Escherichia coli

Protein Expr Purif, 2001 Nov, 23(2), 296 - 300
High-level expression and single-step purification of human tryptophanyl-tRNA synthetase; Xu F et al.; Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL . The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography . The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction . The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture . The His(6) tag at C terminus had little effect on the binding ability of its substrates .

Protein Expr Purif, 2001 Nov, 23(2), 289 - 95
Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin; Mavrangelos C et al.; The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material . We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp . Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression . The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent .

Protein Expr Purif, 2001 Nov, 23(2), 270 - 81
An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer; Grimm KM et al.; A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group . This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response . Our research has focused on developing the purification process for preparing tetramer reagent . Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable . In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration . The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC . The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step . The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate . The tetramer is further purified to remove any excess MHC or avidin components . Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process .

Protein Expr Purif, 2001 Nov, 23(2), 219 - 25
Overproduction, purification, and characterization of beta-1,3-glucanase type II in Escherichia coli; Salazar O et al.; An Escherichia coli recombinant system produced soluble and full-length beta-1,3-glucanase type II (BglII) cloned from the yeast-lytic actinomycete Oerskovia xanthineolytica . The expression system was designed to produce recombinant BglII with a six-histidine peptide fused to the carboxy end of the protein . The expression level was optimized to produce 30% of total protein of E . coli as the recombinant protein, releasing 75% to the extracellular space . The 43-kDa recombinant protein was purified by IMAC to homogeneity and its molecular and biochemical characteristics were studied, showing that there are no important functional differences with those properties described for the BglII purified from O . xanthineolytica .

Metab Eng, 2001 Oct, 3(4), 362 - 79
The organization of metabolic reaction networks . III . Application for diauxic growth on glucose and lactose; Kremling A et al.; A mathematical model to describe carbon catabolite repression in Escherichia coli is developed and in part validated . The model is aggregated from two functional units describing glucose and lactose transport and degradation . Both units are members of the crp modulon and are under control of a global signal transduction system which calculates the signals that turn on or off gene expression for the specific enzymes . Using isogenic mutant strains, our model is validated by a set of experiments . In these experiments, substrate composition of the preculture and of the experimental culture are varied in order to stimulate the system in different ways . With the obtained measurements (three states in the liquid phase and one intracellular component) a part of the model parameters could be estimated . Therefore all experiments could be sufficiently described with a single set of parameters .

Metab Eng, 2001 Oct, 3(4), 313 - 21
Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization; Smolke CD et al.; A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter . DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes . This construct was transformed into Escherichia coli cells harboring the genes for phytoene production . By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene . In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates . These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates .

J Mol Biol, 2001 Oct 26, 313(3), 583 - 92
Crystal structure of the Escherichia coli RNA degradosome component enolase; Kuhnel K et al.; The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site . This closure mimics the conformation of the substrate-bound intermediate . A comparison of the structure of the E . coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme . The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength . The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible .

J Mol Biol, 2001 Oct 26, 313(3), 539 - 57
Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA; Cloutier JF et al.; The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer . Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify . Therefore, we use the NNK analogue 4-{(acetoxymethyl)nitrosamino}-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR) . NNKOAc induced single-strand breaks in a concentration-dependent manner . Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA . Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure . NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine . In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation . Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248 . We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment . Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV . These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA . The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks . These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds .

J Mol Biol, 2001 Oct 26, 313(3), 501 - 10
Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli; Balakrishnan L et al.; A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex . This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment . HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59) . Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase . The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC . Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate . The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential . Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function . The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it . In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e . TolC recruitment was not triggered . Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex .

J Mol Biol, 2001 Oct 26, 313(3), 485 - 99
The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor; Newman JD et al.; The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma(E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1) . These genes encode the periplasmic electron carrier cytochrome c(2) and sigma(E)/ChrR, respectively . Using in vitro transcription assays with purified R . sphaeroides core RNA polymerase and sigma(E), we show that ChrR is sufficient to inhibit sigma(E)-dependent transcription . Inhibition is proposed to proceed through a binding interaction, since sigma(E) and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli . Active preparations of ChrR and the sigma(E)/ChrR complex each contain stoichiometric zinc . Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting sigma(E) activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity . Treatment of ChrR with the thiol-modifying reagent p-hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein . Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding . These data indicate that ChrR is a specific anti-sigma factor of sigma(E) that requires zinc for function . Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria .

Biochem Biophys Res Commun, 2001 Nov 2, 288(3), 499 - 502
Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein; Kamiya H et al.; To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides . Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein . These damaged deoxynucleotides elicit transversion mutations in E . coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H . (1998) J . Biol . Chem . 273, 11069-11074) . These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides .

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12724 - 9
Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway; Hubner A et al.; RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria . To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another . Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B . burgdorferi . In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B . burgdorferi . Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B . burgdorferi's parasitic strategy, host range, and virulence expression.

Anal Biochem, 2001 Nov 1, 298(1), 57 - 61
An FKBP12 binding assay based upon biotinylated FKBP12; Carreras CW et al.; A binding assay was developed for measuring the affinity of FKBP12 ligands . A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase . The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with {3H}FK506 for FKBP12 sites on the plate . The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values . The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules .

Arch Biochem Biophys, 2001 Nov 1, 395(1), 57 - 68
A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function; Scott EE et al.; The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility . Histidine residues were appended to the C-terminus to simplify purification . The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column . Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate . All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation . These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis .

Arch Biochem Biophys, 2001 Nov 1, 395(1), 25 - 31
Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products; Nakamura K et al.; Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism . SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating . One mutant, F209T, showed indole 3-hydroxylation <WT but had a k(cat) for coumarin 7-hydroxylation 13-fold >WT . The double mutant L240C/N297Q consistently produced very blue colonies . Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products . When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants . This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products .

Arch Biochem Biophys, 2001 Nov 1, 395(1), 14 - 20
Effects of lipids on the interaction of SecA with model membranes; Ahn T et al.; The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus . Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1) . When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E . coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E . coli PE . In addition, when the PE of E . coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40% . The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E . coli PE/DOG > E . coli PE > DOPC . These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes .

J Immunol, 2001 Nov 1, 167(9), 5470 - 7
Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen; Binder M et al.; IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals . We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest . Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P . interpunctella larvae . cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated . Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography . Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions . It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel . Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens . Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.

J Immunol, 2001 Nov 1, 167(9), 5273 - 7
Immunization of mice against West Nile virus with recombinant envelope protein; Wang T et al.; West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis . Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli . The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments . Patients with WN virus infection had Abs that recognized the recombinant E protein . C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus . Passive administration of E protein antisera was also sufficient to afford immunity . E protein is a candidate vaccine to prevent WN virus infection.

J Immunol, 2001 Nov 1, 167(9), 5202 - 8
The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r; Kardos J et al.; The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex . Activated C1r then cleaves and activates zymogen C1s . C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity . The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP) . To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli . We successfully renatured the inclusion body proteins . After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other . To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed . Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain . We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s . Therefore, we propose that CCP2 module provides accessory binding sites . Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain . Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure . Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

J Immunol, 2001 Nov 1, 167(9), 5129 - 35
Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2; Parhami-Seren B et al.; Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71 . Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity . Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used . All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain . However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities . When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65 . These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.

J Immunol, 2001 Nov 1, 167(9), 5067 - 76
Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo; Pulendran B et al.; The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood . In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens . We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling . Coinjections of E . coli LPS + OVA or P . gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles . E . coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5 . In contrast, P . gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma . Consistent with these results, E . coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P . gingivalis LPS did not . Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha . Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E . coli LPS, but not P . gingivalis LPS . Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.

J Bacteriol, 2001 Nov, 183(22), 6721 - 5
Colicin A immunity protein interacts with the hydrophobic helical hairpin of the colicin A channel domain in the Escherichia coli inner membrane; Nardi A et al.; The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA) . This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai) . We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP) . We showed that a fusion protein made up of the hydrophobic alpha-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells . This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.

J Bacteriol, 2001 Nov, 183(22), 6684 - 7
The dimerization function of MinC resides in a structurally autonomous C-terminal domain; Szeto TH et al.; Limited proteolysis of the Escherichia coli cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain . We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.

J Bacteriol, 2001 Nov, 183(22), 6645 - 53
Screening of environmental DNA libraries for the presence of genes conferring Na(+)(Li(+))/H(+) antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products; Majernik A et al.; Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc . The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl . Two positive E . coli clones were obtained during the initial screening of 1,480,000 recombinant E . coli strains . Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype . The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family . The insert of pAM3 harbored the DNA region of E . coli K-12 containing nhaA, nhaR, and gef . This region is flanked by highly conserved insertion elements . The sequence identity with E . coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E . coli . The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E . coli and enabled the antiporter-deficient E . coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0 . The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E . coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5 . The maximal activity was observed at pHs 8.3 and 8.6, respectively . The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).

J Bacteriol, 2001 Nov, 183(22), 6538 - 42
Deletion of lolB, encoding an outer membrane lipoprotein, is lethal for Escherichia coli and causes accumulation of lipoprotein localization intermediates in the periplasm; Tanaka K et al.; Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane . In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex . On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo . To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene . At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp . LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane . Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E . coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.

J Bacteriol, 2001 Nov, 183(22), 6532 - 7
Involvement of the N terminus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli; Yang X et al.; Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes . The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA . To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated 'L11) was constructed . Bacteria overexpressing 'L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation . In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response . The overexpressed 'L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA . By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11 . To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis . The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype . These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

Thromb Res, 2001 Aug 1, 103(3), 165 - 72
Thrombotic events revisited in children with acute lymphoblastic leukemia: impact of concomitant Escherichia coli asparaginase/prednisone administration; Nowak-Gottl U et al.; Recently published data suggest that the prothrombin G20210A variant, the TT677 methylenetetrahydrofolate reductase genotype, the factor V G1691A mutation, deficiencies of protein C, protein S, antithrombin, and elevated lipoprotein (a) concentrations were associated with venous thromboembolism in childhood patients treated according to the BFM protocol . To unravel the role of these prothrombotic risk factors and different treatment modalities, the present comparative study was performed in childhood leukemia patients of the same living population . Four hundred and twenty consecutively recruited leukemic children (BFM n=300; COALL n=120) were enrolled in this study with respect to the presence of prothrombotic risk factors and the occurrence of symptomatic venous thrombosis . No significant difference was found in the prevalence rates of thrombotic risk factors in the Caucasian populations studied . Symptomatic venous thromboembolism occurred in 11.6% of BFM patients compared with 2.5% in the COALL treatment group {odds ratio (OR)/95% confidence intervals (CI): 7.7/1.8-32.6; P=.005} . Including age, prothrombotic risk factors, central venous lines, treatment protocols, and anti-leukemic drugs in a logistic regression model, only the concomitant Escherichia coli asparaginase/prednisone administration in leukemic children suffering from a prothrombotic risk factor was found to increase the rate of thrombotic manifestations during leukemia treatment in patients of the same Caucasian origin (OR/95% CI: 34.5/4.39-271.42; P=.0008) . Based on the data presented here, we suggest the use of prednisone and E . coli asparaginase concomitantly administered in a leukemic patient suffering from a prothrombotic risk factor to be responsible for the onset of venous thrombosis in the majority of cases.

Cell, 2001 Oct 19, 107(2), 235 - 46
GroEL/GroES-mediated folding of a protein too large to be encapsulated; Chaudhuri TK et al.; The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered . We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria . We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release . Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution . Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.

Biochem J, 2001 Nov 1, 359(Pt 3), 507 - 16
Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases; Jowsey IR et al.; GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date . Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography . The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2) . Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities . The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme . Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively) . Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences . The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow . Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species . The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

Biochem J, 2001 Nov 1, 359(Pt 3), 497 - 505
Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A; Himpel S et al.; Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr) . In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2 . Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells . Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated . When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321 . A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A . MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A . Enzymic activity was not affected in the DYRK1A-Y111F mutant . The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Inorg Chem, 1997 May 7, 36(10), 2079 - 2083
Methyl Transfer to Mercury Thiolates: Effects of Coordination Number and Ligand Dissociation; Wilker JJ et al.; The complexes {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} and {(C(4)H(9))(4)N}{Hg(SC(6)H(5))(3)} demethylate (CH(3)O)(3)PO as revealed by (1)H, (31)P{(1)H}, and (199)Hg{(1)H} NMR spectroscopy in DMSO-d(6) solution . The products of the {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} reaction are CH(3)SC(6)H(5), (CH(3)O)(2)PO(2)(-), and {Hg(SC(6)H(5))(3)}(-), whereas {Hg(SC(6)H(5))(3)}(-) demethylates (CH(3)O)(3)PO to yield CH(3)SC(6)H(5) and {Hg(SC(6)H(5))(2){(CH(3)O)(2)PO(2)}}(-) . Kinetic and solution studies of {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} reveal a rapid equilibrium between bound and free thiolate . The dissociated thiolate is the nucleophile active toward (CH(3)O)(3)PO . These results imply that the metal center of the inactive mercury derivative of the Escherichia coli Ada DNA alkylation repair protein may comprise a three-coordinate {Hg(S-cysteine)(3)}(-) moiety and an unbound, protonated cysteine (HS-Cys69).

Inorg Chem, 1997 Mar 12, 36(6), 969 - 978
Alkyl Transfer to Metal Thiolates: Kinetics, Active Species Identification, and Relevance to the DNA Methyl Phosphotriester Repair Center of Escherichia coli Ada; Wilker JJ et al.; The Ada protein of Escherichia coli employs a {Zn(S-cys)(4)}(2)(-) site to repair deoxyribonucleic acid alkyl phosphotriester lesions . The alkyl group is transferred to a cysteine thiolate in a stoichiometric reaction . We describe a functional model for this chemistry in which a thiolate of {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} accepts a methyl group from (CH(3)O)(3)PO . The thiolate salt (CH(3))(4)N(SC(6)H(5)) is also active in methyl transfer, but the thiol C(6)H(5)SH fails to react . Conductivity measurements and kinetic studies demonstrate that {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} forms ion pairs in dimethyl sulfoxide (DMSO) solution (K(IP) = 13 +/- 4 M(-)(1)) which exhibit diminished reactivity . The reaction of {Zn(SC(6)H(5))(4)}(2)(-) with (CH(3)O)(3)PO is first order with respect to each reagent . A second-order rate constant for this reaction, k(Zn), was determined to be (1.6 +/- 0.3) x 10(-)(2) M(-)(1) s(-)(1) . From kinetic data and equilibria studies, all reactivity of {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} toward (CH(3)O)(3)PO could be attributed to dissociated thiolate . Metal complexes representing alternative protein sites were prepared and displayed the following kinetic trend of methyl transfer ability: {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} > {(CH(3))(4)N}(2){Co(SC(6)H(5))(4)} approximately {(CH(3))(4)N}(2){Cd(SC(6)H(5))(4)} > {(CH(3))(4)N}{Zn(SC(6)H(5))(3)(MeIm)} > {Zn(SC(6)H(5))(2)(MeIm)(2)}, where MeIm = 1-methylimidazole . These results are consistent with a dissociated thiolate being the active species and suggest that a similar mechanism might apply to alkyl phosphotriester repair by Ada.

Biochemistry, 2001 Oct 30, 40(43), 13079 - 87
Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from Mn-substituted FeSOD and its very high E(m); Vance CK et al.; Fe and Mn are both entrained to the same chemical reaction in apparently superimposable superoxide dismutase (SOD) proteins . However, neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active . We have proposed that the two SOD proteins must apply very different redox tuning to their respective metal ions and that tuning appropriate for one metal ion results in a reduction potential (E(m)) for the other metal ion that is either too low (Fe) or too high (Mn) {Vance and Miller (1998) J . Am . Chem . Soc . 120, 461-467} . We have demonstrated that this is true for Fe-substituted MnSOD from Escherichia coli and that this metal ion-protein combination retains the ability to reduce but not oxidize superoxide . We now demonstrate that the corollary is also true: Mn-substituted FeSOD {Mn(Fe)SOD} has a very high E(m) . Specifically, we have measured the E(m) of E . coli MnSOD to be 290 mV vs NHE . We have generated Mn(Fe)SOD and find that Mn is bound in an environment similar to that of the native (Mn)SOD protein . However, the E(m) is greater than 960 mV vs NHE and much higher than MnSOD's E(m) of 290 mV . We propose that the different tuning stems from different hydrogen bonding between the proteins and a molecule of solvent that is coordinated to the metal ion in both cases . Because a proton is taken up by SOD upon reduction, the protein can exert very strong control over the E(m), by modulating the degree to which coordinated solvent is protonated, in both oxidation states . Thus, coordinated solvent molecules may have widespread significance as "adapters" by which proteins can control the reactivity of bound metal ions.

Biochemistry, 2001 Oct 30, 40(43), 13068 - 78
Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri; Gencic S et al.; Methyl group transfer reactions are essential in methane-forming pathways in all methanogens . The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines . Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2 . Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations . Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+) . Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity . Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center . Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm . EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine . Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate . UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond . Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex . Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found . It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

Biochemistry, 2001 Oct 30, 40(43), 13060 - 7
Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase; Chih HW et al.; Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase . The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding . This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product . Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction . The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate . The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction . The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.

