analyzed in the light of biochemical, structural, and genomic data available for the L-LDH, {LDH-like} L-MalDH, and dimeric L-MalDH . This analysis led to the elaboration of a refined evolutionary scenario of the super-family, in which the selection of L-LDH and the fate of L-MalDH during mitochrondrial genesis are presented.

Eur Biophys J, 2002 Jun, 31(3), 217 - 27 Epub 2002 Feb 19.
Setting up and optimization of membrane protein simulations; Faraldo-Gomez JD et al.; In this paper we describe a method for setting up an atomistic simulation of a membrane protein in a hydrated lipid bilayer and report the effect of differing electrostatic parameters on the drift in the protein structure during the subsequent simulation . The method aims to generate a suitable cavity in the interior of a lipid bilayer, using the solvent-accessible surface of the protein as a template, during the course of a short steered molecular dynamics simulation of a solvated lipid membrane . This is achieved by a two-stage process: firstly, lipid molecules whose headgroups are inside a cylindrical volume equivalent to that defined by the protein surface are removed; then the protein-lipid interface is optimized by applying repulsive forces perpendicular to the protein surface, and of gradually increased magnitude, to the remaining lipid atoms inside the volume occupied by the protein surface until it is emptied . The protein itself may then be inserted . Using the bacterial membrane proteins KcsA and FhuA as test cases, we show how the method achieves the formation of a suitable cavity in the interior of a dimyristoylphosphatidylcholine lipid bilayer without perturbing the configuration of the non-interfacial regions of the previously equilibrated lipid bilayer, even in cases of membrane proteins with irregular geometrical shapes . In addition, we compare subsequent simulations in which the long-range electrostatic interactions are treated via either a cut-off or a particle-mesh Ewald method . The results show that the drift from the initial structure is less in the latter case, especially for KcsA and for the non-core secondary structural elements (i.e . surface loops) of both proteins.

Eur Biophys J, 2002 Jun, 31(3), 172 - 8 Epub 2002 Jan 29.
Sampling the conformational space of membrane protein surfaces with the AFM; Scheuring S et al.; The atomic force microscope acquires topographs of single native membrane proteins at subnanometer resolution . Owing to the high signal-to-noise ratio, such images allow the conformational space of membrane protein surfaces to be sampled . This is demonstrated by topographs of porin OmpF, aquaporin-Z, and bacteriorhodopsin, all recorded at a lateral resolution of <7 A and a vertical resolution of ~1 A . The amplitudes of the domain movements were estimated from a large number of single molecule topographs and the corresponding energy landscapes calculated . To visualize the motion of protein domains, movies were generated by similarity ranking of the observed protein configurations . Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-001-0197-8

J Gen Virol, 2002 Jun, 83(Pt 6), 1483 - 91
Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9; Qanungo KR et al.; Genome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced . In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa . Secondary structure prediction showed the presence of nine alpha-helices in the central and terminal domains with localized similarity to RNA-binding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases . Nucleotide sequences were 99.6% identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases . The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E . coli and polyclonal antibody was raised against the purified protein . Immunoblot as well as immunofluorescence analysis with anti-NSP38 antibody showed that the protein was not present in polyhedra or uninfected cells but was present in AmCPV-infected host midgut cells . NSP38 was expressed in insect cells as soluble protein via a baculovirus expression vector and shown to possess the ability to bind poly(rI)-(rC) agarose, which was competitively removed by AmCPV viral RNA . These results indicate that NSP38 is expressed in virus-infected cells as a non-structural protein . By binding to viral RNA, it may play a role in the regulation of genomic RNA function and packaging.

Science, 2002 May 24, 296(5572), 1466 - 70
Combinatorial synthesis of genetic networks; Guet CC et al.; A central problem in biology is determining how genes interact as parts of functional networks . Creation and analysis of synthetic networks, composed of well-characterized genetic elements, provide a framework for theoretical modeling . Here, with the use of a combinatorial method, a library of networks with varying connectivity was generated in Escherichia coli . These networks were composed of genes encoding the transcriptional regulators LacI, TetR, and lambda CI, as well as the corresponding promoters . They displayed phenotypic behaviors resembling binary logical circuits, with two chemical "inputs" and a fluorescent protein "output." Within this simple system, diverse computational functions arose through changes in network connectivity . Combinatorial synthesis provides an alternative approach for studying biological networks, as well as an efficient method for producing diverse phenotypes in vivo.

J Biol Chem, 2002 Aug 2, 277(31), 28238 - 46 Epub 2002 May 23.
Identification of novel SH3 domain ligands for the Src family kinase Hck . Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1; Scott MP et al.; The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established . However, few in vivo ligands are known for the SH3 domain of Hck . In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937 . Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP) . We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12 . In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration . Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck . We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck . The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck . Our results suggest that these proteins may be novel activators/effectors of Hck.

J Bacteriol, 2002 Jun, 184(12), 3401 - 5
Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus; Verhees CH et al.; The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized . The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg . The P . furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM) . Based on the presence of conserved sequence motifs and the substrate specificity of the P . furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

J Bacteriol, 2002 Jun, 184(12), 3338 - 47
Dam- and OxyR-dependent phase variation of agn43: essential elements and evidence for a new role of DNA methylation; Wallecha A et al.; Phase variation of the outer membrane protein Ag43 in E . coli requires deoxyadenosine methylase (Dam) and OxyR . Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding . Here we report further characterization of agn43 transcription and its regulation . Transcription was initiated from a sigma(70)-dependent promoter at the G residue of the upstream GATC sequence . Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription . Analyses of transcription in vivo of agn'-lacZ with mutated Dam target sequences support this conclusion . Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription . In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR . Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from -16 to +42, which includes the three GATC sequences . Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation

J Bacteriol, 2002 Jun, 184(12), 3260 - 7
In vivo analysis of an essential archaeal signal recognition particle in its native host; Rose RW et al.; The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores . However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons . In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host . As part of this analysis, an hv54h knockout strain was created . In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea . Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H . volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h . These results provide the first in vivo evidence that these components interact in archaea . Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

J Bacteriol, 2002 Jun, 184(12), 3253 - 9
The molybdate-responsive Escherichia coli ModE transcriptional regulator coordinates periplasmic nitrate reductase (napFDAGHBC) operon expression with nitrate and molybdate availability; McNicholas PM et al.; Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium . These changes are mediated by the transcription factors Fnr and NarP, respectively . Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor . This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis . Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent K(d) of about 35 nM at a position centered at -133.5 relative to the start of napF transcription . Although the ModE binding site sequence is similar to other E . coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription . Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo . In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE . These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins . The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.

