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 a