Biochemistry, 2001 Oct 30, 40(43), 13051 - 9
Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator Complex; Braun TF et al.; Bacterial flagella are turned by rotary motors that obtain energy from the membrane gradient of protons or sodium ions . The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes that contain multiple copies of each protein . The complexes conduct ions across the membrane, and couple ion flow to motor rotation by a mechanism that appears to involve conformational changes {Kojima, S., and Blair, D . F . (2001) Biochemistry 40, 13041-13050} . Structural information on the MotA/MotB complex is very limited . MotA has four membrane-spanning segments, and MotB has one . We have begun a targeted disulfide-cross-linking study to probe the arrangement of membrane segments in the MotA/MotB complex, beginning with the single membrane segment of MotB . Cys residues were introduced in 21 consecutive positions in the segment, and disulfide cross-linking was studied in MotA/MotB complexes either in membranes or detergent solution . Most of the Cys-substituted MotB proteins formed disulfide-linked dimers in significant yield upon oxidation . The yield of dimer varied regularly with the position of the Cys substitution, following the pattern expected for a parallel, symmetric dimer of alpha-helices . In a structural model based on the cross-linking experiments, critical Asp32 residues that are believed to facilitate proton movement are positioned on separate surfaces of the MotB dimer and so probably function within two distinct proton channels . Regions accessible to solvent were mapped by measuring the reactivity of introduced Cys residues toward N-ethyl maleimide and a charged methanethiosulfonate reagent . Positions near the middle of the segment were inaccessible to sulhydryl reagents . Positions within 6-8 residues of either end, which includes residues around Asp32, were accessible.

Biochemistry, 2001 Oct 30, 40(43), 13015 - 9
The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli; Sahin-Toth M et al.; Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety {Sahin-Toth, M., Akhoon, K . M., Runner, J., and Kaback, H . R . (2000) Biochemistry 39, 5097-5103} . To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-{(14)C}ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site . NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM . A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca . 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM) . Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM) . No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6 . In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM) . The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions . Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides.

Biochemistry, 2001 Oct 30, 40(43), 12967 - 73
Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies; Mani RS et al.; Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini . This study represents the first systematic examination of the physical properties of this enzyme . The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities . The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538 . The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer . The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies . The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A . These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51 . Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix . Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy.

Biochemistry, 2001 Oct 30, 40(43), 12904 - 12
Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase; Zheng R et al.; Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants . Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E . coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa . Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP . Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1) . The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of {(18)O}AMP produced from (18)O transfer using {carboxyl-(18)O}pantoate . Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction . These data also suggest that both half-reactions are partially rate-limiting . Reverse isotope exchange of {(14)C}-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.

Biochemistry, 2001 Oct 30, 40(43), 12896 - 903
Synthesis and biochemical characterization of a phosphorylated analogue of the response regulator CheB; Saxl RL et al.; CheB is a response regulator protein in the bacterial chemotaxis two-component signal transduction pathway . Methylesterase CheB functions together with methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli . The level of glutamate methylation in turn indirectly controls the direction of flagellar rotation . Like most two-component response regulators, CheB is activated in vivo by phosphorylation of a single aspartate, Asp 56, in its regulatory domain . Extensive biochemical and crystallographic studies have been completed on the inactive, unphosphorylated form of CheB . Because of the inherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isolated for further characterization . We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and chemical modification strategies . Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB . The unique cysteine in the substituted form of CheB was modified by sodium thiophosphate, Na(3)SPO(3), using two sequential disulfide bond exchange reactions . The analogue, D56C/C207S/C309S CheB-SPO(3), contained a thiophosphate group covalently bonded to the protein through a disulfide linkage at residue 56 . Mass spectrometry showed that the protein was singly modified . Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that the analogue had a half-life of 28 days . In in vitro methylesterase assays in the presence of Mg(2+), the analogue exhibited activity equivalent to that of fully phosphorylated C207S/C309S CheB . Thus, D56C/C207S/C309S CheB-SPO(3) is a stable analogue that may be useful for characterization of the active form of CheB.

Biochemistry, 2001 Oct 30, 40(43), 12826 - 32
Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of Trp457; Aoyagi M et al.; To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS(ox); residues 1-498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) binding site located at the dimer interface and in supporting H(4)B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS(ox) dimers (residues 66-498) . In W457F iNOS(ox), all the important hydrogen-bonding and aromatic stacking interactions that constitute the H(4)B binding site and that bridge the H(4)B and heme sites are preserved . In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H(4)B . Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H(4)B binding segment (Ser112-Met114), which might indicate reduced stability of the Trp457 mutant dimers . The Trp457 mutant structures show decreased pi-stacking with bound pterin when the wild-type pi-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A . The reduced pterin pi-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H(4)B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous-dioxy (Fe(II)O(2)) species during catalysis . These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H(4)B binding site and of H(4)B-bound dimers, and in influencing the rate of electron transfer between H(4)B and heme in NOS catalysis.

Biochemistry, 2001 Oct 30, 40(43), 12808 - 18
Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential; Murray JM et al.; Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme . The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms . One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ . To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine . We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme . Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F . Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis . The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ . Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate . At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F . Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step . Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen . The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site.

Biochemistry, 2001 Oct 30, 40(43), 12801 - 7
Folding determinants of LDL receptor type A modules; Koduri V et al.; To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5) . In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other . LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present . Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered . These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold.

Biochemistry, 2001 Oct 30, 40(43), 12772 - 81
Structure of beta-ketoacyl-{acyl carrier protein} reductase from Escherichia coli: negative cooperativity and its structural basis; Price AC et al.; The structure of beta-ketoacyl-{acyl carrier protein} reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit . The comparison of the E . coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein . This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer . FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP) . NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG . Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.

Biochemistry, 2001 Oct 30, 40(43), 12761 - 71
(15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant; Assfalg M et al.; The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy . The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed . (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant . In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame {Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C . M., and Barker, P . D . (2000) Biochemistry 39, 1499-1514} . The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species . The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system . Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2 . Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein . The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species . A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562) . In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing . The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant . It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.

Plant Mol Biol, 2001 Nov, 47(4), 519 - 31
Gene isolation and characterization of two acyl CoA oxidases from soybean with broad substrate specificities and enhanced expression in the growing seedling axis; Agarwal AK et al.; The first committed step in the beta-oxidation of fatty acids is catalyzed by the enzyme acyl-CoA oxidase (ACOX), which oxidizes a fatty acyl-CoA to a 2-trans-enoyl-CoA . To understand the role of beta-oxidation during seedling growth in soybean, two ACOX cDNAs were isolated by screening a seedling library with a DNA fragment obtained by RT-PCR by using degenerate oligonucleotides . The two cDNAs (ACX1;1 and ACX1;2) are 86% identical to each other at the nucleotide and the amino acid level . Their deduced amino acid sequences share significant homology with known acyl-CoA oxidases, including the conserved CGGHGY motif, a putative flavin mononucleotide binding site . In both sequences, the last three amino acids, ARL, represent a putative peroxisome targeting signal . The mRNA and protein of both cDNAs accumulated in all seedling tissues, with relatively stronger expression in the growing seedling axis and hypocotyl, and weaker expression in the cotyledon . Immunolocalization studies indicated that the two proteins were localized in the phloem cells of hypocotyl tissue . The two cDNAs were expressed in Escherichia coli and shown to possess acyl-CoA oxidase activity . With fatty acyl-CoA substrates of varying chain lengths, it was demonstrated that both ACX1;1 and ACX1;2 have broad substrate specificities (C8-C18) . The stronger expression of ACX1;1 and ACX 1;2 in the axis and hypocotyl tissue, the weaker expression in the oil-rich cotyledon tissue, and the broad substrate specificities suggest that the two acyl-CoA oxidases might play a general house-keeping role during soybean seedling growth, such as the turnover of membrane lipids.

Comb Chem High Throughput Screen, 2001 Nov, 4(7), 545 - 52
Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology; Amery L et al.; Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S . Biochem . J., 1999, 340, 561-568) . To identify other unknown peroxisomal enzymes, we further exploited this promising approach . Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1) . Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family . However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate . The recombinant protein interacted with HsPex5p, the human PTS1-binding protein . Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted . Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1 . These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.

Biotechnol Bioeng, 2001 Nov, 76(3), 187 - 92
Towards the development of a minimal cell model by generalization of a model of Escherichia coli: use of dimensionless rate parameters; Browning ST et al.; A model of a minimal cell would be a valuable tool in identifying the organizing principles that relate the static sequence information of the genome to the dynamic functioning of the living cell . Our approach for developing a minimal cell model is to first generalize an existing model of Escherichia coli by expressing reaction rates as ratios to a set of reference parameters . This generalized model is a prototype minimal cell model that will be developed by adding detail to explicitly include each chemical species . We tested the concept of a generalized model by testing the effect of scaling all enzyme-catalyzed reactions in the E . coli model . The scaling has little effect on cellular function for a wide range of kinetic ratios, where the kinetic ratio is defined as the rate of all enzyme-catalyzed reactions in a given model relative to those in the E . coli model .

Biotechnol Bioeng, 2001 Nov 20, 75(4), 492 - 4
Thiosulphate improves yield of hydrogen production from glucose by the immobilized formate hydrogenlyase system of Escherichia coli; Nandi R et al.; Escherichia coli cells with formate hydrogenlyase activity that were immobilized in agar beads produced hydrogen from glucose at the approximate yield of 0.6 mole of hydrogen per mole . Succinate or thiosulphate added to glucose increased hydrogen production two-fold . Thiosulphate did not increase the rate of hydrogen production but reduced the consumption of glucose for the same amount of hydrogen produced compared to control . Oxaloacetate and traces of pyruvate instead of succinate accumulated at the end when thiosulphate was present .

Biotechnol Bioeng, 2001 Nov 20, 75(4), 439 - 50
Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli; DeLisa MP et al.; Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E . coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes . In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E . coli . Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling . Importantly, our findings demonstrate for the first time that recombinant E . coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway .

Biotechnol Bioeng, 2001 Nov 20, 75(4), 387 - 92
The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products; Chamsart S et al.; Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy . Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied . Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer . Comparison has been made with unstressed material subjected to similar holding times . These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained . These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification .

Semin Thromb Hemost, 2001 Oct, 27(5), 437 - 43
Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide; Naggi A et al.; A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described . This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis . The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation . Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds . The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin.

Pharmacogenetics, 2001 Oct, 11(7), 609 - 17
Polymorphism of human Alpha class glutathione transferases; Tetlow N et al.; The recognition of the importance and utility of single nucleotide polymorphisms has generated an interest in the development of new strategies for their identification . Analysis of the Expressed Sequence Tag (EST) database can provide a rapid and efficient means of identifying polymorphisms . Screening of the Alpha class glutathione transferases (GSTs) in the EST database identified 10 putative polymorphisms in the coding region of the GSTA1 and GSTA2 genes, six of which were subsequently verified by sequence analysis . Polymerase chain reaction/restriction fragment length polymorphism analysis revealed the existence of three variants, a silent base substitution, K125K (G365A) in GSTA1, and T112S and E210A in GSTA2, in European Australian, African and Chinese populations . The variant isoforms of GSTA2 were expressed in Escherichia coli, purified, and enzymatically characterized . Modelling of the two GSTA2 polymorphisms into a three-dimensional structure of GSTA2, and characterization of their enzymatic properties, has shown that the structure and function of the wild-type GSTA2-2 isoenzyme is not significantly altered by these polymorphisms . This report demonstrates that analysis of the EST database provides a rapid and efficient means of identifying variant proteins.

Pharmacogenetics, 2001 Oct, 11(7), 597 - 607
Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid; Dai D et al.; Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol) . It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney . In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8 . CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians . Neither occurred in Asians . The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians . CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed . Recombinant CYP2C8*3 was defective in the metabolism of both substrates . The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1 . CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1 . CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1) . Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid . This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.

J Vet Med B Infect Dis Vet Public Health, 2001 Sep, 48(7), 501 - 12
Sows intramammarily inoculated with Escherichia coli influence of time of infection, hormone concentrations and leucocyte numbers on development of disease; Magnusson U et al.; The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland . Ten cross-bred primiparous sows were intramammarily inoculated with living E . coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition . Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made . All seven sows inoculated close to parturition developed a rectal temperature of >39.5 degrees C during the first 48 h post-partum and two of them also showed other signs of clinical disease . In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5 degrees C during the first 48 h post-partum and none of them showed any other sign of clinical discase . There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition . There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol-17beta and 15-ketodihydro-PGF2alpha before inoculation . Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition . In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group . The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of chinical coliform mastitis in the sow.

Appl Biochem Biotechnol, 2001 Jul, 95(1), 23 - 30
Construction, expression, and characterization of recombinant hirudin in Escherichia coli; Bi Q et al.; The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli . Many elements that could affect its expression level were compared . The product was purified to homogeneity via three chromatographic steps--ion exchange, gel filtration, and reverse phase chromatography--on the AKTA Explorer System . The anti-thrombin activity of HV2-K47 is much higher than that of recombinant HV2 . Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins.

Methods Enzymol, 2002, 345, 127 - 40
Expression, purification, and assay of cytosolic (catalytic) domains of membrane-bound mammalian adenylyl cyclases; Hatley ME et al.; The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes . They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha . They have served as critical reagents for the development of plausible models of the catalytic mechanism of the enzyme . They have enabled X-ray crystallographic analysis of adenylyl cyclase; this technique was not approachable with the small quantities of the membrane-bound enzyme available previously . The information obtained by using the soluble domains of adenylyl cyclase has provided templates for description of the behavior of many forms of purine nucleotide cyclases from a variety of species . We now appreciate both adenylyl cyclases and guanylyl cyclases as dimeric enzymes with a 2-fold symmetrical domain arrangement (or pseudosymmetrical in the case of heterodimerization) . The active sites are located at the interface between the two domains, both of which contribute binding surfaces.

Biol Pharm Bull, 2001 Oct, 24(10), 1102 - 7
Thermostability of doubly glycosylated recombinant lysozyme; Hashimoto Y et al.; We prepared a lysozyme mutant (Q41S/R61S) introducing Asn-type glycosylation signal sites by yeast expression system . On purification by cation exchange column at pH 7, three fractions were obtained . Peptide mapping and mass-spectrometry showed the fractions were the derivatives glycosylated at both Asn39 and Asn59, at only Asn39, and not glycosylated . It was revealed that the processing of Asn-linked oligosaccharide at Asn39 and Asn59 occurred independently in yeast cells . The denaturation temperatures of these derivatives by differential scanning calorimetry were 76.0, 68.8, and 67.5 degrees C at pH 3, respectively . The stabilization of glycosylated lysozyme depends on the degree of glycosylation . We concluded that stabilized proteins can be constructed by glycosylation at proper sites . Thermodynamic stabilization by the artificial double glycosylations on a protein has not yet been reported.

Genetika, 2001 Aug, 37(8), 1055 - 62
{Features of functioning of the promoter of microcin C51 promoter under various conditions of Escherichia coli cell growth}; Veselovskii AM et al.; The level of transcription from the promoter of the microcin C51 operon (Pmcc) depends on the growth phase of Escherichia coli cells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase . The functioning of Pmcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lac operon . A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the Pmcc promoter at the exponential phase of growth; the expression of Pmcc-lac during cell entry into the stationary phase was higher under unfavorable medium conditions . The use of composite rich media impaired this feature . The addition of l-leucine (100 micrograms/ml) to the medium decreased the expression of Pmcc-lac in wild-type cells carrying the delta lrp mutation . A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from Pmcc during cell entry into the stationary growth phase . The capacity of the Pmcc promoter and of the wild-type lacZ gene promoter was virtually the same upon IPTG induction . A mutation in the ompR gene did not markedly influence transcription started from Pmcc.

Bioorg Khim, 2001 Sep-Oct, 27(5), 364 - 71
{The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker}; Skosyrev VS et al.; Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed . The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins . These proteins were purified to homogeneity and subjected to limited trypsinolysis . It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules . The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers . Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases . The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.

J Ind Microbiol Biotechnol, 2001 Aug, 27(2), 94 - 103
Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity; Yu CL et al.; The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations . The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene . Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions . In addition, the position of oxidation and the enantiomeric composition of products were characterized . All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions . A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline . Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation . Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity . Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes . A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I) . The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO.

J Biol Chem, 2002 Jan 4, 277(1), 586 - 92 Epub 2001 Oct 18.
The activity of Arabidopsis glycosyltransferases toward salicylic acid, 4-hydroxybenzoic acid, and other benzoates; Lim EK et al.; Benzoates are a class of natural products containing compounds of industrial and strategic importance . In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring . These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned . A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites . Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed . The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid . Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases . All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups . Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring . To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid . The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring . This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants.