J Endotoxin Res, 2002, 8(2), 127 - 33
Cellular mechanism underlying LPS-induced inhibition of in vitro L-leucine transport across rabbit jejunum; Abad B et al.; Lipopolysaccharide (LPS) is a known causative agent of sepsis . In previous studies, we have shown that it reduces L-leucine mediated transport across the rabbit jejunum by about 30% . In this study, the mechanism(s) of LPS inhibition on amino acid transport were analysed in detail . LPS did not inhibit L-leucine transport across brush border membrane vesicles, suggesting the need for an intracellular step . The inhibitory effect of LPS was not altered by the addition of protein kinase A (PKA) inhibitor (IP(20), 10(-7) M) or an analog of cAMP (DB-cAMP, 3 x 10(-4) M), indicating that the PKA signal transduction pathway was not involved in the LPS effect . However, the inhibitory effect of LPS was suppressed by trifluoroperazine (10(-7) M), a Ca(2+)/calmodulin inhibitor and staurosporine (10(-7) M), an protein kinase C (PKC) inhibitor . Likewise, LPS inhibition disappeared in media without calcium . These results suggest that LPS could inhibit the intestinal uptake of L-leucine across the small intestine in vitro by intracellular processes related to calcium, involving PKC and calmodulin protein.

J Endotoxin Res, 2002, 8(2), 99 - 107
Protective effects of isohelenin, an inhibitor of nuclear factor kappaB, in endotoxic shock in rats; Sheehan M et al.; Recent in vitro studies have shown that isohelenin, a sesquiterpene lactone, inhibits the NF-kappaB pathway . This study examines the effect of isohelenin in endotoxic shock induced by administration of Escherichia coli endotoxin in male Wistar rats . A group of rats received isohelenin (2 mg/kg intraperitoneally) 15 min before endotoxin . In vehicle-treated rats, administration of endotoxin caused severe hypotension, which was associated with a marked hyporeactivity to norepinephrine and acetylcholine in ex vivo aortas . Elevated levels of plasma nitrate/nitrite, metabolites of nitric oxide (NO), were also found . These inflammatory events were preceded by cytosolic degradation of inhibitor-kappaBalpha (IkappaBalpha) and activation of nuclear factor-kappaB (NF-kappaB) in the lung within 15 min of endotoxin administration . Treatment with isohelenin resulted in hemodynamic improvement and reduced plasma levels of NO metabolites . Nuclear translocation of NF-kappaB was inhibited by isohelenin treatment in the lung, whereas degradation of IkappaBalpha was unchanged . In a separate set of experiments, treatment with isohelenin significantly improved survival in mice challenged with endotoxin . We conclude that isohelenin exerts beneficial therapeutic effects during endotoxic shock through inhibition of NF-kappaB.

J Oral Rehabil, 2002 May, 29(5), 401 - 7
Human peripheral blood monocytes versus THP-1 monocytes for in vitro biocompatibility testing of dental material components; Heil TL et al.; Monocytes play a central role in the response of tissues to biomaterials . Monocytic cell lines such as the THP-1 cell line have been used extensively as models for primary monocytes (directly from blood) in biocompatibility research . However, little information exists about the appropriateness of these cell lines as models . Thus, the current study compared the biological response of both primary peripheral blood monocytes (PBMs) and the THP-1 cell line to four common components of dental materials known to be released into the oral environment: nickel ions, 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), and 2,2-bis{4(2-hydroxy-3-methacryloloxy)-phenyl}propane (Bis-GMA) . Comparisons were made by constructing dose-response curves for each type of monocyte and the four components . The 50% cytotoxicity values (TC50 values) were then statistically compared . In addition, the response of the monocytes to the materials with and without lipopolysaccharide (LPS) stimulation were assessed by measuring TNF-alpha secretion from the monocytes . The results showed that the PBMs were 5-10 times less sensitive than the THP-1 monocytes to these dental components, but that both cell lines ranked the components identically . TNF-alpha secretion from both types of monocytes often showed similar trends, although some inconsistent results were noted . The current study supports the use of THP-1s as a model for ranking the cytotoxicity of components of dental biomaterials . Furthermore, the secretory activity of PBMs appears to be generally well represented by the THP-1s . However, sufficient differences between these cell types exist to recommend confirmation of any critical results obtained with THP-1s using PBMs.

Lett Appl Microbiol, 2002, 34(6), 417 - 21
Effect of diethylsulphoxide on growth, survival and ion exchange of Escherichia coli; Markarian SA et al.; AIMS: To study the effect of diethylsulphoxide (DESO) on Escherichia coli growth, survival and ionic exchange in comparison with dimethylsulphoxide (DMSO) . METHODS AND RESULTS: Bacterial survival was estimated by counting colony-forming units and by the most probable number (five-tube) technique; the K+ and H+ transport and H(2) formation were determined electrochemically . Diethylsulphoxide at concentrations between 0.01 and 0.5% (w/v) stimulated and above 5% decreased the anaerobic growth rate and survival . 2H+ : K+ exchange and H(2) formation were lost at 5% DESO . At 0.05% DESO the kinetic characteristics of H+ : K+ exchange and H(2) formation were typical for Delta micro (H(+)) -dependent TrkA uncoupled with F(0)F(1) under respiration . CONCLUSIONS: Diethylsulphoxide at low concentrations serves as an electron acceptor for an anaerobic respiratory chain stimulating bacterial growth and survival through the modulation of H+ : K+ exchange and H(2) formation activity . The effects of DESO were more pronounced than those of DMSO . SIGNIFICANCE AND IMPACT OF THE STUDY: Diethylsulphoxide determines essential biological and therapeutic properties that make its application preferable.

Mol Microbiol, 2002 Jun, 44(5), 1331 - 9
Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp; Grompone G et al.; Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction . RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability . In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively . In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit . DSB formation was independent of RecA, RecQ and the helicase function of PriA . In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability . In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable . We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.

Eur J Biochem, 2002 May, 269(10), 2630 - 7
Caged O(2) . Reaction of cytochrome bo(3) oxidase with photochemically released dioxygen from a cobalt peroxo complex; Ludovici C et al.; We developed the synthesis of the caged oxygen donor (micro-peroxo)(micro-hydroxo)bis{bis(bipyridyl)cobalt(III)} complex (HPBC) as nitrate salt, which has, compared with the perchlorate-form described previously {MacArthur, R., Sucheta, A., Chong, F.F . & Einarsdottir, O . (1995) Proc . Natl Acad . Sci . USA, 92, 8105-8109}, greatly enhanced solubility . Now, the quantum efficiency of the photolytical release of dioxygen was determined to be 0.4 per photon at a laser wavelength of 308 nm, which was used to observe biological reactions . The X-ray structure of HPBC has been solved, and the molecular interactions of photochemically generated oxygen with cytochrome oxidase were investigated with optical and FT-IR spectroscopy: it acts as acceptor of electrons transferred from prereduced cytochrome bo(3), the heme-copper oxidase from Escherichia coli . FT-IR spectra revealed typical absorbance difference changes in the carbonyl region of cytochrome bo(3), supported by bandshifts due to solvent isotope exchange and by assignment using site-directed mutants . IR difference spectra of the photooxidation reaction using the caged oxygen compound, and of the photoreduction reaction using the caged electron donor FMN, have inverted shapes . The spectroscopic signals of carboxyl groups are thus equivalent in both reactions: the use of chemically produced oxygen allows the observation of the ongoing molecular changes of cytochrome bo(3) oxidase under quasi-physiological conditions.

Eur J Biochem, 2002 May, 269(10), 2567 - 73
Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits; Wehrle F et al.; The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli . Both recombinant subunits a and b were purified via Ni(2+) chelate affinity chromatography . A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes . The F(o) liposomes catalysed (22)Na(+) uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N'-dicyclohexylcarbodiimide (DCCD) . In the absence of a membrane potential the Fo complexes catalysed (22)Na(+)(out)/Na(+)(in)-exchange . After F(1) addition the F(1)F(o) complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na(+) pumping, and ATP hydrolysis, which was inhibited by DCCD . Functional F(o) hybrids were reconstituted with recombinant subunits a and b from P . modestum and c(11) from Ilyobacter tartaricus . These Fo hybrids had Na(+) translocation activities that were not distinguishable from that of P . modestum F(o).

Eur J Biochem, 2002 May, 269(10), 2516 - 26
A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor; Hortner M et al.; Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells . Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades . The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins . Recently, the feedback inhibitor suppressor of cytokine signaling-3 (SOCS-3), also referred to as cytokine-inducible SH2-containing protein 3 (CIS-3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR-associated Janus kinase 2 (Jak2) {Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I . & Yoshimura, A . (2000) J . Biol . Chem 275, 29338-29347} . In this study tyrosine 401 was identified as a binding site for SOCS-3 on the EpoR . Here we show that human SOCS-3 binds to pY401 with a Kd of 9.5 microm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS-3 but with a ninefold higher affinity than we found for the previously reported motif pY401 . In addition, SOCS-3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS-3 binding . Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS-3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS-3 on the EpoR.

Eur J Biochem, 2002 May, 269(10), 2491 - 7
The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity; Bhattacharya SK et al.; DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability . This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases . Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures . Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus . Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245-287) to the active site of human DNA ligase I . To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35 . The purified HNBB-truncated protein has a molecular mass of approximately equal 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity . These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted . Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.

Eur J Biochem, 2002 May, 269(10), 2473 - 84
The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans; Cogez V et al.; Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium . Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues . A wild-type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography . One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC . opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis . Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis . In contrast, inactivation of a gene similar to ndvB, encoding the OPG-glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides . Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit . Expression of opgIHC in E . coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.

J Nat Prod, 2002 May, 65(5), 737 - 9
Horiolide, a novel norditerpenoid from Indian ocean soft coral of the genus Sinularia; Radhika P et al.; A novel norditerpenoid, horiolide (1), has been isolated from an Indian Ocean soft coral of the genus Sinularia . The structural elucidation was achieved by a study of its spectral characteristics . This compound is structurally characterized by a new carbon skeleton having one six-membered cyclohexane ring bearing an isopropylene moiety, a carbonyl group, and one seven-membered ring attached to a five-membered lactone moiety.

Res Vet Sci, 2002 Apr, 72(2), 163 - 7
Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig; Garside LH et al.; The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced . The gene complemented the aroA mutation in Escherichia coli AB2829 . A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A . pleuropneumoniae by allelic replacement . Intratracheal infection of susceptible pigs with A . pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease . The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals . The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route . However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response .

Cytokine, 2002 Mar 7, 17(5), 227 - 33
Structure function analysis of interleukin 7: requirement for an aromatic ring at position 143 of helix D; vanderSpek JC et al.; The residues located at the carboxyl terminus of helix D in interleukin-7 (IL-7) have previously been targeted as important for recruitment and binding to the gamma chain component of the IL-7 receptor (IL-7R) . In this study, Trp 143 of helix D was mutated to His, Phe, Tyr and Pro and these mutants, along with a W143A mutant previously described, were studied to determine the effects on activation of DNA synthesis and binding affinity to IL-7R positive 2E8 cells . The W143F and W143Y mutants were similar to wild type IL-7 in their binding properties and retained 85% and 74% of their activating properties, respectively . In contrast, the W143H mutant possessed a lower binding affinity and a corresponding decrease in activation, the W143A mutant possessed an over 100-fold decreased binding affinity and some residual activation activity and the W143P mutant possessed a greatly decreased binding affinity and did not activate . These results strongly suggest an aromatic residue is required at position 143 for IL-7R binding and subsequent signal transduction .

Artif Cells Blood Substit Immobil Biotechnol, 2002 Mar, 30(2), 83 - 98
The hemodynamic effects of diaspirin cross-linked hemoglobin in dopamine-resistant endotoxic shock in swine; Freilich E et al.; As the blood substitute Diaspirin Cross-linked Hemoglobin (DCLHb) has potent vasopressor activity, we assessed its hemodynamic effects in a clinically relevant dopamine-resistant endotoxic shock model in swine . In a randomized and controlled study, E . coli LPS was administered to anesthetized and invasively monitored swine . Group I (n = 3) control pigs were not resuscitated . Groups II (n = 5) and III (n = 6) pigs received dopamine (DA) after MAP decreased 30%, and hetastarch and DCLHb, respectively, after dopamine-resistance occurred . Progressive hemodynamic decline occurred in Group I pigs . DA failed to restore MAP to baseline . However, 0% and 67% of pigs also treated with heta-starch and DCLHb, respectively, achieved temporary restoration of baseline MAP (p = 0.03), prompting a reduction in the dose of DA in 0% of hetastarch vs . 50% of DCLHb treated pigs . Except for increased MPAP and decreased heart in DCLHb treated pigs (p<0.001), hemodynamics and survival were not different (p>0.05) . In conclusion, although DCLHb exacerbated pulmonary hypertension and did not improve O2 utilization or survival, because DCLHb restored MAP to baseline and had a dopamine sparing effect, further investigation of DCLHb's hemodynamic effects in adrenergic agent-resistant endotoxemia is warranted.