J Biol Chem, 2002 Jan 4, 277(1), 593 - 601 Epub 2001 Oct 18.
Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase; Moradpour D et al.; The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention . Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp . This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyprotein or subgenomic replicons . Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected . mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level . The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells . The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV . More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.

J Biol Chem, 2001 Dec 7, 276(49), 46225 - 9 Epub 2001 Oct 18.
Asymmetric recognition of DNA local distortion . Structure-based functional studies of eukaryotic Msh2-Msh6; Drotschmann K et al.; Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA . A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically . A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer . We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair . Purified Msh2-Msh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity . Moreover, wild-type Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond . The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone contacts . The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation.

J Biotechnol, 2001 Dec 28, 92(2), 195 - 204
Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer; Stegmann E et al.; For the first time gene cloning systems have been developed for Amycolatopsis japonicum . Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A . japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer . The direct transformation procedure was modified to introduce DNA . The most important parameter for an efficient DNA uptake was the age of the culture . Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies . Further, conditions for transformation of A . japonicum protoplasts were established . The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar . The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation . The plasmid was genetically stable, and could easily be reisolated from A . japonicum . We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible . The plasmid pSET152 integrated in the A . japonicum chromosome . A titre of 2.4 x 10(-4) exconjugants per recipient was obtained.

J Biotechnol, 2001 Dec 28, 92(2), 179 - 86
Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747; Ragnitz K et al.; The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized . Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A . aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60% . Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant . All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields . Therefore, the enzyme was highly purified and used in immobilization experiments . The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q . The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide . Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.

J Biotechnol, 2001 Dec 28, 92(2), 133 - 58
Modeling of inducer exclusion and catabolite repression based on a PTS-dependent sucrose and non-PTS-dependent glycerol transport systems in Escherichia coli K-12 and its experimental verification; Wang J et al.; We used genetically engineered sucrose positive Escherichia coli K-12 derivatives as a model system for the modeling and experimental verification of regulatory processes in bacteria . These cells take up and metabolize sucrose by the phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (Scr-PTS) . Expression of the scr genes, which cluster in two different operons (scrYAB and scrK), is negatively controlled by the ScrR repressor . Additionally, expression of the scrYAB operon, but not of the scrK operon is positively controlled by the cAMP-CRP complex . Modeling of sucrose transport and metabolism through the Scr-system and of the scr gene expression has been performed using a modular and object-orientated new approach . To verify the model and identify important model parameters we measured in a first set of experiments induction kinetics of the scr genes after growth on glycerol using strains with single copy lacZ operon fusions in the scrK or scrY genes, respectively . In a second set of experiments an additional copy of the complete scr-regulon was integrated into the chromosome to construct diplogenotic strains . Differences were observed in the induction kinetics of the cAMP-CRP-dependent scrY operon compared to the cAMP-CRP independent scrK operon as well as between the single copy and the corresponding diplogenotic strains.

Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 5046 - 50
Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold; Stallings WC et al.; 5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate . The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques . The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution . The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet . Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices . The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal . A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described . The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments . The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1446 - 50
Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants; Hanley-Bowdoin L et al.; The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (AL1) that is required for viral DNA replication . We have constructed transgenic Nicotiana benthamiana plants in which the AL1 open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter . The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-AL1 construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against AL1 protein overproduced in Escherichia coli . Six of nine transgenic lines complemented a TGMV A variant with a mutation in AL1 when coinoculated with the B component of the TGMV genome . Single- and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A . These results establish that the transgenic plants express functional AL1 protein and show that this viral protein is not only required, but sufficient, for single- and double-stranded replication of TGMV DNA in the presence of host proteins . These results also show that the AL1 protein is not by itself a determinant of disease or pathogenesis.

J Biol Chem, 2001 Dec 28, 276(52), 49371 - 7 Epub 2001 Oct 17.
The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity; Poplawski A et al.; The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication . The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme . The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type . In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli . The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme . The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity . These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.

Eur J Biochem, 2001 Oct, 268(20), 5430 - 8
Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans; Laurent V et al.; Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes . In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme . The C . elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues . The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa . Nematodes transformed with a promoter reporter construct, W03G9.4:GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression . yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa . The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide) . The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 microM), a selective inhibitor of mammalian AP-P . A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate . The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin . We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.

Eur J Biochem, 2001 Oct, 268(20), 5270 - 7
Liberation of the intramolecular interaction as the mechanism of heat-induced activation of HSP90 molecular chaperone; Tanaka E et al.; The molecular chaperone function of HSP90 is activated under heat-stress conditions . In the present study, we investigated the role of the interactions in the heat-induced activation of HSP90 molecular chaperone . The preceding paper demonstrated two domain-domain interactions of HtpG, an Escherichia coli homologue of mammalian HSP90, i.e . an intra-molecular interaction between the N-terminal and middle domains and an intermolecular one between the middle and C-terminal domains . A bacterial two-hybrid system revealed that the two interactions also existed in human HSP90alpha . Partners of the interaction between the N-terminal and middle domains of human HSP90alpha could, but those between the middle and C-terminal domains could not, be replaced by the domains of HtpG . Thus, the interface between the N-terminal and middle domains is essentially unvaried from bacterial to human members of the HSP90-family proteins . The citrate synthase-binding activity of HtpG at an elevated temperature was solely localized in the N-terminal domain, but HSP90alpha possessed two sites in the N-terminal and other domains . The citrate-synthase-binding activity of the N-terminal domain was suppressed by the association of the middle domain . The complex between the N-terminal and middle domains is labile at elevated temperatures, but the other is stable even at 70 degrees C . Taken together, we propose the liberation of the N-terminal client-binding domain from the middle suppressor domain is involved in the temperature-dependent activation mechanism of HSP90 molecular chaperone.

Eur J Biochem, 2001 Oct, 268(20), 5258 - 69
Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone; Nemoto TK et al.; In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 . Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e . Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha . A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions . Thus, HtpG consists of three domains, i.e . Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90 . The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions . As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer . Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C . The C-terminal two-thirds of domain A, i.e . Asp116-Arg336, were sufficient for the binding to domain B . We finally propose the domain organization of an HtpG dimer.

Res Microbiol, 2001 Sep, 152(7), 663 - 9
Amount and redox state of cytoplasmic, membrane and periplasmic proteins in Escherichia coli redox mutants; de Crouy-Chanel A et al.; We analyzed the amount and redox state of cytoplasmic, membrane and periplasmic proteins in Escherichia coli mutants deficient in thioredoxin, thioredoxin reductase, glutathione and DsbA, by observing the electrophoretic profile of bacterial extracts after in vivo labelling with monobromobimane . Our results show that these mutations affected not only the amount and the redox state of proteins localized in the same compartment as the deficient oxidoreductase, but also those of the proteins localized in other compartments . These results concord with the hypothesis that there is a link between the redox reactions that occur in the cytoplasm and the periplasm.

Anesthesiology, 2001 Oct, 95(4), 928 - 32
Ketamine inhibits endotoxin-induced shock in rats; Taniguchi T et al.; BACKGROUND: Cytokines and nitric oxide are believed to participate importantly in the pathogenesis of endotoxin-induced shock . Several investigators have documented that ketamine attenuates production of cytokines and nitric oxide in endotoxemia, but little is known concerning hemodynamic effects of the drug in this state . The objective of the current study was to assess the potential modifying effects of ketamine in endotoxemia . METHODS: The authors randomly assigned 40 rats to one of four equal groups: endotoxin alone, receiving Escherichia coli endotoxin (15 mg/kg, administered intravenously); saline control, receiving saline only; ketamine alone, receiving ketamine (10 mg x kg(-1) x h(-1), administered intravenously); pretreatment, with ketamine administration initiated before the endotoxin exposure; and posttreatment, with ketamine initiated 2 h after endotoxin . During the 5 h after endotoxin injection, hemodynamics, acid-base status, and plasma concentrations of tumor necrosis factor alpha and interleukin 6 were assessed in each group . RESULTS: Endotoxin injection produced progressive hypotension, metabolic acidosis, and a large increase in the plasma cytokine concentrations . This hemodynamic and cytokine responses to endotoxin were completely abolished in the pretreatment group and modestly suppressed in the posttreatment group . in the absence of endotoxin, ketamine did not modify these responses . CONCLUSION: Ketamine administration inhibited hypotension, metabolic acidosis, and cytokine responses in rats injected with endotoxin . The results suggest that judicious use of ketamine as an anesthetic agent may offer advantages in endotoxemia.

Chem Pharm Bull (Tokyo), 2001 Oct, 49(10), 1359 - 61
An antifungal cadinanolide from Pseudoelephantopus spicatus; Ragasa CY et al.; A new sesquiterpene lactone was obtained from the chloroform extract of Pseudoelephantopus spicatus . Its structure was elucidated by extensive one dimentional (1D) and 2D NMR spectroscopy and mass spectrometry . It was found to exhibit moderate antifungal activity against C albicans and A . niger, and low activity against T . mentagrophytes, S . aureus, E . coli and P . aeruginosa . It was inactive against B . subtilis.

Chem Pharm Bull (Tokyo), 2001 Oct, 49(10), 1299 - 303
Structural and thermodynamic behavior of eukaryotic initiation factor 4E in supramolecular formation with 4E-binding protein 1 and mRNA cap analogue, studied by spectroscopic methods; Shen X et al.; The structural and thermodynamic behavior of the complex formation of eIF4E with either or both mRNA cap analogue (m7GTP, m7GpppA, or m7GpppG) and 4EBP1 has been investigated by spectroscopic measurements . Although the circular dichroism (CD) spectrum of eIF4E was little affected by the association with any cap analogue, the association constant of eIF4E with m7GpppA/G, estimated from the fluorescence quenching, was about 10 times larger than that with m7GTP . The van't Hoff analyses showed that the m7GpppA/G binding is enthalpy-driven with a large negative deltaH(o), and this is in contrast with the entropy-driven binding of m7GTP, where the positive deltaS(o) is large enough to overcome an increase of deltaH(o) . This different behavior obviously originates in the interaction of the second nucleotide in m7GpppA with eIF4E, suggesting the importance of the nucleotide sequence linked to the m7Gppp terminal moiety, in addition to the specific interaction with the m7G base, for the recognition of mRNA cap structure by eIF4E . On the other hand, the CD spectra indicated that the binding of 4EBP1, an endogenous eIF4E-regulatory protein without having any defined secondary structure, shifted the m7GTP- or m7GpppA/G-bound eIF4E to an irregular structure, although such a structural change was not observed for eIF4E alone . The association constant of 4EBP1 with m7GTP- or m7GpppA/G-bound eIF4E was by two orders of magnitude larger than that with eIF4E alone . These results suggest the close interrelation in the supramolecular formation of 4EBP-eIF4E-mRNA cap structure.

Int J Oncol, 2001 Nov, 19(5), 1015 - 20
A novel treatment of human malignant gliomas in vitro and in vivo: FADD gene transfer under the control of the human telomerase reverse transcriptase gene promoter; Komata T et al.; Telomerase activity has a close relationship with malignancies in many cell types and it is tightly regulated at the transcriptional level of human telomerase reverse transcriptase (hTERT) . Utilizing the hTERT promoter, the authors developed a gene delivery system of Fas associated protein with death domain (FADD) (hTERT/FADD) . FADD is a protein which plays an important role in the apoptotic pathway of Fas . Over-expression of FADD induces apoptosis in the cells regardless of Fas expression on the cell surface . We hypothesized that we might be able to restrict the expression of FADD in malignant glioma cells if we use the gene transfer system under the control of hTERT promoter . This study was designed to investigate whether the hTERT/FADD construct induces apoptosis effectively in malignant glioma cells while keeping normal cells intact . First, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, we confirmed that hTERT mRNA was expressed in human malignant glioma cells (U373-MG, A172 and GB-1), but not in cultured astrocytes (TEN) or fibroblasts (MRC5) . After transient transfection with the hTERT/FADD construct, a significant number of FADD-positive cells and apoptotic cells were detected in hTERT-positive malignant glioma cells . In contrast, hTERT-negative astrocytes and fibroblasts remained intact . Furthermore, subcutaneously implanted U373-MG tumors treated with the hTERT/FADD construct reduced in volume significantly compared to the conrol treatment (p=0.0001) . Gene transfer of FADD under the control of the hTERT promoter may be a novel and promising therapy to kill hTERT-positive malignant glioma cells while sparing normal brain cells lacking hTERT.

Protein Sci, 2001 Nov, 10(11), 2228 - 40
Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations; Izadi-Pruneyre N et al.; The three-dimensional structure of apo-neocarzinostatin (apo-NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven-stranded antiparallel beta-sandwich, very similar to the immunoglobulin folding domain . We investigated the backbone dynamics of apo-NCS by (13)C-NMR relaxation measurements and molecular dynamics simulation . Model-free parameters determined from the experimental data are compared with a 1.5-nsec molecular simulation of apo-NCS in aqueous solution . This comparison provides an accurate description of both local and collective movements within the protein . This analysis enabled us to correlate dynamic processes with key interactions of this beta-protein . Local motions that could be relevant for the intermolecular association with the ligand are also described.

J Biol Chem, 2002 Jan 4, 277(1), 445 - 54 Epub 2001 Oct 16.
HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins; Verhagen AM et al.; Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac . We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin . Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP . HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs . HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death . The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity . Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.

Vet Immunol Immunopathol, 2001 Nov, 83(1-2), 115 - 22
Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs; Tang L et al.; Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses . However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited . In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc) . The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans . Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

Radiat Res, 2001 Nov, 156(5 Pt 1), 495 - 502
Effect of exposure to 900 MHz radiofrequency radiation on intrachromosomal recombination in pKZ1 mice; Sykes PJ et al.; Radiofrequency (RF) radiation emitted from mobile phones is not considered to be directly genotoxic, but it may have downstream effects on cellular DNA . We studied the effect of 4 W/kg pulsed 900 MHz RF radiation on somatic intrachromosomal recombination in the spleen in the pKZ1 recombination mutagenesis model . Somatic intrachromosomal recombination inversion events were detected in spleen tissue of pKZ1 mice by histochemical staining for E . coli beta-galactosidase protein in cells in which the lacZ transgene has undergone an inversion event . pKZ1 mice were exposed daily for 30 min to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms for 1, 5 or 25 days . Three days after the last exposure, spleen sections were screened for DNA inversion events . There was no significant difference between the control and treated groups in the 1- and 5-day exposure groups, but there was a significant reduction in inversions below the spontaneous frequency in the 25-day exposure group . This observation suggests that exposure to RF radiation can lead to a perturbation in recombination frequency which may have implications for recombination repair of DNA . The biological significance of a reduction below the spontaneous frequency is not known . The number of mice in each treatment group in this study was small (n = 10 or n = 20) . Therefore, repetition of this study with a larger number of animals is required to confirm these observations.

Acta Neuropathol (Berl), 2001 Oct, 102(4), 306 - 12
Cerebrovascular damage in young rabbits after intravenous administration of Shiga toxin 2; Mizuguchi M et al.; Acute encephalopathy associated with Shiga toxin-producing Escherichia coli (STEC) primarily affects children . To elucidate the age-dependent vulnerability of the central nervous system (CNS), we injected Shiga toxin 2 (Stx2) intravenously to young rabbits and examined the clinical and pathological effects on the CNS . Although neurological disorders caused by Stx2 were similar between young and adult rabbits, the dose required to produce them in the young was one third of that required for the adults . Vascular lesions appeared as early as 24 h after injection in the young, but not at all in the adult . Arteriolar changes, such as hydropic swelling of the endothelial cells and karyorrhexis of the medial cells, were specific to the CNS of young animals . Evidence for apoptosis of vascular cells was scarce because DNA strand breaks and activation of caspases-3 and -9 were absent in the vast majority . Given our results, we conclude that the cerebral blood vessels of immature brains are more vulnerable to Stx2 than those of adults in the rabbit.

Transplantation, 2001 Oct 15, 72(7), 1237 - 40
Defibrotide for the treatment of veno-occlusive disease after liver transplantation; Mor E et al.; BACKGROUND: Veno-occlusive disease (VOD) after liver transplantation is associated with acute rejection and poor outcome . The use of antithrombotic and thrombolytic agents is limited by their toxicity . Defibrotide is a polydeoxyribonucleotide with thrombolytic and antithrombotic properties and no systemic anticoagulant effect . METHODS: Defibrotide, 35-40 mg/kg/day, was administered intravenously for 21 days on a compassionate-use basis to two patients aged 66 and 49 years . VOD had developed 6 weeks and 4 months after orthotopic liver transplantation for hepatitis C and hepatitis B infection, respectively . VOD was diagnosed clinically by findings of weight gain (8.5% and 16%), ascites, jaundice (serum bilirubin 5.4 mg/dl and 21.7 mg/dl), and severe coagulopathy (in one patient), and histologically by the presence of hemorrhagic centrilobular necrosis and fibrous stenosis of the hepatic venules . One of the patients had received azathioprine as part of the immunosuppressive regimen . There was no evidence of acute cellular rejection histologically . RESULTS: After 3 weeks of defibrotide administration, the first patient showed complete clinical resolution of the VOD, and serum bilirubin level normalized . He is alive 6 months after transplantation . The second patient, treated at a later stage of disease, showed marked improvement in the coagulopathic state, but there was no resolution of the VOD . He died 2 months later of multiorgan failure due to Escherichia coli sepsis . Neither patient had side effects from the drug . CONCLUSIONS: Defibrotide is a promising drug for the treatment of VOD after liver transplantation and needs to be evaluated in large, prospective studies.