Novartis Found Symp, 2002, 245, 51 - 61; discussion 61-5, 165-8
The structure of GlpF, a glycerol conducting channel; Fu D et al.; The passage of water or small neutral solutes across the cell membrane in animals, plants and bacteria is facilitated by a family of homologous membrane channels, variously known as aquaporins though perhaps more correctly as aquaglyceroporins . The glycerol facilitator (GlpF) is a 28 kDa aquaglyceroporin that catalyses transmembrane diffusion of glycerol and certain linear polyhydric alcohols in Escherichia coli . X-ray crystallographic analysis of GlpF to 2.2 A resolution revealed an alpha-barrel structure, surrounded by six full-length transmembrane helices and two half-spanning helices that are joined head-to-head in the middle of the membrane . These helices are arranged to a quasi twofold manner relative to the central membrane plane, where highly conserved residues make helix-to-helix contacts that stabilize the relative position and orientation of the helices in the structure . This sequence-structure correlation suggests that the evolutionary divergence of aquaporins and aquaglyceroporins is constrained by a conserved structural framework within which specialized function may be developed . Three glycerol molecules were resolved in the central channel through the GlpF monomer, thereby defining a transmembrane channel for glycerol permeation . The structure of glycerol GlpF complex provides insight into the chemical basis for transmembrane selective permeability.

Mol Plant Microbe Interact, 2002 Apr, 15(4), 380 - 7
Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine; Davis JM et al.; To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals . PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4 . Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities . However, other fractions contained enzyme activities that increased markedly after elicitor treatment . The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f . sp . pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms . Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge . Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase . These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms . This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Amino Acids, 2002, 22(1), 15 - 26
Protein metabolism during an acute phase response in chickens; Barnes DM et al.; Fractional rates of liver, muscle, plasma and acute phase portein synthesis were measured in chickens injected with saline or E . coli lipopolysaccharide (LPS) . Male Single Comb White Leghorns were infused with a primed constant infusion of 15N-L-methionine and 2H5-L-phenylalanine into the portal vein for 2 h . Changes in plasma amino acid enrichment were similar for both amino acids reaching an apparent plateau by the 30 min sampling time . The enrichment of plasma protein-bound amino acid was measurable after 1 h of isotope infusion and increased linearly over 2h . LPS injection decreased free phenylalanine enrichment in the carotid artery (50%), and reduced tissue free methionine enrichment in the liver, pectoralis, and gastrocnemius by 16, 41, and 31% respectively . Isotopic enrichment of phenylalanine in liver protein, plasma protein and hemopexin increased in LPS injected birds relative to control birds . Fractional rates of muscle protein synthesis were not affected by LPS injection, however, liver protein, plasma protein, and hemopexin fractional synthesis rates increased 141, 161 and 266% respectively compared with untreated animals.

Zoolog Sci, 2002 Jan, 19(1), 43 - 8
Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian, Halocynthia roretzi; Matsumoto M et al.; Spermatozoa bind to the vitelline coat in the ascidians and many other animals . The binding of sperm in Halocynthia roretzi is mediated by a sperm alpha-L-fucosidase and complementary-L-fucosyl residues of glycoproteins in the vitelline coat . cDNA clones for alpha-L-fucosidase were isolated from growing testis mRNA . It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H . roretzi sperm alpha-L-fucosidase . A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W . 52.4 kDa) . The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence . The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E . coli . The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner . These data suggest that sperm-egg binding is mediated by the sperm alpha-L-fucosidase, HrFuc'ase in the ascidian, H . roretzi.

Microb Ecol, 2001 Aug, 42(2), 208 - 214
Increase in the Culturable Cell Number of Escherichia coli during Recovery from Saline Stress: Possible Implication for Resuscitation from the VBNC State; Ohtomo R et al.; The change in colony-forming units (CFUs) in saline-stressed Escherichia coli cultures was examined . Exposure of E . coli cells to high saline stress decreased the number of CFUs significantly . When the culture was relieved from the stressful condition, the number of CFUs returned within 2 hr to the same level as before the stress . This recovery in the number of CFUs seemed to be independent of DNA synthesis and cell division, because the same phenomenon was also observed in the presence of nalidixic acid . However, the total cell number enumerated by microscopy was the same before and after relief from saline stress . This phenomenon was considered to be the result of the resuscitation of cells that had been in the VBNC state during saline stress . Our method, in which we examined CFU recovery in the presence of a DNA synthesis inhibitor, might be useful for further VBNC study.

Biochem J, 2002 Jun 1, 364(Pt 2), 555 - 62
Sterol 14alpha-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family; Lamb DC et al.; The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14alpha-demethylases (CYP51) . The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli . Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL) . Reconstitution experiments with E . coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanosterol . An S . coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential . The role of this CYP in bacteria is intriguing . No sterol product was detected in non-saponifiable cell extracts of the parent S . coelicolor A3(2) strain or of the mutant . S . coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces . We propose that it is a member of a new CYP51 subfamily.

Biochem J, 2002 Jun 1, 364(Pt 2), 527 - 35
Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry; Blakey D et al.; The Amt family of high-affinity ammonium transporters is a family of integral membrane proteins that are found in archaea, bacteria, fungi, plants and animals . Furthermore, the family has recently been extended to humans with the recognition that both the erythroid and non-erythroid Rhesus proteins are also ammonium transporters . The Escherichia coli AmtB protein offers a good model system for the Amt family and in order to address questions relating to both its structure and function we have overproduced a histidine-tagged form of the protein (AmtB6H) and purified it to homogeneity . We examined the quaternary structure of AmtB6H (which is active in vivo) by SDS/PAGE, gel-filtration chromatography, dynamic light scattering and sedimentation ultracentrifugation . The protein was resistant to dissociation by SDS and behaved as a stable oligomer on SDS/PAGE . By equilibrium desorption chromatography we determined the mass ratio of dodecyl beta-D-maltoside to AmtB in the detergent-solubilized complex to be 1.03+/-0.03, and this allowed us to calculate, from analytical-ultracentrifugation data, that AmtB purifies as a trimer.

Biochem J, 2002 Jun 1, 364(Pt 2), 457 - 63
Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life; Facchin S et al.; The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown . We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein . Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties . Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE . A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity . This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

Biochem J, 2002 Jun 1, 364(Pt 2), 441 - 7
Ascorbate up-regulates MLH1 (Mut L homologue-1) and p73: implications for the cellular response to DNA damage; Catani MV et al.; We have found previously that ascorbic acid (vitamin C), as well as acting as a radical scavenger, may modulate the expression of several genes {i.e . fra-1, glutathione S-transferase Pi (GSTpi) and Mut L homologue-1 (MLH1)} in human keratinocytes . In the present paper, we demonstrate that MLH1, as well as its downstream target p73, can be positively modulated by this antioxidant vitamin, indeed, up-regulation of the two mRNAs was observed after just 2 h, and increased further up to 16 h of treatment . Modulation of MLH1 and p73 gene expression improves cellular susceptibility to apoptosis triggered by the DNA-damaging agent cisplatin . Indeed, in ascorbate-supplemented cells, increased cisplatin-induced apoptosis was seen, involving activation of the MLH1/c-Abl/p73 signalling cascade . Our results were further confirmed by studies performed on genetically defined mutants, i.e . mouse embryo fibroblasts derived from knock-out animals for c-Abl or p53, as well as human colon carcinoma cell lines deficient in MLH1 . The increased sensitivity to cisplatin observed in ascorbate-loaded cells appeared to be dependent exclusively on MLH1 and c-Abl expression, and independent of p53 . These data suggest a potential mechanism accounting for the anti-carcinogenic and anti-cancer activities of vitamin C.

Biochem Soc Trans, 2002 Apr, 30(2), 133 - 40
Functions and interplay of the tRNA-binding sites of the ribosome; Marquez V et al.; The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids . The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e . an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins . Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites . In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals . These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA.EF-Tu.GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA(2).mRNA complex during translocation and the maintenance of the reading frame.

Biochem Soc Trans, 2002 Apr, 30(2), 47 - 51
Substrate channelling in 2-oxo acid dehydrogenase multienzyme complexes; Perham RN et al.; Heteronuclear NMR spectroscopy and other experiments indicate that the true substrate of the E1 component of 2-oxo acid dehydrogenase complexes is not lipoic acid but the lipoyl domain of the E2 component . E1 can recognize the lipoyl-lysine residue as such, but reductive acylation ensues only if the domain to which the lipoyl group is attached is additionally recognized by virtue of a mosaic of contacts distributed chiefly over the half of the domain that contains the lipoyl-lysine residue . The lipoyl-lysine residue may not be freely swinging, as supposed hitherto, but may adopt a preferred orientation pointing towards a nearby loop on the surface of the lipoyl domain . This in turn may facilitate the insertion of the lipoyl group into the active site of E1, where reductive acylation is to occur . The results throw new light on the concept of substrate channelling and active-site coupling in these giant multifunctional catalytic machines.

J Infect Dis, 2002 Jun 1, 185(11), 1681 - 3 Epub 2002 May 17.
Natural history of enteroaggregative and enterotoxigenic Escherichia coli infection among US travelers to Guadalajara, Mexico; Adachi JA et al.; The natural history of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E . coli (ETEC) infection was studied among 40 US travelers who provided weekly stool samples for 4 weeks after arrival in Mexico . At enrollment, 5 subjects were colonized by EAEC and 3 by ETEC . During the first 2 weeks after enrollment, 12 developed EAEC diarrhea, 7 developed ETEC diarrhea (5 with mixed EAEC/ETEC diarrhea), 13 had EAEC colonization, and 7 had ETEC colonization . During the third and fourth weeks, 4 experienced EAEC diarrhea, 2 experienced ETEC diarrhea (1 with mixed EAEC/ETEC diarrhea), 31 had EAEC colonization, and none had ETEC colonization . Plasmid DNA analysis showed a high degree of heterogeneity among EAEC isolates . Symptomatic EAEC infection occurred early after arrival in Guadalajara, Mexico, and was as common as ETEC infection . Asymptomatic EAEC infection was recurrent.

J Pharmacol Exp Ther, 2002 Jun, 301(3), 945 - 52
Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6; Sridar C et al.; Tamoxifen is primarily used in the treatment of breast cancer . It has been approved as a chemopreventive agent for individuals at high risk for this disease . Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes . The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated . The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner . Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH . The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min . The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification . The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration . The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity . A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis . Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Genes Dev, 2002 May 15, 16(10), 1260 - 70
Regulation of sigma factor competition by the alarmone ppGpp; Jishage M et al.; Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp . We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation . An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70) . Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp . Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells . Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

J Biol Chem, 2002 Aug 16, 277(33), 29455 - 9 Epub 2002 May 21.
Molecular basis of the globoside-deficient P(k) blood group phenotype . Identification of four inactivating mutations in the UDP-N-acetylgalactosamine: globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase gene; Hellberg A et al.; The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, P(k), and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood . Individuals with the rare but clinically important erythrocyte phenotypes P(1)(k) and P(2)(k) lack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriated Escherichia coli . As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed . To define the molecular basis of the P(k) phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-beta-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase or globoside synthase activity . Homozygosity for different nonsense mutations (C(202) --> T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P(2)(k) phenotype . Two individuals with P(1)(k) and P(2)(k) phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase . We conclude that crucial mutations in the globoside synthase gene cause the P(k) phenotype.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 105 - 10
Site-directed gene disruption in Xylella fastidiosa; da Silva Neto JF et al.; As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella . Vector pSP3 was constructed by ligating to the E . coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3 . Transformation experiments have shown that pSP3 replicates stably in Xylella . When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture . These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 55 - 60
Architectural requirements for optimal activation by tandem CRP molecules at a class I CRP-dependent promoter; Tebbutt J et al.; The Escherichia coli cyclic AMP receptor protein (CRP) activates transcription at target promoters by interacting with the C-terminal domain of the RNA polymerase alpha subunit . We have constructed a set of promoters carrying tandem DNA sites for CRP with one site centred at position -61.5 and the other site located at different upstream positions . Optimal CRP-dependent activation of transcription is observed when the upstream DNA site for CRP is located at position -93.5 or at position -103.5 . Evidence is presented to suggest that activation by the upstream-bound CRP molecule is due to interaction with the C-terminal domain of the RNA polymerase alpha subunit.

FEBS Lett, 2002 May 22, 519(1-3), 66 - 70
Functional identification of the cDNA coding for a wheat endo-1,4-beta-D-xylanase inhibitor; Elliott GO et al.; Using expressed sequence tag data, we obtained a full-length cDNA encoding a wheat protein inhibitor of xylanases (XIP-I) . The 822 bp open reading frame encoded a protein of 274 amino acids with a molecular mass of 30.2 kDa, in excellent agreement with the native protein . Expression in Escherichia coli confirmed that the cDNA encoded a functional endo-1,4-beta-D-xylanase inhibitor . Its deduced amino acid sequence exhibited highest similarity to sequences classified as class III chitinases, but the inhibitor did not exhibit chitinase activity . This is the first full-length cDNA sequence that encodes a novel class of protein which inhibits the activity of endo-1,4-beta-D-xylanases.