Glycoconj J, 2001 Mar, 18(3), 231 - 43
Different glycosphingolipid composition in human neutrophil subcellular compartments; Karlsson A et al.; The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay . Several organelle-related differences in glycosphingolipid content were observed . The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils . Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules . Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions . A non-acid triglycosylceramide, recognized by Gal(alpha)4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules . Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane . Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.

J Virol, 2001 Nov, 75(22), 10969 - 78
Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol); Gerber K et al.; The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase . We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol) . Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J . B . Flanegan and D . Baltimore, Proc . Natl . Acad . Sci . USA 74:3677-3680, 1977; A . V . Paul, J . H . van Boom, D . Filippov, and E . Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations . HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template . The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U) . The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU . This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands . The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction . Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

J Biol Chem, 2001 Dec 7, 276(49), 45505 - 8 Epub 2001 Oct 15.
The Phe-X-Glu DNA binding motif of MutS . The role of hydrogen bonding in mismatch recognition; Schofield MJ et al.; The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch . Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed . The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays . The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS . Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue . A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo . DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.

FEBS Lett, 2001 Oct 12, 506(3), 286 - 90
Identification of an Arabidopsis cDNA encoding a lipoyltransferase located in plastids; Wada M et al.; In plant cells, the pyruvate dehydrogenase (PDH) complex that requires lipoic acid as an essential coenzyme is located in plastids and mitochondria . The enzyme complex has to be lipoylated in both organelles . However, the lipoyltransferase located in plastids has not been reported . In this study, an Arabidopsis thaliana LIP2p cDNA for a lipoyltransferase located in plastids has been identified . We have shown that this cDNA encodes a lipoyltransferase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoyltransferase activity, and that LIP2p is targeted into chloroplasts . These findings suggest that LIP2p is located in plastids and responsible for lipoylation of the plastidial PDH complex.

Zhonghua Gan Zang Bing Za Zhi, 2001 Aug, 9(4), 223 - 5
{Studies on mimotopes of hepatitis C virus E1 protein}; Cao F et al.; OBJECTIVE: To study the B-cell epitope of E1 protein of hepatitis C virus . METHODS: By induction of IPTG, the E.coli M15 strains harboring the pQE30-HCVe118 expressed truncated C-terminal HCV E1 protein (Pte1) . The proteins were purified with preparative electrophoreses system, which captured anti-E1 IgG in HCV (+) sera . By applying the antibodies as selective molecular 12 mers random peptide libraries were panned, and positive clones were obtained by ELISA . Amino acid sequences of display peptide were compared with that of HCV E1 protein . RESULTS: The purified HCV E1 proteins could react specifically with partly anti-HCV sera by ELISA . Among 10 phage display peptides, 6, 6, 2 were the most homologous to HCV E1 protein at position 320-336aa, 251-263aa, 225-248, respectively . CONCLUSIONS: There exist multiple B-cell epitopes in HCV E1 protein . At least one preponderant epitope is mapped at residues 320-336 of HCV E1 protein.

Biochemistry, 2001 Oct 23, 40(42), 12562 - 74
Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state; Lebedev N et al.; Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli . The results obtained with an individual stereoisomer of the substrate {C8-ethyl-C13(2)-(R)-protochlorophyllide} demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site . The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH) . Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme . At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation . Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme . In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide . We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.

Biochemistry, 2001 Oct 23, 40(42), 12497 - 504
Dehydration is catalyzed by glutamate-136 and aspartic acid-135 active site residues in Escherichia coli dTDP-glucose 4,6-dehydratase; Gross JW et al.; The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and rereduction . The enzyme contains the tightly bound coenzyme NAD(+), which mediates the dehydrogenation and rereduction steps of the reaction mechanism . In this study, we have determined that Asp135 and Glu136 are the acid and base catalysts, respectively, of the dehydration step . Identification of the acid catalyst was performed using an alternative substrate, dTDP-6-fluoro-6-deoxyglucose (dTDP-6FGlc), which undergoes fluoride ion elimination instead of dehydration, and thus does not require protonation of the leaving group . The steady-state rate of conversion of dTDP-6FGlc to dTDP-4-keto-6-deoxyglucose by each Asp135 variant was identical to that of wt, in contrast to turnover using dTDP-glucose where differences in rates of up to 2 orders of magnitude were observed . These results demonstrate Asp135's role in protonating the glucosyl-C6(OH) during dehydration . The base catalyst was identified using a previously uncharacterized, enzyme-catalyzed glucosyl-C5 hydrogen-solvent exchange reaction of product, dTDP-4-keto-6-deoxyglucose . Base catalysis of this exchange reaction is analogous to that occurring at C5 during the dehydration step of net catalysis . Thus, the decrease in the rate of catalysis ( approximately 2 orders of magnitude) of the exchange reaction observed with Glu136 variants demonstrates this residue's importance in base catalysis of dehydration.

J Mol Biol, 2001 Oct 12, 313(1), 99 - 109
Structure of a variant of lac repressor with increased thermostability and decreased affinity for operator; Bell CE et al.; A single amino acid substitution, K84L, in the Escherichia coli lac repressor produces a protein that has substantially increased stability compared to wild-type . However, despite the increased stability, this altered tetrameric repressor has a tenfold reduced affinity for operator and greatly decreased rate-constants of inducer binding as well as a reduced phenotypic response to inducer in vivo . To understand the dramatic increase in stability and altered functional properties, we have determined the X-ray crystal structures of a dimeric repressor with and without the K84L substitution at resolutions of 1.7 and 3.0 A, respectively . In the wild-type dimer, K84-11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent . In the K84L-11 substituted protein there is reorientation of the N-subdomains, which allows the leucine to become deeply buried at the monomer-monomer interface . This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion pairing, and van der Waals interactions at the monomer-monomer interface . The lysine residue at position 84 appears to exert its key effects by destabilizing the "optimal" conformation of the repressor, effectively loosening the dimer interface and allowing the repressor to adopt the conformations necessary to function as a molecular switch .

J Mol Biol, 2001 Oct 12, 313(1), 1 - 12
MicF: an antisense RNA gene involved in response of Escherichia coli to global stress factors; Delihas N et al.; The micF gene is a stress response gene found in Escherichia coli and related bacteria that post-transcriptionally controls expression of the outer membrane porin gene ompF . The micF gene encodes a non-translated 93 nt antisense RNA that binds its target ompF mRNA and regulates ompF expression by inhibiting translation and inducing degradation of the message . In addition, other factors, such as the RNA chaperone protein StpA also play a role in this regulatory system . Expression of micF is controlled by both environmental and internal stress factors . Four transcriptional regulators are known to bind the micF promoter region and activate micF expression . The crystal structure of one these transcriptional activators, Rob, complexed with the micF promoter has been reported . Here, we review new developments in the micF regulatory network .

J Food Prot, 2001 Oct, 64(10), 1510 - 4
Combined effect of water activity and pH on the inhibition of Escherichia coli by nisin; Cerrutti P et al.; The Doehlert design and surface response methodology were used to study the influence of pH and water activity (aw) on Escherichia coli inhibition by nisin . Combining stress factors at levels where they are not inhibitory by themselves, a reduction of E . coli survival fraction can be achieved with lower nisin doses than in a single nisin treatment . For all the pH values assayed, a synergistic effect of aw and nisin concentration was detected, and the isoresponse lines showed the existence of an area of maximum inhibition . Factors that reduced viable cell counts by 4 to 5 log cycles were 1,000 to 1,400 IU of nisin per ml at pH 5.5 to 6.5 and a water activity of 0.97 and 0.98 . The addition of different ionic and nonionic solutes to control aw suggested that the effect of aw in the inhibitory action of nisin on E . coli cells was not solute-specific . The use of the Doehlert experimental design was effective to determine the optimal combination of stress factors, as well as to point out the most important variables that affected E . coli inhibition.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 718 - 23
Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems; Guerrero SA et al.; The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids . Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems . As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M . tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here . Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme . Both proteins yield crystals under similar conditions . The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.

Chin Med J (Engl), 1999 Aug, 112(8), 691 - 7
A recombinant multi-epitope, multi-stage malaria vaccine candidate expressed in Escherichia coli; Li M et al.; OBJECTIVE: To construct and evaluate a recombinant multi-epitope, multistage malaria vaccine candidate expressed in Escherichia coli (E . coli) . METHODS: A hybrid gene (HGF) encoding several putative immunodominant T or T/B epitopes from MSP-1, MSP-2, Pf155/RESA of Plasmodium falciparum (P . falciparum) and two immune-stimulating epitopes from interleukin-1 and tetanus toxin was synthesized . Two copies of HGF and a copy of gene encoding Pattaroyo's Spf66 were connected together to construct a sandwich hybrid gene HGFSP . The gene was cloned into an expression vector pWR450-I for production of a fusion protein with beta-galactosidase . Efficacy of this vaccine candidate in inducing specific immunity against malaria parasites was evaluated . RESULTS: Immunization of different species of animals with purified recombinant peptide showed that the peptide was able to induce remarkable antibody response to the immunized peptide as well as falciparum malaria parasites . The epitopes included in the construct could induce antibodies against the intact parasite proteins as demonstrated by western blotting, indicating the epitopes retained their antigenicity in the new peptide construct . Antibodies from animals immunized with recombinant HGFSP peptide exhibited good ability in inhibition of the in vitro growth of malaria parasites, augmentation of phagocytosis of the parasites or infected RBC by phagocytes, and facilitation of antibody dependent cell mediated cytotoxicity to the cultured malaria parasites . CONCLUSION: The recombinant peptide seems to be a potential candidate which is valuable for further investigation.

Ugeskr Laeger, 2001 Oct 1, 163(40), 5541 - 2
{Hemorrhagic diarrhea with competing etiologies}; Jacobsen N et al.; A case history of a 41-year-old woman with bloody diarrhoea is described . A right-sided hemicolectomy was performed and histological findings showed pseudomembranous colitis . The woman was infected with verotoxin-producing E . coli O103:H2 . She had taken an overdose of ergotamine and was using a contraceptive containing oestrogen . Each of these have previously been described as being the cause of bloody diarrhoea, but we suggest the combination as the aetiology in the present case.

Nucleic Acids Res . 2001 Oct 15;29(20):E98.
Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro; Ferguson HA et al.; c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation . To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro . Full-length human c-Fos and c-Jun were expressed in Escherichia coli . The expression of c-Fos was dependent on a helper plasmid that encodes rare (Arg)tRNAs . Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies . A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos . The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system . The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators.

Nucleic Acids Res, 2001 Oct 15, 29(20), 4166 - 78
UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker; Meng W et al.; The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) . We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination . Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription . Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element . Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element . Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

J Biol Chem, 2001 Dec 21, 276(51), 47930 - 6 Epub 2001 Oct 12.
Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling; Lee EY et al.; It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition . To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis . More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP . The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-{gamma-(32)P}azidoguanosine 5'-triphosphate (8-N(3)-GTP) . Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-{gamma-(32)P}GTP was significantly decreased in the presence of 300 microm GTP . Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP . The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH . Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP . These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.

J Biol Chem, 2001 Dec 14, 276(50), 47029 - 37 Epub 2001 Oct 12.
Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB; Schaeffer ML et al.; Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents . Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors . In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M . tuberculosis FASII system . KasA and KasB were expressed in E . coli and purified by affinity chromatography . Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP . Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons . Activity of KasA and KasB increased with use of M . tuberculosis AcpM, suggesting that structural differences between AcpM and E . coli ACP may affect their recognition by the enzymes . Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin . These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

Med Microbiol Immunol (Berl), 2001 Sep, 189(4), 225 - 9
Antibodies to Lassa virus Z protein and nucleoprotein co-occur in human sera from Lassa fever endemic regions; Gunther S et al.; It is not known whether the small 11-kDa Z protein of Lassa virus is immunogenic during human Lassa virus infection . To obtain evidence for the existence of an antibody response and to test the suitability of these antibodies for serosurveys, sera from Lassa fever endemic regions (Guinea and Nigeria, n = 75) were tested for co-reactivity to Z protein and nucleoprotein (NP) . Sera from a non-endemic region (Uganda, n = 50) served as a specificity control . Z protein and NP were expressed in Escherichia coli, affinity-purified, and used as antigen in Western blot . Indirect immunofluorescence (IIF) with Lassa virus-infected cells was performed for comparison . Due to high unspecific reactivity of the African sera, Western blot testing was performed with a 1:1,000 serum dilution . Under these conditions, none of the control sera but 12% of the sera from endemic regions co-reacted with both Z protein and NP . Reactivity to Z protein was significantly associated with NP reactivity (P < 10(-6)) . NP and Z protein-specific antibodies were co-detected in 33% of the IIF-positive sera and in 5% of the IIF-negative sera (P = 0.001) . These data provide evidence for appearance of antibodies to Z protein and NP following Lassa virus infection . A recombinant blot for detection of both antibody specificities seems to be specific but less sensitive than IIF.

Int J Legal Med, 2001 Aug, 115(1), 16 - 22
Trajectory reconstruction from trace evidence on spent bullets . I . Deposits from intermediate targets; Karger B et al.; Contact of a fired bullet with an intermediate target of sufficient resistance commonly causes the bullet to ricochet, fragment or perforate together with a deviation in trajectory . The transfer of intermediate target material to bullets and subsequent detection on the bullet surface after recovery from a bullet collector, were examined using a scanning electron microscope and an energy-dispersive X-ray spectrometer (SEM/EDS) . A total of 76 gunshots (9 mm Luger FMJ RN bullets) were fired at various intermediate targets and at combinations of intermediate targets and tissue located in line . Elements already present on unfired bullets and elements from the bullet collector, the jacket, the charge and primer could be consistently detected as a "background" . Abundant deposits of "fragile" (brittle) materials such as concrete, flat glass, asphalt and gypsum board could be visualised on every bullet by SEM . The transfer dynamics involved a direct imprint of target material on the bullet surface and thus preferential locations at the tip but also indirect deposition over the entire surface ("powder effect") . X-ray microanalysis demonstrated matching spectra of the elemental composition of these deposits and of the targets contacted . After perforation of "ductile" (flexible) materials such as wood and car body parts, the deposits on the bullets did not show characteristic spectra . If multi-layered car metal targets were hit, few and uncharacteristic fragments were scattered over the bullet surface and titanium indicative of paint-work could be determined on only a minority of bullets . The elemental composition of wood itself was heterogeneous but the fibrous morphology of the deposits was typical . The SEM/EDS findings in gunshots including subsequent perforation of tissue were similar . In particular, the trace evidence primarily transferred to the bullets was not eliminated by secondary contact and the determination of the fragile target materials was not affected . So when a person is killed or injured by a gunshot, the presence of a ricochet and the target material can be determined . This possibility needs to be considered before an evidential bullet is cleaned or contaminated.

Cell Stress Chaperones, 2001 Jul, 6(3), 201 - 8
Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana; Lin BL et al.; The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily . While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170 . Gene structure and genome organization suggest duplication in the origin of some genes . The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.

Ukr Biokhim Zh, 2001 Jan-Feb, 73(1), 24 - 8
{Effect of destruction of Escherichia coli cells on the catalytic ability of catalase}; Semchyshin HM et al.; The possibility for investigation of catalase (CAT) activity under the conditions of intact E . coli cells was estimated . This approach is based on the possibility of hydrogen peroxide freely cross biological membranes . CAT activity of native cells had a broad maximum between pH values 4.5 and 7.5 . Desintegration of cells by freezing--thawing and ultrasonication indicated that there were two CAT activity peaks at pH values about 3.5 and 7.0 . Activity of CAT with acid pH-optimum decreased at cell desintegration, but one with neutral pH-optimum was rather stable under this procedure . The enzyme in native conditions was less sensitive to the inhibition by high concentrations of hydrogen peroxide than its counterpart from destroyed cells . Activity of CAT in native and desintegrated cell preparations had different sensitivity to heating and inhibition by reduced glutathione, but it was inhibited by azide similarly . Difference in the CAT properties of native and desintegrated bacteria preparations may be explained by different possibility to penetrate cell membrane by reagents and/or by possible modification of the enzyme properties at destruction of native microenvironment.

Cell Mol Biol Lett, 2001, 6(3), 571 - 85
Studies of the erythrocyte spectrin tetramerization region; Park S et al.; Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers . We have prepared model peptides to study the tetramerization region . Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization . Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner . Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle . A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed . Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle . Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner . This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association . Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis . NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T . A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.