FEBS Lett, 2002 May 22, 519(1-3), 16 - 22
The determinants of the oligomeric structure in Hsp16.5 are encoded in the alpha-crystallin domain; Koteiche HA et al.; The determinants of the oligomeric assembly of Hsp16.5, a small heat-shock protein (sHSP) from Methanococcus jannaschii, were explored via site-directed truncation and site-directed spin labeling . For this purpose, subunit contacts around the two-, three- and four-fold symmetry axes were fingerprinted using patterns of proximities between nitroxide spin labels introduced at selected sites . The lack of change in this fingerprint in an N-terminal truncation of the protein demonstrates that the interactions are encoded in the alpha-crystallin domain . In contrast, the truncation of the N-terminal domain of Mycobacterium tuberculosis Hsp16.3, a bacterial sHSP with an equally short N-terminal region, results in the dissociation of the oligomer to a trimer . These results, in conjunction with those from previous truncation studies in mammalian sHSP, suggest that as the alpha-crystallin domain evolved to encode a smaller basic unit than the overall oligomer, the control of the assembly and dynamics of the oligomeric structure became encoded in the N-terminal domain.

Biochemistry, 2002 May 28, 41(21), 6824 - 33
Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG; Chen L et al.; MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan . Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain . Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains . We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate . Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG . Using synthetic substrate analogues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first . This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.

Biochemistry, 2002 May 28, 41(21), 6679 - 87
Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site; Alam M et al.; Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins . The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression . Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity . Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme . A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified . Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme . CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding . Human TGH was glycosylated in the insect cells . Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme . Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein . A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).

Biochemistry, 2002 May 28, 41(21), 6660 - 7
Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein; Dong A et al.; Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E . coli . It is activated by cAMP binding, which leads to some yet undefined conformational changes . These changes do not involve significant redistribution of secondary structures . A potential mechanism of activation is a ligand-induced change in structural dynamics . Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy . Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions . While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively . To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain . Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP . The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.

Biochemistry, 2002 May 28, 41(21), 6640 - 50
Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662; Leesnitzer LM et al.; In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain . GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively . Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay . Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification . Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification . This cysteine is conserved among all three PPARs . In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma . The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation . GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha . Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays . Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta . Corepressor binding was decreased . In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding . The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences . The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.

Shock, 2002 May, 17(5), 427 - 32
Hypotension during septic shock does not correlate with exhaled nitric oxide in anesthetized rat; Pedoto A et al.; Sepsis is characterized by hypotension, acidosis, and increased nitric oxide (NO) production . The role of NO in the development of sepsis-related hypotension is still unclear . The relationship among exhaled nitric oxide (ENO), arterial blood pressure (BP), and pH after administration of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) was investigated in anesthetized rats . Forty-three adult male Sprague-Dawley rats were randomized into five groups: group 1 (C, n = 8) received normal saline; group 2 (LPS-I, n = 8) received Escherichia coli (LPS) 10 mg/kg intravenously (i.v.); group 3 (LPS-h, n = 10) received 100 mg/kg LPS i.v.; group 4 (n = 9) was treated with 100 mg/kg i.v . aminoguanidine (AG) 1 h after receiving 100 mg/kg i.v . LPS; group 5 (TNFalpha, n = 8) received 1 microg recombinant rat TNFalpha i.v. . ENO, BP, and pH were measured every 30 min for 4 h whereas arterial blood gases and pH were measured every hour . LPS administration induced a dose-related increase in ENO and a dose-related decrease in BP and pH . AG blocked the increase in ENO after LPS but had minimal effect on BP and pH . TNFalpha administration increased ENO without changing BP and pH . In LPS-treated rats, no significant correlation was found between ENO and BP (r2 = 0.13, P= ns) . However, there was a significant correlation between pH and BP (r2 = 0.7, P < 0.01) . Our results suggest that, in this animal model, ENO may not be a key mediator in the development of systemic hypotension during sepsis, while acidosis may significantly contribute to it.

Shock, 2002 May, 17(5), 404 - 10
Cardiac response to nitric oxide synthase inhibition using aminoguanidine in a rat model of endotoxemia; Vona-Davis L et al.; This study evaluates the effect of aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase (iNOS), on the prevention of cardiac depression in acute endotoxemia . Cardiac performance was evaluated after 4 h of exposure to endotoxin . Rats (n = 5) were selected randomly to receive, by intraperitoneal injection, one of four treatments: saline, LPS (lipopolysaccharide, E . coli, 4 mg/kg, AG (aminoguanidine 100 mg/kg), and LPS + AG at various times . AG and saline treatments were administered 30 min before LPS and at 1 and 3 h after LPS injection . Hearts were perfused using the Langendorff isolated perfusion system and a balloon-tipped catheter was placed into the left ventricle to measure left ventricular developed pressure (LVDP) . Myocyte contractile function was assessed with electrical field stimulation and video microscopy . Tissue was immunostained for the expression of iNOS and for nitrotyrosine, a byproduct of protein nitration by peroxynitrite . Perfused hearts from LPS-treated rats exhibited a 57% decrease (P < 0.05) in LVDP compared to saline-treated animals . No improvement in ventricular function was observed with the administration of AG . Similarly, cardiac myocytes prepared from LPS-treated animals demonstrated a significant (P < 0.05) reduction in percent and velocity of shortening and this effect was unaltered with the same dose of AG . AG administration significantly reduced serum nitrite/nitrate levels (P < 0.05) in endotoxemic rats to control levels . Localized expression of iNOS in the myocardium was lessened with AG treatment and was not associated with peroxynitrite formation in this model of endotoxemia . The results indicate that AG given in vivo before and after endotoxin (at a concentration sufficient to decrease NO production) did not reduce cardiac depression . We conclude that selective inhibition of iNOS and the reduction of NO production do not prevent cardiac dysfunction at an early stage in an acute model of endotoxemia.