Science, 2001 Oct 12, 294(5541), 369 - 74
Observation of covalent intermediates in an enzyme mechanism at atomic resolution; Heine A et al.; In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments . Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates . Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism . In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished . A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

Plant Physiol, 2001 Oct, 127(2), 655 - 64
Multiple, distinct isoforms of sucrose synthase in pea; Barratt DH et al.; Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum) . The genes have distinct patterns of expression in different organs of the plant, and during organ development . Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties . Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms . Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1 . One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95% . The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected . The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.

J Biol Chem, 2001 Dec 14, 276(50), 47094 - 9 Epub 2001 Oct 11.
Phe71 is essential for chaperone-like function in alpha A-crystallin; Santhoshkumar P et al.; Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K . K., Kumar, R . S., Kumar, G . S., and Quinn, P . T . (2000) J . Biol . Chem . 275, 3767-3771) . In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein . There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins . However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein . Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments . Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins . The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase . However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included . These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin . Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.

Infect Immun, 2001 Nov, 69(11), 6951 - 61
Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor; Kirby AE et al.; Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor . Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide . Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans . Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR . Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR . A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR . When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell . Sigma competition experiments in E . coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity . Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system . Fur titration assays indicated that expression of rhuI was probably Fur dependent.

Infect Immun, 2001 Nov, 69(11), 6839 - 45
Cytotoxic necrotizing factor from Escherichia coli induces RhoA-dependent expression of the cyclooxygenase-2 Gene; Thomas W et al.; Cytotoxic necrotizing factor 1 (CNF) is a toxin produced by some isolates of Escherichia coli that cause extraintestinal infections . CNF can initiate signaling pathways that are mediated by the Rho family of small GTPases through a covalent modification that results in constitutive activation . In addition to regulating the assembly of actin stress fibers and focal adhesion complexes, RhoA can also regulate gene expression at the level of transcription . Here we demonstrate for the first time, by using a luciferase-based reporter system, that the transcription of cyclooxygenase-2 (COX-2) is strongly upregulated in NIH 3T3 fibroblasts treated with CNF and that this effect is dependent upon the activation of RhoA by the toxin . Subsequent protein tyrosine phosphorylation events modulate the induction, but the transcription signal is not mediated by Rho-associated kinase (p160/ROCK) and so must rely upon another effector that is activated by RhoA . CNF therefore induces COX-2 expression via a RhoA-dependent signaling pathway that diverges from the pathway that regulates cytoskeletal rearrangements in response to RhoA activation.

Infect Immun, 2001 Nov, 69(11), 6785 - 95
Type III secretion-dependent cell cycle block caused in HeLa cells by enteropathogenic Escherichia coli O103; Nougayrede JP et al.; Rabbit enteropathogenic Escherichia coli (EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation . We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms . We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis . A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content . This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation . In contrast, intimin and Tir mutants retained the antiproliferative effect . The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition . Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells . Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1) . Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13(suc1)-agarose beads . This shows that Cdk1 is implicated in the G2/M arrest caused by EPEC strain E22.

Infect Immun, 2001 Nov, 69(11), 6625 - 32
Characterization of FasG segments required for 987P fimbria-mediated binding to piglet glycoprotein receptors; Choi BK et al.; The 987P fimbriae of enterotoxigenic strains of Escherichia coli bind to both glycoprotein and glycolipid receptors on the brush borders of piglet enterocytes . A mutation in lysine residue 117 of the adhesive subunit FasG {fasG(K117A)} previously shown to abrogate 987P binding to the lipid receptor sulfatide did not affect the interaction with the glycoprotein receptors . Both the fimbriae and the FasG subunits of the wild type and the fasG(K117A) mutant bound to the glycoprotein receptors, confirming that lysine 117 was not required for binding to the glycoprotein receptors . Truncated FasG molecules were used to identify domains required for glycoprotein receptor recognition . At least two segments which did not include lysine117, namely, residues 211 (glutamine) to 220 (serine) and 20 (aspartic acid) to 41 (serine), were shown to be involved in the FasG-glycoprotein receptor interactions by ligand-blotting assays . Changing isoleucine 217 or leucine 215 of FasG to alanine abolished the property of a truncated FasG fusion protein to inhibit 987P recognition of its glycoprotein receptors . Thus, the K117 residue of FasG is required only for binding to the glycolipid receptor, whereas the newly identified hydrophobic residues of the FasG subunit are required specifically for the recognition of the glycoprotein receptor . Taken together, our data indicate that different residues of the FasG adhesin are important in 987P fimbrial binding to sulfatide and glycoprotein receptors, suggesting different mechanisms of interaction.

Infect Immun, 2001 Nov, 69(11), 6573 - 9
Enteropathogenic Escherichia coli virulence factor bundle-forming pilus has a binding specificity for phosphatidylethanolamine; Khursigara C et al.; The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells . While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified . We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding . In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid . Strains which did not express BFP did not bind PE, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101 . E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes . Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69 . Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

EMBO J, 2001 Oct 15, 20(20), 5802 - 11
Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition; Duval-Valentin G et al.; IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected . Transposase synthesis is usually driven by a weak promoter, p(IRL), in the left end (IRL) . Circle junction formation creates a strong promoter, p(junc), with a -35 sequence located in the right end and the -10 sequence in the left . p(junc) assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration . Insertion results in p(junc) disassembly and a return to the low p(IRL)- driven transposase levels . We demonstrate that p(junc) plays an important role in regulating IS911 transposition . Inactivation of p(junc) strongly decreased IS911 transposition when transposase was produced in its natural configuration . This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate . We have also investigated other members of the IS3 and other IS families . Several, but not all, IS3 family members possess p(junc) equivalents, underlining that the regulatory mechanisms adopted to fine-tune transposition may be different.

Virus Res, 2001 Nov 28, 80(1-2), 23 - 31
Expression kinetics of the transcript and product of the UL28 homologue of bovine herpesvirus 1; Desloges N et al.; We report that the bovine herpesvirus 1 (BHV1) UL28 ORF, a homologue of the herpes simplex virus (HSV) UL28 gene, represents a functional gene encoding a viral specific protein . The BHV1 UL28 ORF, located at positions 53058-->55538 of the viral genome, encodes a viral specific transcript of 3.4 kb detected at 6 h post-infection (p.i.) after which its levels accumulated up to 12 h p.i . and then remained constant up to 24 h p.i . Transcription of the BHV1 UL28 was determined to initiate 95 bases upstream from the ORF's initiating codon, which corresponds to 33 nucleotides downstream from a putative TATA box . A BHV1 UL28 specific antiserum, generated against a T7-Tag/UL28 fusion protein expressed in E . coli, specifically reacted with a 100 kDa protein in Western blots of BHV1-infected protein cell lysates . The expression kinetics of the protein was delayed by 6 h relative to that of its transcript suggesting that the gene is regulated at the translational level . In contrast to the HSV and pseudorabies virus UL28 genes, which belong to viral genes of the early (beta) class, that of BHV1 was unambiguously classified as a gamma2 gene . Further studies will be required to determine whether these kinetic differences have any functional implications.

Bioorg Med Chem, 2001 Nov, 9(11), 2885 - 93
Comparative QSAR studies on bibenzimidazoles and terbenzimidazoles inhibiting topoisomerase I; Mekapati SB et al.; Terbenzimidazoles that inhibit topoisomerase are of interest as anticancer drugs . We have reviewed the literature and have developed 13 quantitative structure-activity relationships (QSARs) on cleaving DNA or inhibiting the growth of tumor cell cultures . The results are correlated with octanol/water partition coefficients or molecular refractivity . Suggestions have been made for the development of improved derivatives.

Trends Microbiol, 2001 Oct, 9(10), 494 - 500
The Sec protein-translocation pathway; Mori H et al.; The Sec machinery (or translocase) provides a major pathway of protein translocation from the cytosol across the cytoplasmic membrane in bacteria . The SecA ATPase interacts dynamically with the SecYEG integral membrane components to drive the transmembrane movement of newly synthesized preproteins . This pathway is also used for integration of some membrane proteins and the Sec translocase interacts with other cellular components to achieve its cellular roles . The detailed protein interactions involved in these processes are being actively studied and a structural understanding of the protein-conducting channel has started to emerge.

No To Shinkei, 2001 Sep, 53(9), 881 - 5
{Cerebral arteritis and cerebritis caused by subdural empyema: two cases report}; Horie N et al.; We report two cases of cerebral angitis and cerebritis caused by subdural empyema . A 22-year-old man, who complained of a headache and high fever, suddenly developed unconsciousness and right hemiparesis . CT and MRI demonstrated left subdural empyema with diffuse brain swelling . CT angiography showed diffuse narrowing of the left internal carotid artery, middle cerebral artery, and anterior cerebral artery . Although we performed craniotomy, continuous irrigation with drainage, systemic injection of antibiotics for subdural empyema, antiplatelet therapy, and hyperbaric oxygen therapy for angitis, his condition remained unchanged . A 67-year-old man who had previously undergone burr hole surgery presented to our hospital for the treatment of scalp infection . He suddenly developed unconsciousness and right hemiparesis . CT and MRI demonstrated left subdural empyema with diffuse brain swelling, but MR angiography did not show any abnormal findings . Hemiparesis improved after the surgery and systemic injection of the antibiotics . Subdural empyema with sinusitis or meningitis around the skull base sometimes causes cerebral angitis . We considered that the angiographical evaluation for the subdural empyema was necessary to detect angitis.

Plant Cell, 2001 Oct, 13(10), 2319 - 31
The Arabidopsis TAG1 transposase has an N-terminal zinc finger DNA binding domain that recognizes distinct subterminal motifs; Mack AM et al.; The in vitro DNA binding activity of the Arabidopsis Tag1 transposase (TAG1) was characterized to determine the mechanism of DNA recognition . In addition to terminal inverted repeats, the Tag1 element contains four different subterminal repeats that flank a transcribed region encoding a 729-amino acid protein . A single site-specific DNA binding domain is located near the N terminus of TAG1, between residues 21 and 133 . This domain binds specifically to the AAACCC and TGACCC subterminal repeats, found near the 5' and 3' ends of the element, respectively . The ACCC sequence within these repeats is critical for recognition because mutations at positions 3, 5, and 6 abolished binding, yet the first two bases also are important because substitutions at these positions decreased binding by up to 90% . Weak interaction also occurs with the terminal inverted repeats, but no binding was observed to the other two 3' subterminal repeat regions . Sequence analysis of the TAG1 DNA binding domain revealed a C(2)HC zinc finger motif . Tests for metal dependence showed that DNA binding activity was inhibited by divalent metal chelators and greatly enhanced by zinc . Furthermore, mutation of each cysteine residue predicted to be a metal ligand in the C(2)HC motif abolished DNA binding . Together, these data show that the DNA binding domain of TAG1 specifically binds to distinct subterminal repeats and contains a zinc finger.

Plant Cell, 2001 Oct, 13(10), 2191 - 209
The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis; Ishiguro S et al.; The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening . The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds . We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites . DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1-specific manner, and DAD1-green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts . These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis . DAD1 promoter::beta-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments . A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.

J Biol Chem, 2001 Dec 14, 276(50), 47702 - 8 Epub 2001 Oct 10.
Complex of transfer-messenger RNA and elongation factor Tu . Unexpected modes of interaction; Zvereva MI et al.; Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 +/- 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA . Ribosomes that are stalled either in a coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA . Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features . Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA.EF-Tu.GTP complex . tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu.GDP using a zero-length cross-link . The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3' end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present . Two cross-linking sites with EF-Tu.GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu.GDP.

Toxicon, 2001 Nov, 39(11), 1673 - 80
The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli; Boquet P; The cytotoxic necrotizing factor 1, from uropathogenic Escherichia coli, is the paradigm of Rho-GTPases-activating bacterial toxins . CNF1 is a MW 108kDa A-B protein toxin divided into three domains which are implicated in the three steps of the intoxication process . The N-terminal domain contains the cell receptor function and binds with high affinity to a cell receptor not yet identified . Binding of the toxin is followed by its internalization by endocytosis and its transport into late endosomes . The middle toxin domain contains two hydophobic helices which allow translocation of the toxin across the membrane upon acidification in late endosomes . Finally the carboxy-terminal domain of CNF1 is an enzyme which deamidates Rho-GTP-binding proteins (Rho, Rac and Cdc42) glutamine 63 (for Rho) or glutamine 61 (for Rac and Cdc42) . Deamidation of glutamine 63/61 blocks the intrinsic or the GTPase activating protein (GAP)-induced hydrolysis of GTP leading to the permanent activation of the GTPase . Activation of Rho GTPases by CNF1 induces a profound reorganization of the cell actin cytoskeleton . By its properties on Rho GTPases CNF1 is to date an invaluable tool for cell biology studies.

Toxicon, 2001 Nov, 39(11), 1619 - 27
Escherichia coli cytotoxic necrotizing factors and Bordetella dermonecrotic toxin: the dermonecrosis-inducing toxins activating Rho small GTPases; Horiguchi Y; Escherichia coli cytotoxic necrotizing factors (CNFs) and Bordetella dermonecrotic toxin (DNT) have been recently found to comprise a novel family of dermonecrosis-inducing toxins which activate the small GTPases of the Rho family . They are single chain polypeptides consisting of an N-terminal domain responsible for binding to target cells and a C-terminal catalytic domain . CNFs (CNF1 and 2) and DNT share in the catalytic domain about 30% identical residues and a consensus sequence where the catalytically active center Cys resides . Both toxins deamidate Rho and other members of the Rho family, Rac and Cdc42, at Gln in the switch II region, which plays an important role in their GTPase activity . DNT, in addition, catalyzes a cross-link of the Gln of the GTPases with ubiquitous polyamines such as putrescine, spermidine, and spermine . The deamidation and the polyamination result in abrogation of the GTPase activity, and in addition, the polyamination endows Rho with the ability to interact with a downstream effector, ROCK, in a GTP-independent manner . These effects render the GTPases constitutively active, which underlies the toxicities of CNFs and DNT.

Int J Parasitol, 2001 Nov, 31(13), 1435 - 40
Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense; Boulange A et al.; The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2) . While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes . Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised . Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain) . These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences . It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Cell, 2001 Oct 5, 107(1), 79 - 89
Structural analysis of DNA replication fork reversal by RecG; Singleton MR et al.; The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells . RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass . We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork . The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal . We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart . In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.

Gene, 2001 Aug 8, 273(2), 267 - 74
Cloning and characterization of a new soluble murine J-domain protein that stimulates BiP, Hsc70 and DnaK ATPase activity with different efficiencies; Kroczynska B et al.; Hsp70s perform many functions in the cell through their ATPase activity that is stimulated by a genuine partner that contains a highly conserved so called J-domain . Here we report the cloning and characterization of a new J-domain protein named MmDjC7 . The complete cDNA encodes a putative soluble 22 kDa protein that contains a conserved J-domain, but lacks the G/F- and C-rich regions found in the bacterial Escherichia coli DnaJ . Northern analysis revealed that mmDjC7 mRNA (0.9 kb) is most abundant in the heart and liver tissues . Recombinant hexahistidine tagged MmDjC7 (25 kDa) was efficiently expressed in E . coli and purified to homogeneity . MmDjC7 stimulates the ATPase activity of murine BiP, Hsc70 and E . coli DnaK, albeit with very different molar ratios that vary from 1:2 (for BiP/MmDjC7) to 1:10 (for DnaK/MmDjC7) . MmDjC7 thus appears to be a new J-domain protein that can possibly interact with more than one Hsp70.

Gene, 2001 Aug 8, 273(2), 259 - 65
Cooperative effects by the initiation codon and its flanking regions on translation initiation; Stenstrom CM et al.; The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end . AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes . The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency . We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli . One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed . The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found . The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+) . The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs . The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Biochem Biophys Res Commun, 2001 Oct 19, 288(1), 121 - 8
Characterization of thermal stability of the Escherichia coli Fapy-DNA glycosylase; Kuznetsov SV et al.; Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence . Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degrees C . Heat aggregation is preceded by tertiary conformational changes of Fpg . However, the secondary structure of the fraction that does not aggregate remains unchanged up to approximately 60 degrees C . The kinetics of heat aggregation occurs with an activation enthalpy of approximately 21 kcal/mol . The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above approximately 3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins . With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of approximately 3.6 kcal/mol at 25 degrees C . An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg . A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented .

Arch Biochem Biophys, 2001 Oct 15, 394(2), 156 - 60
Allosteric transition and substrate binding are entropy-driven in glucosamine-6-phosphate deaminase from Escherichia coli; Bustos-Jaimes I et al.; Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants . The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model . This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E . coli GlcN6P deaminase . The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors .

Photochem Photobiol, 2001 Sep, 74(3), 489 - 94
Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry; Sudo Y et al.; Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis . ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm . The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone . The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured . The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species . From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1999 Jul, 15(4), 301 - 4
{The effect of intestinal ischemia/reperfusion on increased sensitivity to endotoxin and its potential mechanism}; Yao Y et al.; OBJECTIVE: To investigate the effect of intestinal ischemia/reperfusion injury (I/R) on increased sensitivity to endotoxin and its potential mechanism(s) . METHODS: Sprague-Dawley rats underwent 45 minutes of superior mesenteric artery occlusion followed by reperfusion . Twelve hours after reperfusion, endotoxin (Escherichia coli LPS, 1.5 mg/kg) was injected intravenously and rats were killed 12 hours later for measurement of organ function parameters . Also, in vitro study was performed to determine LPS-induced tumor necrosis factor (TNF)release in whole blood . RESULTS: LPS injection after I/R resulted in marked hemodynamic dysfunction and multiple organ damage(P < 0.01), while no significant or only minor changes in organ function parameters were observed in both I/R and LPS groups . In whole blood, monoclonal antibody to CD14 significantly blocked the release of TNF at low LPS concentration (< or = 10 ng/ml), and this response was dose dependent . Moreover, TNF release in LPS-stimulated whole blood obtained after I/R was inhibited to a significantly greater degree than that in baseline blood samples or sham-operation group(P < 0.05-0.01) . CONCLUSION: Splanchnic artery occlusion followed by reperfusion could lead to increased sensitivity to the subsequent LPS challenge, which may be associated with a shift toward CD14-dependent mechanism(s).

Chin Med J (Engl), 1999 Feb, 112(2), 162 - 5
Augmentation of antitumor effect of adenovirus-mediated CD suicide gene therapy by cotransfer of interleukin 2 gene in melanoma-bearing mice; Ju DW et al.; OBJECTIVE: To investigate the antitumor effect of combined adenovirus encoding E . coli cytosine deaminase (AdCD) and adenovirus encoding murine interleukin 2 (AdIL-2) on murine melanoma . METHODS: C57BL/6 mice were inoculated s.c . with B16F10 melanoma cells and 3 days later received injections of AdCD and/or AdIL-2 at the site of tumor inoculation followed by administration of 5-flurocytosine (5FC) 300 mg/kg per day for 10 days . RESULTS: Mice receiving AdCD/5FC/AdIL2 therapy developed tumors more slowly and survived much longer when compared with mice treated with AdCD/5FC, AdIL2, AdlacZ/5FC, or PBS . Immunological analysis illustrated that combined treatment could enhance NK activity and CTL activity . Flow cytometry demonstrated that AdCD/5FC/AdIL2 therapy increased the expression of MHC-1 and CD80 molecules on freshly isolated tumor cells . The CD4+ and CD8+ T cell infiltration in the tumor increased significantly after the combined therapy . CONCLUSIONS: Our data showed that combined transfer of CD suicide gene and IL-2 gene could inhibit the tumor growth more significantly . The increased specific and non-specific antitumor immunity might be responsible for the enhanced therapeutic effect.

Oncogene, 2001 Sep 20, 20(42), 6066 - 72
Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methy1-6-phenolimidazo {4,5-b} pyridine (PhIP) synergistically affect mutagenesis in mouse colon; Zhang S et al.; Msh2 deficiency and food-borne carcinogen PhIP have been implicated as genetic and environmental factors, respectively, in human colon carcinogenesis . It is not clear whether loss of one or both alleles of Msh2 gene increases the mutational sensitivity in colon when exposed to environmental carcinogens . In the current study, Msh2(+/-)/lacI and Msh2(-/-)/lacI double transgenic mice were treated with PhIP and mutations in the lacI gene were studied in the colon . The spontaneous mutation frequency (MF) is approximately eightfold higher in Msh2(-/-) mice than in Msh2(+/+) mice, while Msh2(+/-) mice display similar levels of spontaneous mutation as the Msh2 wild type mice . PhIP induced a significant increase in MF in all genotypes of mice . However, induced MF is much higher in Msh2(-/-) mice compared to Msh2(+/+) and Msh2(+/-) mice . Msh2(+/-) mice displayed an increased level of G:C>T:A transversions and -1 frameshifts upon PhIP treatment . In contrast, loss of both Msh2 alleles mainly results in increased frequency of G:C>A:T transitions when exposed to PhIP . These results suggest that a defect in mismatch repair may result in an enhanced sensitivity from exposure to a dietary carcinogen . It also provides insight into interaction between genetic and environmental factors in human carcinogenesis.

Gene Ther, 2001 Oct, 8(19), 1443 - 9
Overexpression of human insulin-like growth factor-I promotes new tissue formation in an ex vivo model of articular chondrocyte transplantation; Madry H et al.; Articular cartilage, the tissue that forms the gliding surface of joints, has a poor regenerative capacity . Insulin-like growth factor-I (IGF-I) is a polypeptide that is anabolic and mitogenic for cartilage . Transfection of articular chondrocytes with an expression plasmid vector containing the cDNA for human IGF-I under the control of the cytomegalovirus promoter/enhancer led to expression of the transgene and synthesis of biologically relevant amounts of IGF-I protein . Transplantation of transfected articular chondrocytes on to the surface of articular cartilage explants led to the formation of a new tissue layer on the cartilage explant surface . The new tissue was characterized by the presence of type II collagen and proteoglycan and by the absence of type I collagen, consistent with hyaline-like cartilage . The tissue formed by the chondrocytes expressing IGF-I was thicker and contained more cells than controls transfected with an expression plasmid vector containing the Escherichia coli (E . coli) beta-galactosidase (lacZ) gene . Transplantation of articular chondrocytes that overexpress human IGF-I also increased DNA synthesis and the synthesis of glycosaminoglycans by the underlying explant cartilage chondrocytes . These results identify a mechanism by which IGF-I may simultaneously promote chondrogenesis and shift cartilage homeostasis in an anabolic direction . The data further suggest that therapeutic growth factor gene transfer may be applicable to articular cartilage.

Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11991 - 6 Epub 2001 Oct 02.
Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA; Sengupta J et al.; S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome . It has a pivotal role in stabilizing the mRNA on the ribosome . Thus far, S1 has eluded structural determination . We have identified the S1 protein mass in the cryo-electron microscopic map of the Escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1 . According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data . Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion . The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit . Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine-Dalgarno sequence, explains the protein's role in the recognition of the 5' region of mRNA.

J Biol Chem, 2001 Dec 14, 276(50), 47690 - 4 Epub 2001 Oct 09.
Lipoprotein sorting signals evaluated as the LolA-dependent release of lipoproteins from the cytoplasmic membrane of Escherichia coli; Terada M et al.; Escherichia coli lipoproteins are anchored to either the inner or outer membrane through fatty acyl chains covalently attached to an N-terminal cysteine . Aspartate at position 2 functions to retain lipoproteins in the inner membrane, although the retention is perturbed depending on the residue at position 3 . We previously revealed that LolCDE and LolA play critical roles in this lipoprotein sorting . To clarify the sorting signals, the LolA-dependent release of lipoprotein derivatives having various residues at positions 2 and 3 was examined in spheroplasts . When the residue at position 3 was serine, only aspartate at position 2 caused the retention of lipoproteins in spheroplasts . We then examined the release of derivatives having aspartate at position 2 and various residues at position 3 . Strong inner membrane retention occurred with a limited number of species of residues at position 3 . These residues were present at position 3 of native lipoproteins having aspartate at position 2, whereas residues that inhibited the retention were not . It was also found that a strong inner membrane retention signal having residues other than aspartate at position 2 could be formed through the combination of the residues at positions 2 and 3 . These results indicate that the inner membrane localization of native lipoproteins is ensured by the use of a limited number of strong inner membrane retention signals.

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 109 - 19
Expression, purification, immunological characterization and application of Escherichia coli-derived hepatitis C virus E2 proteins; Liu J et al.; The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells . Anti-E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees . Therefore E2 has become a major target for the development of anti-HCV vaccines . Two E2 fragments {amino acids (aa) 450-565 and aa 385-565} derived from a subtype 1b HCV genome were expressed as N-terminally hexahistidine-tagged proteins in Escherichia coli and purified to over 85% purity . Both proteins were specifically recognized by homologous hepatitis-C-patient's serum on Western blotting, suggesting that these E . coli-derived E2 proteins displayed E2-specific antigenicity . E2-116 (aa 450-565) elicited strong antibody responses in BALB/c mice and rabbits . Rabbit antiserum raised against renatured E2-116 (R(E2-116R)) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting . E2-181 (aa 385-565) reacted with 40% of anti-HCV(+) patients' sera in ELISA . R(E2-116R) and E2-181 were successfully used in preliminary modified vaccinia virus Ankara- and DNA-based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice . The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 85 - 92
Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp . Bac Kan 8-98; Chi PV et al.; We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp . Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin . The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3) . The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture . The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS . It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM . The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline.

Mol Ther, 2001 Oct, 4(4), 365 - 71
High-level, beta-catenin/TCF-dependent transgene expression in secondary colorectal cancer tissue; Lipinski KS et al.; There is an urgent need for improved therapies for inoperable metastatic colon cancer . Gene-directed enzyme prodrug therapy (GDEPT) using adenovirus vectors works well in preclinical models of this disease, but successful clinical application is hampered by an inability to construct vectors that express at high levels in infected tumor cells but not in infected normal cells . Constitutive activation of beta-catenin-dependent gene expression is almost certainly a key causative event in the genesis of colon and some other cancers . Here we have exploited this oncogenic defect to design a synthetic promoter, CTP1, that, in contrast to currently available tumor-selective promoters, is both highly active in cancer cells and highly cancer-cell-specific . CTP1 directs high-level beta-galactosidase expression in freshly isolated biopsies of secondary colon cancer, but is not detectably active in associated normal liver tissue . We also demonstrate that CTP1 can direct high-level, tumor-specific therapeutic gene expression in vivo . Intratumoral injection of an adenovirus vector encoding Escherichia coli nitroreductase driven by CTP1 efficiently sensitized SW480 xenografts to the prodrug CB1954, whereas systemic vector and prodrug administration produced no apparent signs of toxicity . CTP1 may form the basis for effective, targeted gene therapy of metastatic colon cancer and other tumors with deregulated beta-catenin/T cell factor.

Mol Ther, 2001 Oct, 4(4), 347 - 55
Antitumor activity of cationic lipid complexed with immunostimulatory DNA; Rudginsky S et al.; We previously reported that treatment of intraperitoneal tumors with complexes of cationic lipid and noncoding plasmid DNA leads to the development of a specific, cytotoxic T-cell response correlating with the rejection of established tumor cells as well as subsequent tumor re-challenge . Here, focusing on an intraperitoneal AB12 mesothelioma model, we show that the anticancer effects of the lipid:DNA complex are associated with DNA containing immunostimulatory CpG motifs . Complexes prepared with cationic lipid and bacterial plasmid DNA, Escherichia coli genomic DNA fragments, or synthetic immunostimulatory CpG oligodeoxynucleotides provided a substantial survival benefit, whereas eukaryotic DNA and methylated bacterial DNA had little or no therapeutic activity . Alternative inflammatory stimuli such as thioglycolate, poly(I:C), and incomplete or complete Freund's adjuvant failed to reproduce the antitumor activity obtained with the lipid:DNA complex . The innate immune response triggered by lipid:DNA complexes led to the development of a systemic immune response against tumor cells that allowed animals to reject tumors not only at the intraperitoneal treatment site, but also at a distal subcutaneous site . These data demonstrate that immunostimulatory DNA complexed with cationic lipid is a potent inducer of innate and adaptive immune responses against tumor cells and represents a potentially useful tool in the immunotherapy of cancers for which tumor-associated antigens have not been identified.

Mol Genet Metab, 2001 Sep-Oct, 74(1-2), 264 - 72
Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia; Lai K et al.; We previously identified a missense mutation at amino acid 135 of human galactose 1-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was substituted for a serine (TCG), S135L . This mutation was common in black patients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts . However, there was residual GALT activity and protein in their leukocytes, and they had near normal total body {13C}galactose oxidation to 13CO2 in breath . To evaluate the biochemical mechanism(s) producing these effects, we overexpressed hGALT proteins with site-directed mutations in this nonconserved amino acid in a GALT-minus Escherichia coli . Enzyme activities detected in bacterial lysates overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H135, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respectively . Only the threonine substitution (S135T) had significant enzyme activity in these lysates . There was also decreased abundance of all mutant proteins in the lysates exposed to bacterial proteolysis during preparation and analysis . This added the variable of bio-instability to analysis of enzyme activities in lysates . To further characterize the catalytic role of serine at amino acid 135 and to differentiate bio-instability from impaired catalysis by the leucine substitution, we purified wild-type and L135-hGALT proteins to homogeneity and analyzed identical amounts of enzyme protein . We found that the apparent Vmax of the purified L135-hGALT protein was significantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 micromol glucose 1-phosphate released/min/mg hGALT protein with no increase in KM for galactose 1-phosphate for the second displacement . The first displacement reaction, although three orders of magnitude slower, was similar between the wild type and L135-hGALT . We conclude that a hydroxyl group on amino acid 135 is required for the catalysis of uridyl transfer from UDP-glucose to UDP-galactose in the presence of galactose 1-phosphate, and plays a role in the bio-stability of hGALT .

Biol Chem, 2001 Aug, 382(8), 1235 - 43
Functional dissection of trigger factor and DnaK: interactions with nascent polypeptides and thermally denatured proteins; Schaffitzel E et al.; In Escherichia coli, the ribosome-associated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypeptides . Here we investigated the functional relationship of TF and DnaK by comparing various functional properties of both chaperones . First, we analyzed the ability of TF and DnaK to associate with nascent polypeptides and full-length proteins released from the ribosome . Toward this end, we established an E . coli based transcription/translation system containing physiological ratios of TF, DnaK and ribosomes . In this system, TF can be crosslinked to nascent polypeptides of sigma32 . No TF crosslink was found to full-length sigma32, which is known to be a DnaK substrate . In contrast, DnaK crosslinked to both nascent and full-length sigma32 . DnaK crosslinks critically depended on the type of chemical crosslinker . Crosslinks represent specific substrate-chaperone interactions since they relied on the association of the nascent polypeptides with the substrate binding pocket of DnaK . While DnaK is known to be the major chaperone to prevent protein aggregation under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heat-sensitive phenotype of a deltadnaK52 mutant in vivo . These data indicate that TF and DnaK show strong differences in their ability to prevent aggregation of denatured proteins and to associate with native like substrates, but share the ability to associate with nascent polypeptides.

Eur J Immunol, 2001 Oct, 31(10), 2986 - 96
Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds; Ostergaard Pedersen L et al.; The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides . MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis . Here, we present an efficient solution to this problem . Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions . In contrast to common practice, the molecules were not reduced during the purification . The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding . This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s) . Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically . Several denatured MHC-I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding . The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre-oxidized recombinant MHC-I.

Eur J Immunol, 2001 Oct, 31(10), 2960 - 9
Influence of enterotoxin on mucosal intranet: selective inhibition of extrathymic T cell development in intestinal intraepithelial lymphocytes by oral exposure to heat-labile toxin; Kim JK et al.; We tested the possibility that heat-labile enterotoxin of Escherichia coli (LT) affects the development of extrathymic T cells in the intraepithelial lymphocyte (IEL) compartment . After oral administration of LT, the number of extrathymic CD8alphaalpha+ IEL was selectively and significantly diminished when compared with the corresponding cells in phosphate-buffered saline-fed control mice . To clarify the mechanism behind this selective reduction of CD8alphaalpha+ IEL, we analyzed the expression of essential cytokines and their corresponding receptors for the mucosal intranet formed by intestinal epithelial cells (IEC) and IEL . The expression levels of stem cell factor, interleukin (IL)-7, and IL-15 in IEC, and their corresponding receptors, i . e . c-kit, IL-7 receptor, and IL-15 receptor, in CD8alphaalpha+ IEL were reduced following oral feeding with LT . These findings suggest that LT negatively regulates development of CD8alphaalpha+ IEL via the disruption of mucosal intranet-associated cytokine and cytokine receptors, which are required for the development and/or expansion of extrathymically developed T cells . Further, LT-induced destruction of the mucosal intranet resulted in the impairment of IEC generation via an increase of apoptosis.

J Immunol, 2001 Oct 15, 167(8), 4693 - 700
Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies; Jotwani R et al.; Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined . We show that in human CP tissues, CD1a(+) immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83(+) mature dendritic cells (DCs) specifically infiltrate the CD4(+) lymphoid-rich lamina propria . In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response . Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1beta, PGE(2)) and Th (IL-10, IL-12) cytokines . Interestingly, the IL-10:IL-12 ratio elicited from P . gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs . This may account for the significantly (p < 0.05) lower proliferation of autologous CD4(+) T cells and reduced release of IFN-gamma elicited by P . gingivalis-pulsed DCs . Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P . gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.

J Biol Chem, 2001 Dec 7, 276(49), 46678 - 84 Epub 2001 Oct 08.
In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus; Thibeault D et al.; The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins . Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature . NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent . To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties . We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain . The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography . The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027) . NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product . This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.

J Bacteriol, 2001 Nov, 183(21), 6491 - 3
Transcription-induced cytosine-to-thymine mutations are not dependent on sequence context of the target cytosine; Beletskii A et al.; We showed previously that transcription of a plasmid-borne kan allele increases C-to-T mutations in the nontranscribed strand . Using two new plasmid-borne kan alleles, one cmp allele, and a chromosomal kan allele, we found in this study that transcription-induced mutations are not limited to specific genes, alleles, or locations and are likely to be a general property of transcript elongation in Escherichia coli.

J Bacteriol, 2001 Nov, 183(21), 6487 - 90
Interaction of MutS and Vsr: some dominant-negative mutS mutations that disable methyladenine-directed mismatch repair are active in very-short-patch repair; Lieb M et al.; In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T-G mismatches that result from the deamination of 5-methylcytosines in DNA to C-G . The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair . Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair . Some mutS mutations (class I) did not lower VSP repair in a mutS(+) background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS(+) . Other plasmid-borne mutS mutations (class II) and mutS(+) decreased VSP repair in the mutS(+) background . Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.

J Bacteriol, 2001 Nov, 183(21), 6315 - 23
Regulation of rRNA transcription correlates with nucleoside triphosphate sensing; Barker MM et al.; We have previously shown that the activity of the Escherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo . We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription . To test this model, we have measured the ATP requirements for transcription from 11 different rrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo . The seven rrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation) . In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo . We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation . We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).

J Bacteriol, 2001 Nov, 183(21), 6305 - 14
Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli; Hirvonen CA et al.; The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS . In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters . We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity . Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents . The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter . Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1 . Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter . These studies have implications for the control of gene expression of unlinked multigene families.

J Bacteriol, 2001 Nov, 183(21), 6151 - 8
Molecular cloning and functional analysis of the MutY homolog of Deinococcus radiodurans; Li X et al.; The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein . Expressed MutY(Dr) is able to complement E . coli mutY mutants but not mutM mutants to reduce the mutation frequency . The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are . Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E . coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch . However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride . This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA . The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec) . Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec) . Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.

FEBS Lett, 2001 Oct 5, 506(2), 143 - 8
Membrane interactions and self-association of the TatA and TatB components of the twin-arginine translocation pathway; De Leeuw E et al.; The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin-arginine signal peptides . The essential Tat pathway components TatA, TatB and TatC are shown to be integral membrane proteins . Upon removal of the predicted N-terminal transmembrane helix TatA becomes a water-soluble protein . In contrast the homologous TatB protein retains weak peripheral interactions with the cytoplasmic membrane when the analogous helix is deleted . Chemical crosslinking studies indicate that TatA forms at least homotrimers, and TatB minimally homodimers, in the native membrane environment . The presence of such homo-oligomeric interactions is supported by size exclusion chromatography.

FEBS Lett, 2001 Oct 5, 506(2), 108 - 12
The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli; Li ZY et al.; To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co-expression . When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies . Co-expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control . Co-expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell . Co-expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl-prolyl cis/trans isomerase activity was 1% of wild-type, gave the same results as wild-type TF . This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.

Biochemistry, 2001 Oct 16, 40(41), 12407 - 11
Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles; Dreier J et al.; Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems . Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain . These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate . In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA . Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme . Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases . The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled . The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme . Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable . Sequence comparison with the E . coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S . coelicolor enzyme . The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal . The implications of this unexpected finding are discussed.

Biochemistry, 2001 Oct 16, 40(41), 12329 - 38
Amino acid substitution at position 99 affects the rate of CRP subunit exchange; Baker CH et al.; We investigated the characteristics of CRP having amino acid substitutions at position 99 . Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex {Garcia, A . E., and Harman, J . G . (1996) Protein Sci . 5, 62-71} showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP . To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F) . Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP . Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity . There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro . The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP . Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface . The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP . The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.

Plasmid, 2001 Sep, 46(2), 86 - 94
Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis; Pomerantsev AP et al.; pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328 . pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var . japonica . The replicon of pOM1 includes the ori region and the repA gene . The ori region, located upstream of the repA gene includes two sets of 31- and 13-bp direct repeats (DR), with AT-rich regions preceding each of the DRs . Two putative promoters of the repA gene were found connected with the DR regions . A 40-kDa protein was encoded by the repA gene and found essential for replication . Expression of the tetC gene is regulated by an Escherichia coli sigma(70)-like promoter and is dependent on the F . tularensis strain and its environment .

Korean J Parasitol, 2001 Sep, 39(3), 241 - 6
Determination of antigenic domain in GST fused major surface protein (Nc-p43) of Neospora caninum; Son ES et al.; The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins . The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T; OFR without signal sequence and C-terminal hydrophobic sequence, S; N-terminal 2/3 parts of S, A; C-terminal 2/3 parts, P; N-terminal 1/3 part, X; middle 1/3 part, Y; and C-terminal 1/3 part, Z, respectively . The DNA fragments were cloned into pGEX-4T vector . Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T, 66 kDa for S, 52 kDa for A, 53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE . The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot . These findings suggest that the antigenic domain of Nc-p43 of N . caninum may be localized in the C-terminal 2/3 parts . Together with A19 clone in SAG1 of Toxoplasma gondii (Nam et al., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species.

Biotechnol Bioeng, 2001 Nov 5, 75(3), 285 - 91
Analysis of an engineered sulfate reduction pathway and cadmium precipitation on the cell surface; Wang CL et al.; We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium . To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced . Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide . In this study, observations of various E . coli clones were combined with an analysis of kinetic and transport phenomena . This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation . An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow .

J Cell Sci, 2001 Sep, 114(Pt 17), 3189 - 98
Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium; Schutze J et al.; Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level . Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex . In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B . Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF . Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequences found in Protostomia and Deuterostomia . However, the C-terminal portion of the sponge sequence lacks the SH3 domain characteristic for amphiphysin/BIN1 . The polypeptide with a calculated size of 47 kDa was expressed in Escherichia coli . The recombinant, soluble 36 kDa putative AF was prepared and found to compete with the AF complex-associated adhesion protein of the AF-Fraction 6B for the binding sites at the cell surface . Furthermore, the recombinant putative AF was recognized by the antibody used to screen the cDNA library by western blotting . In addition, there is evidence that the recombinant putative AF binds to the G . cydonium galectin . It is concluded that the putative G . cydonium AF--a further autapomorphic molecule characteristic for Metazoa--binds to the AR present on the cell surface in association with the homologous galectin.

J Biol Chem, 2001 Dec 14, 276(50), 47508 - 11 Epub 2001 Oct 05.
ATP synthase F(1) sector rotation . Defective torque generation in the beta subunit Ser-174 to Phe mutant and its suppression by second mutations; Iko Y et al.; Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm . A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported . The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala . These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.

J Biol Chem, 2001 Dec 7, 276(49), 46004 - 10 Epub 2001 Oct 04.
Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family; Chang HW et al.; The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities . The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain . Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D . R., and Koonin, E . V . (1999) Nucleic Acids Res . 27, 1223-1242) . One is the E . coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5'-terminated strand of double-stranded DNA . We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA . Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE . These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins . These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.

Chem Biol, 2001 Oct, 8(10), 997 - 1010
Exploring the impact of different thioesterase domains for the design of hybrid peptide synthetases; Schwarzer D et al.; BACKGROUND: A large number of pharmacologically important peptides are synthesized by multifunctional enzymes, the nonribosomal peptide synthetases (NRPSs) . The thioesterase (Te) domain at the C-terminus of the last NRPS catalyzes product cleavage by hydrolysis or complex macrocyclization . Recent studies with excised Te domains and peptidyl-S-N-acetyl cysteamine substrate substitutes led to substantial insights in terms of cyclization activity and substrate tolerance of these enzymes . Their use in engineered hybrid NRPSs is an interesting but yet only little explored target for approaches to achieve new structural diversity and designed products . RESULTS: To study the capability of various Te domains to function in hybrid NRPSs, six different Te domains that catalyze different modes of termination in their natural systems were fused to a bimodular model NRPS system, consisting of the first two modules of tyrocidine NRPS, TycA and ProCAT . All Te domains were active in hydrolyzing the enzymatically generated dipeptide substrate D-Phe-Abu from the NRPS template with, however, greatly varying turnover rates . Two Te domains were also capable of hydrolyzing the substrate D-Phe-Pro and partially cyclized the D-Phe-Abu dipeptide, indicating that in an artificial context Te domains may display hydrolytic and cyclization activities that are not easily predictable . CONCLUSIONS: Te domains from heterologous NRPSs can be utilized for the construction of hybrid NRPSs . This is the first comparative study to explore their influence on the product pattern . The inherent specificity and regioselectivity of Te domains should allow control of the desired product cleavage, but can also lead to other modes of termination potentially useful for generating structural diversity . Our results provide the first data for choosing the proper Te domain for a particular termination reaction.

Chem Biol, 2001 Oct, 8(10), 951 - 65
A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins; Speight RE et al.; BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function . Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene . RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50 . Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round . The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections . CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display . The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Trends Biochem Sci, 2001 Oct, 26(10), 597 - 604
Macromolecular crowding: obvious but underappreciated; Ellis RJ; Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules . Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers . The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study . The stimulation of protein aggregation by crowding might account for the existence of molecular chaperones that combat this effect . Positive results of crowding include enhancing the collapse of polypeptide chains into functional proteins, the assembly of oligomeric structures and the efficiency of action of some molecular chaperones and metabolic pathways.

Int J Biol Macromol, 2001 Oct 22, 29(3), 175 - 80
Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I; Roche CJ et al.; To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone . A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity . At the scissile phosphate there was little effect on the cleavage rate . At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10 . At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer . The results demonstrate the importance of backbone contacts between DNA substrate and E . coli topoisomerase I.

Liver, 2001 Oct, 21(5), 309 - 19
Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor-alpha in galactosamine-sensitized mice; Osawa Y et al.; BACKGROUND/AIMS: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-alpha . METHODS: Seven-week-old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF-alpha (0.5 microg/mouse) . Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting . Activities of caspases were measured using chromogenic peptides as substrates . Ceramide content was determined using Escherichia coli diacylglycerol kinase . RESULTS: Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF-alpha (GalN/TNF-alpha), but not GalN or TNF-alpha alone . Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-alpha . In a cell-free system, processing of procaspases-9 and -3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF-alpha-treated mice, but not in that from control mice . Processing of procaspase-3 was inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2 . In a reconstitution assay system, procaspase-9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-alpha . Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation . CONCLUSION: Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-alpha-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role . Ceramide may be implicated in this apoptotic process.

Eur J Biochem, 2001 Oct, 268(19), 5157 - 66
Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens; Martensen I et al.; The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP-Neu5Ac hydroxylase . The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning . Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found . The unusual characteristics of this hydroxylase raise questions about its evolution . As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme . Despite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxylase from the starfish A . rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength . In order to shed light on the structural basis for these differences, the primary structure of CMP-Neu5Ac hydroxylase from A . rubens has been determined by PCR and cDNA-cloning techniques, using initial sequence information from the mouse enzyme . The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa . The deduced amino-acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C-terminus was some 60 residues longer . This extension consists of a terminal hydrophobic region, which may mediate membrane-binding, and a preceding hydrophilic sequence which probably serves as a hinge or linker . The identity of the ORF was confirmed by expression of active CMP-Neu5Ac hydroxylase in E . coli at low temperatures.

Eur J Biochem, 2001 Oct, 268(19), 5037 - 44
Cloning, expression, purification and characterization of patatin, a novel phospholipase A; Hirschberg HJ et al.; Patatin is the major protein constituent of potato tubers and displays broad esterase activity . The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins . From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag . This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha . The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity . Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers . The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles . The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms . Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)) . Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad {1} . Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad . To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2 . Identification of active site residues was based on the observation of correctly folded but inactive variants . This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

Brief Bioinform, 2001 Sep, 2(3), 223 - 32
Simultaneous modelling of metabolic, genetic and product-interaction networks; Juty NS et al.; The creation of cell models from annotated genome information, as well as additional data from other databases, requires both a format and medium for its distribution . Standards are described for the representation of the data in the form of Document Type Definitions (DTDs) for XML files . Separate DTDs are detailed for genetic, metabolic and gene product-interaction networks, which can be used to hold information on individual subsystems, or which may be combined to create a whole cell DTD . In the execution of this work, a fifth DTD was also created for a metabolite thesaurus, which allows incorporation of metabolite synonyms and generic nomenclature data into the models . A gene-regulation classification scheme was also created, to facilitate incorporation of gene regulatory information in an efficient manner . The work is described with particular reference to the metabolic network of Escherichia coli, which contains 808 individual enzymes . The assignment of confidence levels to these data, through the use of Gene Ontology evidence codes, is highlighted . In silico investigations may now be performed using the mathematical simulation workbench, DBsolve, which incorporates the facility to introduce data directly from XML.

Mol Genet Genomics, 2001 Sep, 266(1), 126 - 32
A scytalone dehydratase gene from Ophiostoma floccosum restores the melanization and pathogenicity phenotypes of a melanin-deficient Colletotrichum lagenarium mutant; Wang HL et al.; Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin . We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning . Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases . The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported . Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity . The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis . These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C . lagenarium.

J Chromatogr B Biomed Sci Appl, 2001 Oct 5, 762(1), 77 - 86
Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli; Kolodny N et al.; The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E . coli . The protein was purified in a two-step process that was rapid and reproducible . E . coli cells were grown to a high density before induction for 1 h . Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl . The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea . The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E . coli proteins and reduce endotoxin (to 10 EU/50 microg) . Yield was 20 mg of PfCS protein from 10 g of wet cell paste . The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml.

Science, 2001 Oct 5, 294(5540), 165 - 8
Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation; LaRiviere FJ et al.; Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis . Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body . The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated . Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.

Science, 2001 Oct 5, 294(5540), 158 - 60
Conversion of a peroxiredoxin into a disulfide reductase by a triplet repeat expansion; Ritz D et al.; Pathways for the reduction of protein disulfide bonds are found in all organisms and are required for the reductive recycling of certain enzymes including the essential protein ribonucleotide reductase . An Escherichia coli strain that lacks both thioredoxin reductase and glutathione reductase grows extremely poorly . Here, we show that a mutation occurring at high frequencies in the gene ahpC, encoding a peroxiredoxin, restores normal growth to this strain . This mutation is the result of a reversible expansion of a triplet nucleotide repeat sequence, leading to the addition of one amino acid that converts the AhpC protein from a peroxidase to a disulfide reductase . The ready mutational interconversion between the two activities could provide an evolutionary advantage to E . coli.

Curr Opin Microbiol, 2001 Oct, 4(5), 586 - 94
Adaptive mutations, mutator DNA polymerases and genetic change strategies of pathogens; McKenzie GJ et al.; "Adaptive" or "stationary-phase" mutation is a collection of stress responses promoting mutations, some of which are advantageous . In 2000 and 2001, in Escherichia coli, adaptive gene amplification was documented, and a parallel adaptive point-mutation mechanism was linked to the error-prone DNA polymerase, DinB (pol IV) . We suggest that DinB homologues may contribute to adaptive strategies of pathogens, including antigenic variation.

Curr Opin Microbiol, 2001 Oct, 4(5), 558 - 64
The circle is broken: telomere resolution in linear replicons; Kobryn K et al.; Linear DNA molecules with covalently closed hairpin ends (telomeres) exist in a wide variety of organisms . Telomere resolution, a DNA breakage and reunion reaction in which replicated telomeres are processed into hairpin ends, is now known to be a common theme in poxviruses, Borrelia burgdorferi and Escherichia coli phage N15 . Candidate proteins that may perform this reaction have recently been identified in poxviruses . Moreover, the first purification and definitive identification of a telomere resolvase has been reported for phage N15 . This protein is the prototype for a new class of DNA enzyme that performs a unique reaction . Advances in the study of telomere resolution in poxviruses, B . burgdorferi and E . coli phage N15 are discussed.

Gene, 2001 Sep 19, 275(2), 241 - 52
Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins; Zhang A et al.; Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein) . The fusion protein expression can be further improved by fusion to a mini-intein, i.e . an intein that lacks an endonuclease domain . We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments . After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system . In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein . The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction . A total of 17 target proteins were tested in this intein-GFP fusion system . Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity . The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence . The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.

Vaccine, 2001 Oct 15, 20 Suppl 1, S42 - 4
Pre-clinical and clinical investigation of the safety of a novel adjuvant for intranasal immunization; Glueck R; Nasalflu Berna is a trivalent influenza virus vaccine for active immunization against influenza by the nasal route . It consists of influenza virosomes which are formulated from inactivated influenza strains and heat-labile toxin from aseptic Escherichia coli bacteria strain, as an adjuvant (HLT) . The results of preclinical studies in ferrets, baboons, minipigs, mice and rabbits are presented here, and issues concerning route of administration, mechanism of action (preventing the disease and halting further spread of the disease), and the specific safety issues of the adjuvant itself (possible neurological activity of HLT) are examined . No clinical signs were detected in the animals, and hematological values were in the normal range . In particular, there was no evidence of any systemic adverse reaction, including sensitization to the test substances, and no evidence of possible neurological activity of the HLT.Further clinical studies in humans conducted over five influenza seasons using this virosome-formulated intranasal vaccine, elicited high levels of influenza-specific hemagglutination inhibition IgG antibody titers to the strains incorporated in the administered vaccine . In addition, IgA antibodies were also elicited in the nasal mucosa, and in the saliva . In addition to the systemic IgG antibody titers, the nasal mucosal IgA antibody response may provide additional local protection by the inhibition of viral replication and further spread in the respiratory tract . Nasalflu was well tolerated by most of the vaccinated subjects, both in terms of nasal symptoms and possible vaccination-mediated systemic symptoms . Both local and systemic symptoms were primarily mild, with only an occasional subject reporting moderate intensity . Out of four serious adverse events seen during the clinical development, only one was thought to be remotely related to the test vaccine.Nasalflu, developed by the Swiss Serum and Vaccine Institute, is a novel, highly immunogenic and safe influenza subunit vaccine which is easily administered as a nasal spray . This new route of administration is likely to increase compliance to vaccination, and could become an important tool to promote vaccination in population groups which show high resistance to vaccination.