Shock, 2002 May, 17(5), 399 - 403
Effect of curcuminoids as anti-inflammatory agents on the hepatic microvascular response to endotoxin; Lukita-Atmadja W et al.; Curcuminoids, derived from the plant Curcuma domestica Val., have been shown to be free radical scavengers that suppress the production of superoxide by macrophages and potent anti-inflammatory agents that inhibit the lipopolysacharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and the activation of nuclear factor (NF)-kappaB in human monocytic derived cells . The present study was undertaken to determine the efficacy of curcuminoids in inhibiting the hepatic microvascular inflammatory response elicited by LPS . BALB/C mice were gavaged intragastricly with curcuminoids {40 mg/kg body weight (bw) or 80 mg/kg bw} 1 h before intravenous injection of LPS (Escherichia coli, O111:B4, 100 microg/kg bw) . The liver was examined 2 h after LPS injection using in vivo microscopic methods . LPS-treated mice showed significantly increased phagocytic activity of centrilobular Kupffer cells . The numbers of leukocytes adhering to the sinusoidal wall and swollen endothelial cells increased significantly in both the periportal and centrilobular regions, concomitant with a reduction in the numbers of sinusoids containing flow . Pretreatment with curcuminoids at the doses of 40 mg/kg bw or 80 mg/kg bw to endotoxemic mice significantly reduced the phagocytic activity of Kupffer cells, the numbers of adhering leukocytes and swollen endothelial cells . As a result, the number of sinusoids containing flow was increased in animals treated with 40 mg/kg curcuminoids and restored to control levels with 80 mg/kg curcuminoids . Neutrophil sequestration was reduced when measured in sections stained with naphtol AS-D chloroacetate esterase technique . These results demonstrate that curcuminoids are effective in suppressing the hepatic microvascular inflammatory response to LPS and may be a natural alternative anti-inflammatory substance.

Chang Gung Med J, 2002 Mar, 25(3), 201 - 6
Acanthamoeba keratitis presenting as dendritic keratitis in a soft contact lens wearer; Yeung EY et al.; Acanthamoeba keratitis is a rare cause of corneal infection in Taiwan, which can result in devastating visual outcomes . A 37-year-old woman, who wore soft contact lenses, suffered from severe pain in her left eye . Biomicroscopy revealed dendritic keratitis, radial keratoneuritis, and fine keratic precipitates on her cornea . Culture, using non-nutrient agar plate seeded with Escherichia coli, resulted in heavy growth of Acanthamoeba . The inpatient treatment, including topical neomycin-polymyxin B and metronidazole (0.5%) eyedrops, oral ketoconazole, and then oral prednisolone, successfully controlled the corneal infection . The best-corrected visual acuity was 0.9 without any evidence of recurrence of infection after 21 months of follow up . Acanthamoeba keratitis can present as dendritic keratitis, which mimics herpes simplex infection, thus, delays appropriate treatment . Early diagnosis and judicious treatment are essential for restoring the vision and avoiding the subsequent need of penetrating keratoplasty.

RNA, 2002 May, 8(5), 659 - 70
Efficient aminoacylation of the tRNA(Ala) acceptor stem: dependence on the 2:71 base pair; Beuning PJ et al.; Specific aminoacylation by aminoacyl-tRNA synthetases requires accurate recognition of cognate tRNA substrates . In the case of alanyl-tRNA synthetase (AlaRS), RNA duplexes that mimic the acceptor stem of the tRNA are efficient substrates for aminoacylation in vitro . It was previously shown that recognition by AlaRS is severely affected by a simple base pair transversion of the G2:C71 pair at the second position in the RNA helix . In this study, we determined the aminoacylation efficiencies of 50 variants of the tRNA(Ala) acceptor stem containing substitutions at the 2:71 position . We find that there is not a single functional group of the wild-type G2:C71 base pair that is critical for positive recognition . Rather, we observed that base-pair orientation plays an important role in recognition . In particular, pyrimidine2:purine71 combinations generally resulted in decreased aminoacylation efficiency compared to the corresponding purine:pyrimidine pair . Moreover, the activity of a pyrimidine:purine variant could be partially restored by the presence of a major groove amino group at position 71 . In an attempt to understand this result further, dielectric continuum electrostatic calculations were carried out, in some cases with additional inclusion of van der Waals interaction energies, to determine interaction potentials of the wild-type duplexAla and seven 2:71 variants . This analysis revealed a positive correlation between major groove negative electrostatic potential in the vicinity of the 3:70 base pair and measured aminoacylation efficiency.

RNA, 2002 May, 8(5), 647 - 58
In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron; Guo F et al.; In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons . The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained . The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro . Random mutation libraries were constructed to identify active intron variants in E . coli . All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron . Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E . coli . Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo . In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration . The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration . Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed . This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.

RNA, 2002 May, 8(5), 612 - 25
Crosslinking of 4.5S RNA to the Escherichia coli ribosome in the presence or absence of the protein Ffh; Rinke-Appel J et al.; Radioactively labeled 4.5S RNA containing statistically distributed 4-thiouridine residues in place of normal uridine was prepared by T7 transcription . The ability of this modified 4.5S RNA to form a complex with the protein Ffh was demonstrated by a gel shift assay . The modified 4.5S RNA, with or without Ffh, was added to Escherichia coli ribosomes under various conditions, and crosslinking from the thiouridine residues was induced by irradiation at 350 nm . The crosslinked ribosomal components were analyzed by our standard procedures . Two clearly defined types of crosslinking were observed . The first was a crosslink to 23S rRNA, which was entirely dependent both on the presence of Ffh and a nascent protein chain in the 50S subunit . This crosslink was localized to nt approximately 2828-2837 of the 23S rRNA, from position 84 of the 4.5S molecule . The second type of crosslinking, to the 30S ribosomal subunit, was independent of the presence of Ffh, and was found both with vacant 70S ribosomes or isolated 30S subunits . Here the crosslink was localized to the 3'-terminal region of the 16S rRNA, from positions 29-50 of the 4.5S RNA . Cross-linking to ribosomal protein S1 was also observed . The known crystal structure of the protein Ffh/4.5S RNA fragment complex was extrapolated by computer modeling so as to include the whole 4.5S molecule, and this was docked onto the ribosome using the crosslinking data . The results are discussed in terms of multiple functions and binding sites of the 4.5S RNA.

RNA, 2002 May, 8(5), 572 - 8
Surprising flexibility of leader RNA determinants for r-protein L4-mediated transcription termination in the Escherichia coil S10 operon; Zengel JM et al.; Escherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins . Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader . The process requires transcription factor NusA . Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control . We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE . As expected, regulation was optimal when all 7 Us were maintained in the U4CGU3 sequence at the termination site . However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region . Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect . Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic . The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 383 - 7
{The hemolytic site of the basic phospholipase A(2) from Agkistrodon halys pallas}; Jiao HM et al.; The gene of the basic phospholipase A(2) from Agkistrodon halys Pallas (BPLA(2) )was mutated site-directedly by polymerase chain reaction (PCR) and the residue Arg(34) of the encloding protein was substituted by Glu and Gln respectively.The mutant gene has been cloned into the expression vector pBLMVL2 and has been expressed in E.coli RR1 effectively . The protein was produced as insoluble inclusion bodies . After partial purification, the inclusion bodies were denatured and renatured into active form, and the renatured recombinant protein was purified by gel-filtration . The expression product has the same enzymatic activity as the denatured-refolded BPLA(2) and its hemolytic activity dropped distinctly, which suggest that the basic residue Arg(34) of BPLA(2) is a crucial amino acid residue during the process of hemolysis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 347 - 52
{Molecular cloning, expression and characterization of the clones encoding soluble TF mutants}; Yu M et al.; Tissue factor (TF) plays an important role in the pathogenesis of atherosclerotic, sepsis and disseminated intravascular coagulation (DIC) . TF pathway is therefore an attractive therapeutic target in a number of disease states . Here two TF mutants were developed and named MCsTF and MFsTF, in which the amino acids of active sites were mutated . Both of them were expressed in E.coli and used to inhibit TF pathway through competitive FVII/VIIa binding with TF . The results indicated that rMCsTF almost lost all activities of FX activation and procoagulation, and rMFsTF lost 90% activity . The specific catalytic constant ( k ( cat )/ K (m)) of FX activation by the complex formed by FVIIa with rMCsTF or rMFsTF were 2.0% and 3.7%, respectively, compared to that of rsTF . The inhibition effects of the mutants were studied in vitro, and it appeared that the prothrombin time were prolonged in a dose-dependent manner . Therefore, these mutants of TF may become new kind of specific inhibitors of TF pathway, as a promising drug for the treatment of patients with over-expression of TF.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 311 - 7
{Cloning and expression of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain)}; Jin YM et al.; A 1949 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA with 3 pair of primers that were designed according to published SmGCP gene encoding gynecophoral canal protein of Schistosoma mansoni and SjGCP1 gene encoding the conservative region of gynecophoral canal protein of Schistosoma japonicum . Sequence analysis indicated that this fragment, named SjGCP, with 85% identity to SmGCP, contained a complete open reading frame (ORF) of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain).The amino acid sequence shared 83.7% identity with gynecophoral canal protein of Schistosoma mansoni . This fragment was cloned into the expression vector pET28c(+) and subsequently expressed in Escherichia coli . SDS-PAGE revealed that the molecular weight of this expressed product was 80 kD . Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

J Histochem Cytochem, 2002 Jun, 50(6), 863 - 73
Optimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen; Kaur R et al.; We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy . Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM) . Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods . The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM . Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling . The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible . Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.

J Biol Chem, 2002 Jul 26, 277(30), 27528 - 34 Epub 2002 May 17.
Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase . Teaching an old dog new tricks; Thoden JB et al.; UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and UDP-Glc during normal galactose metabolism . The mammalian form of the enzyme, unlike its Escherichia coli counterpart, can also interconvert UDP-GalNAc and UDP-GlcNAc . One key feature of the epimerase reaction mechanism is the rotation of a 4-ketopyranose intermediate in the active site . By comparing the high resolution x-ray structures of both the bacterial and human forms of the enzyme, it was previously postulated that the additional activity in the human epimerase was due to replacement of the structural equivalent of Tyr-299 in the E . coli enzyme with a cysteine residue, thereby leading to a larger active site volume . To test this hypothesis, the Y299C mutant form of the E . coli enzyme was prepared and its three-dimensional structure solved as described here . Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and UDP-GalNAc . These studies have revealed that, indeed, this simple mutation did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial enzyme with minimal changes in its three-dimensional structure . Specifically, although the Y299C mutation in the bacterial enzyme resulted in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than 230-fold.

J Biol Chem, 2002 Jul 26, 277(30), 27109 - 19 Epub 2002 May 17.
Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin; Kamikubo Y et al.; The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1) . In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance . Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153) . Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities . The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing . Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21) . Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity . The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB . These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.

J Biol Chem, 2002 Jul 26, 277(30), 27412 - 22 Epub 2002 May 17.
Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains . Studies on the PH domains of phospholipase C delta 1 and p130; Varnai P et al.; The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP) . The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips . In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP . The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains . By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells . These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 1823 - 30
CAU-1, a subclass B3 metallo-beta-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome; Docquier JD et al.; The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-beta-lactamases of subclass B3 . An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C . crescentus type strain DSM4727 . Expression studies confirmed the metallo-beta-lactamase activity of its product, CAU-1 . The enzyme produced in E . coli was purified by two ion-exchange chromatography steps . CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (> 9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric . Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed . Affinities for the various beta-lactams were poor overall (K(m) values were always > 100 microM and often > 400 microM) . The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3 . In C . crescentus, the CAU-1 enzyme is produced independently of beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure . The CAU-1 enzyme is the first example of a metallo-beta-lactamase in a member of the alpha subdivision of the class Proteobacteria:

Chem Res Toxicol, 2002 May, 15(5), 686 - 91
Kinetics of paraquat and copper reactions with nitroxides: the effects of nitroxides on the aerobic and anoxic toxicity of paraquat; Goldstein S et al.; The toxicity of paraquat (PQ2+) is attributed to intracellularly formed PQ*+, O(2)*-, H(2)O(2), and secondary.OH radicals generated through Fenton-like reactions . Yet, no antidote for PQ2+ toxicity in human has been found also due to poor cell permeability of many common antioxidants that remove toxic species predominantly extracellularly . Cell-permeable nitroxides, which scavenge xenobiotic-derived deleterious radicals and detoxify redox-active metal ions, would be expected to ameliorate PQ2+ toxicity . We have studied using pulse radiolysis the kinetics of the reactions of 2,2,6,6-tetramethyl-piperidinoxyl (TPO) and 4-OH-TPO with PQ*+ and CuIIL(2) (L = 1,10-phenanthroline, 2,2'-bipyridyl) in the absence and presence of DNA . We found that the rate constant for the reaction of PQ*+ with the nitroxides is about 4 orders of magnitude lower than that with O(2) . In addition, the rate of the reaction of the nitroxides with CuIL(2) decreases as {DNA} increases, which suggests that nitroxides react significantly slower with bound metal ions . These results explain the failure of 4-OH-TPO to protect bacterial and mammalian cells from PQ2+ toxicity under air . In contrast, the rate of the reaction of PQ*+ with CuIIL(2) was unaffected by DNA . Furthermore, copper toxicity has been attributed mainly to CuI and was observed predominantly for cells subjected to anoxic conditions . It implied that nitroxides would be effective protectants if PQ2+ induces toxicity also under anoxia . Surprisingly, we found that PQ2+ toxicity under anoxia was even greater than that under air, and under these conditions 4-OH-TPO protected the cells from PQ . These results indicate that the mechanism underlying the anoxic toxicity of PQ2+ differs from that operating in the presence of oxygen, and that reduced transition metal ions are most probably the species responsible for PQ2+ anoxic toxicity.






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