Trends Biotechnol, 2001 Oct, 19(10), 406 - 11
RNase P: from biological function to biotechnological applications; Cobaleda C et al.; The M1 RNA subunit of Escherichia coli RNase P is a ribozyme responsible for the catalytic activity of the complex . It removes the 5' leader sequence from tRNA precursors to form mature tRNAs . M1 recognizes its target mainly on the basis of its structure and this allows the design of modified ribozymes engineered to destroy other molecules without the need for special sequences in the targeted mRNAs . M1 is thus an ideal tool to eliminate the tumourigenic chimeric messengers created after chromosomal translocations . These results have direct implications for cancer therapeutics and molecular biology in general.

Vet Immunol Immunopathol, 2001 Oct, 82(3-4), 229 - 44
Molecular, cellular, and functional characterization of chicken cytokines homologous to mammalian IL-15 and IL-2; Lillehoj HS et al.; DNA sequence analysis of a chicken interleukin (IL)-15 cDNA identified a 187 amino acid open reading frame encoding a protein with a predicted molecular weight of 21,964Da, two potential N-linked glycosylation sites, four highly conserved Cys residues, two out-of-frame AUG initiation codons in the 5' untranslated region, and an unusually long (66 amino acid) signal peptide such that the expected size of the mature protein is 14,462Da . Chicken IL-15 and IL-2 were compared with regard to their molecular, cellular, and functional characteristics . The predicted amino acid sequences of both chicken cytokines showed greater homologies with mammalian IL-15s compared with mammalian IL-2s . Northern hybridization and RT-PCR demonstrated chicken IL-15 gene transcripts in a wide variety of tissues and cell types while the chicken IL-2 gene was expressed only in concanavalin A (con A)-activated spleen cells . Both recombinant cytokines stimulated the growth of spleen T-cells and enhanced the activity of natural killer (NK) cells in vitro . Subcutaneous injection with an expression plasmid encoding IL-15 increased the percentage of CD3+ spleen T-lymphocytes whereas injection of an IL-2 cDNA augmented CD3+, CD4+, CD8+, T-cell receptor (TCR)1+, and TCR2+ T-cells . Collectively, these results indicate that chicken IL-15 and IL-2 are T-cell growth factors potentially capable of enhancing cell-mediated immunity in vivo.

Vet Immunol Immunopathol, 2001 Oct, 82(3-4), 183 - 92
Molecular cloning and functional characterization of bottlenose dolphin (Tursiops truncatus) tumor necrosis factor alpha; Shoji Y et al.; Bottlenose dolphin tumor necrosis factor alpha (doTNF-alpha) cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the nucleic and deduced amino acid sequences were determined . The sequence of the cDNA clones shows that doTNF-alpha has an open reading frame of 699bp encoding 233 amino acids . The nucleic acid sequence of doTNF-alpha indicates 90, 88, 87, and 79% similarity with the cattle, pig, human, and mouse TNF-alpha gene, respectively . Based on the analysis of human and mouse TNF-alpha molecules, doTNF-alpha is processed to a mature protein with 157 amino acids . The 233 amino acids precursor has a hydrophobic region that could serve as a transmembrane domain . The recombinant doTNF-alpha expressed in Escherichia coli as a glutathione S-transferase fusion protein reacted with anti-human TNF-alpha antibody and exerted cytotoxity to the TNF-alpha sensitive murine cell line L929.

Structure (Camb), 2001 Aug, 9(8), 669 - 78
X-ray crystal structure of proto-oncogene product c-Rel bound to the CD28 response element of IL-2; Huang DB et al.; BACKGROUND: The proto-oncogene product c-Rel is a Rel/NF-kappaB family transcription factor that plays a critical role in lymphoid cell development and mediates CD28-induced expression of interleukin 2 (IL-2) . The CD28 response element (CD28RE) in the IL-2 enhancer is nonameric and similar to the kappaB DNA target sites recognized by p65 homodimers . RESULTS: We have determined and refined the X-ray crystal structure of the c-Rel homodimer complexed to the CD28RE DNA site, 5'-AGAAATTCC-3', to 2.85 A resolution . The c-Rel homodimer binds CD28RE in a mode similar to that observed in the p65/IL-8 kappaB crystallographic complex . Binding studies reveal that the c-Rel homodimer recognizes the CD28RE with higher affinity as compared to other canonical kappaB sequences despite the nonconsensus A:T base pair at the 5' end of the CD28RE . Preferential recognition of the CD28RE by c-Rel results from the direct contacts between the protein and the DNA as well as intrasubunit interactions between the beta(f)-beta(g) loop in the dimerization domain and the DNA-contacting loop L1 of the N-terminal domain . Not only do these loops have different conformations in other Rel/DNA crystallographic complexes, but they also contain two of the five oncogenic point mutations found in v-Rel . CONCLUSIONS: The current structure indicates that a non-DNA-contacting loop in the dimerization domain and the DNA-contacting loop L1 may play critical roles in defining affinity and specificity . Two amino acid changes in these segments may account for the differential DNA binding by v-Rel as compared to that of c-Rel.

Biotechnol Prog, 2001 Sep-Oct, 17(5), 963 - 9
Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope; Wangsa-Wirawan ND et al.; Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM) . The inclusion bodies were attached to the AFM tip by covalent bonds . Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH . Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces . It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory . The effects of pH are significantly stronger for the inclusion body compared to the air bubble . This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies . It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.

Biotechnol Prog, 2001 Sep-Oct, 17(5), 897 - 906
Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography; Sunasara KM et al.; This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E . coli . protein (ECP) impurities . The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem . Several operational parameters were investigated to study their effects on the yield of the displacement process . The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification . Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step . The process was successfully scaled-up with respect to particle and column diameter . The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.

Nitric Oxide, 2001, 5(5), 442 - 52
Titration of low K(d) binding sites: binding of arginine analogs to nitric oxide synthases; Smith SM et al.; Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS) . Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d) . An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites . The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM) . N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower .

Biochem Biophys Res Commun, 2001 Oct 12, 287(5), 1125 - 8
Mutant of LolA, a lipoprotein-specific molecular chaperone of Escherichia coli, defective in the transfer of lipoproteins to LolB; Miyamoto A et al.; The outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane as a water-soluble complex with LolA and then transferred to the outer membrane receptor, LolB . LolA thus plays a critical role in the sorting and outer membrane localization of lipoproteins . To dissect the LolA function, the highly conserved residues were subjected to random mutagenesis, followed by selection for a growth defect . LolA(R43L), one of mutants thus constructed, possessed Leu in place of Arg at position 43 and caused accumulation of the LolA(R43L)-lipoprotein complex in the periplasm . LolA(R43L) was as active as wild-type LolA as to the release of lipoproteins from spheroplasts . In marked contrast, the transfer of lipoproteins from LolA(R43L) to LolB was completely inhibited, indicating that Arg at position 43 of LolA is involved in the lipoprotein transfer reaction .

Plant Mol Biol, 2001 Oct, 47(3), 389 - 98
Temporal and spatial expression patterns of TUB9, a beta-tubulin gene of Arabidopsis thaliana; Cheng Z et al.; Transgenic plants carrying chimeric genes composed of segments of the 5'-flanking region of the Arabidopsis 9-tubulin gene (TUB9) fused to the coding region of the beta-glucuronidase (GUS) gene of Escherichia coli were used to investigate the temporal and spatial patterns of TUB9 expression . Chimeric genes that contained at least 800 bp of TUB9 5'-flanking DNA were expressed primarily in floral tissues, with high levels of expression observed in pollen, elongating pollen tubes and ovules . The expression of the reporter genes in ovules ceased at the time of fertilization . In situ hybridization was used to verify that the reporter gene expression in pollen of transgenic plants is representative of the patterns of expression of the endogenous TUB9 gene . In situ hybridization also provided new insight into TUB9 transcript accumulation in ovules . The possible role of TUB9 and the functional implication of the largely non-overlapping expression patterns of tubulin genes are discussed.

Plant Mol Biol, 2001 Oct, 47(3), 367 - 78
The transcript abundance of GmGT-2, a new member of the GT-2 family of transcription factors from soybean, is down-regulated by light in a phytochrome-dependent manner; O'Grady K et al.; A new member of the GT-2 family of transcription factors, GmGT-2, was isolated from soybean while screening a cDNA library with a protein binding site (D1) in the promoter of Aux28, a member of the Aux/IAA family of auxin-responsive genes . GmGT-2 possesses various primary amino acid sequence characteristics common to all GT-2 factors thus far isolated, including sequence identity in the twin trihelix DNA-binding domains . Recombinant GmGT-2 expressed in Escherichia coli binds oligotetramers of both D1 and various GT-boxes . However, unlike other known members of the GT-2 family, GmGT-2 message levels are down-regulated by light in a phytochrome-dependent manner . Evidence is presented that the expression levels of Aux28 mRNA are also down-regulated by phytochrome . These results and other referenced data implicate the possible convergence of phytochrome and auxin signaling pathways.

J Hum Genet, 2001, 46(10), 566 - 71
Identification of GFAT1-L, a novel splice variant of human glutamine: fructose-6-phosphate amidotransferase (GFAT1) that is expressed abundantly in skeletal muscle; Niimi M et al.; Glutamine:fructose-6-phosphate amidotransferase (GFAT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway, which plays an important role in hyperglycemia-induced insulin resistance . To evaluate the role of GFAT1 expression, we analyzed the expression profiles of GFAT1 mRNA in various human tissues using reverse transcriptase-polymerase chain reaction . We report here the identification and cDNA cloning of a novel GFAT1 splice variant expressed abundantly in skeletal muscle and heart . This subtype, designated GFAT1-L, contains a 54-bp insertion within the GFAT1 coding sequence . Recombinant GFAT1-L protein possessed functional GFAT activities and biochemical characteristics similar to GFAT1 . Previously, GFAT1 was considered a simplex enzyme . The identification of a novel GFAT1 subtype possessing functional enzymatic activity and tissue-specific expression should provide additional insight into the mechanism of skeletal muscle insulin resistance and diabetes complications.

Biol Neonate, 2001, 80(3), 186 - 92
CD14 receptor expression and lipopolysaccharide-induced cytokine production in preterm and term neonates; Bessler H et al.; CD14 expression and the capacity of mononuclear cells (MC) from preterm and term neonates to secrete the proinflammatory cytokines interleukin (IL) 1 beta, tumor necrosis factor alpha and IL-6 in response to lipopolysaccharide (LPS) was investigated and compared to that of adults . MC were incubated with various doses of LPS, and the cytokine level in the supernatants was tested . CD14 receptors on MC and the intensity of their expression were analyzed . MC of preterm and term neonates and adults responded to LPS with low, medium and high proinflammatory cytokine production, respectively . CD14 expression was lowest in preterm infants, intermediate in term infants and highest in adults . The difference between term and preterm neonates for both parameters was significant . The results suggest a possible correlation between the lower expression of CD14 receptor on neonatal cells and the reduced secretion of proinflammatory cytokines by these cells . This decreased production may possibly contribute to the low ability of neonates to develop fever .

Mol Cell Biol, 2001 Nov, 21(21), 7268 - 76
Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity; Esposti MD et al.; Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver . Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c . The mechanism of Bid relocation to mitochondria was unclear . We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria . First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylglycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum . Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c . Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids . We discuss how this new role of Bid may relate to its proapoptotic action.

J Biol Chem, 2001 Dec 14, 276(50), 47227 - 32 Epub 2001 Oct 03.
The conformation of the epsilon- and gamma-subunits within the Escherichia coli F(1) ATPase; Hausrath AC et al.; F(1) is the water-soluble portion of the ubiquitous F(1)F(0) ATP synthase . Its structure includes three alpha- and three beta-subunits, arranged as a hexameric disc, plus a gamma-subunit that penetrates the center of the disc akin to an axle . Recently Hausrath et al . (Hausrath, A . C., Gruber, G., Matthews, B . W., and Capaldi, R . A . (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 13697-13702) obtained an electron density map of E . coli F(1) at 4.4-A resolution in which the coiled-coil alpha-helices of the gamma-subunit could be seen to extend 45 A from the base of the alpha(3)beta(3) hexamer . Subsequently the structure of a truncated form of the E . coli gamma-subunit in complex with epsilon has been described (Rodgers, A . J . W., and Wilce, M . C . J . (2000) Nat . Struct . Biol . 7, 1051-1054) . In the present study the 4.4-A resolution electron density map of E . coli F(1) is re-evaluated in light of the newly available data on the gamma- and epsilon-subunits . It is shown that the map of the F(1) complex is consistent with the structure of the isolated subunits . When E . coli F(1) is compared with that from beef heart, the structures of the E . coli gamma- and epsilon-subunits are seen to be generally similar to their counterparts in the bovine enzyme but to undergo major shifts in position . In particular, the two long, coiled-coil alpha-helices that lie along the axis of F(1) both unwind and rotate . Also the epsilon-subunit rotates around the axis by 81 degrees and undergoes a net translation of about 23 A . It is argued that these large-scale changes in conformation reflect distinct functional states that occur during the rotation of the gamma-subunit within the alpha(3)beta(3) hexamer.

Cancer Res, 2001 Oct 1, 61(19), 7110 - 7
Methylated CpG dinucleotides are the preferential targets for G-to-T transversion mutations induced by benzo{a}pyrene diol epoxide in mammalian cells: similarities with the p53 mutation spectrum in smoking-associated lung cancers; Yoon JH et al.; A large fraction of the p53 mutations in lung cancers from smokers are G-to-T transversions, a type of mutation that is infrequent in lung cancers from nonsmokers and in most other tumors . Previous studies have indicated that there is an association between G-to-T transversion hotspots in lung cancers and sites of preferential formation of polycyclic aromatic hydrocarbon adducts along the p53 gene . p53 codons containing methylated CpG sequences are preferential targets for formation of adducts by (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (BPDE) . To assess the role of CpG methylation in induction of mutations by BPDE, we analyzed BPDE mutagenesis in three CpG methylated target genes: a supF shuttle vector and the cII and lacI transgenes in embryonic mouse fibroblasts . After methylation of the shuttle vector at all CpG sequences, 42% of all G-to-T transversions were at CpG sites compared with 23% in unmethylated DNA . In the cII transgene, which is methylated at CpG sequences in vivo, 83 of 147 (56%) of the BPDE-induced mutations were G-to-T transversions, and 58% (48 of 83) of all G-to-T transversions occurred at methylated CpG sequences . In the lacI gene, 68% (75 of 111) of the BPDE-induced mutations were G-to-T events, and 58 of 75 (77%) of these occurred at methylated CpG sequences . The occurrence of transversion hotspots at methylated CpGs correlated with high levels of BPDE adducts formed at such sites . This situation mirrors the one in the p53 gene in lung cancers from smokers where 236 of 465 (51%) of the G-to-T transversions occurred at methylated CpG sites . These findings further strengthen a link between polycyclic aromatic hydrocarbons present in cigarette smoke and lung cancer mutations and provide evidence that mutational processes other than C-to-T transition mutations can occur selectively at methylated CpG sequences.

Cancer Res, 2001 Oct 1, 61(19), 7002 - 8
Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor; Lu D et al.; Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 {or fms-like tyrosine kinase receptor (Flt-1)} and VEGF receptor 2 {or kinase insert domain-containing receptor (KDR)} . Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis . We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis . Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF) . The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography . The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF . Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells . Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.

Mutat Res, 2001 May 10, 485(4), 339 - 44
Effects of low iron conditions on the repair of DNA lesions induced by Cumene hydroperoxide in Escherichia coli cells; Asad LM et al.; In the present study, we evaluated the sensitivity of different Escherichia coli strains to Cumene hydroperoxide (CHP) treatment under distinct conditions of Fe2+ availability . Our results showed that the pretreatment with an iron chelator (dipyridyl) protects all the tested strains against CHP toxic effects, but it was not sufficient to abolish the CHP induced mutagenesis . On the other hand, simultaneous pretreatment with both dipyridyl and neocuproine (copper chelator) leads to a complete protection against CHP mutagenic effects . Our data suggest the participation of copper ion in the CHP mutagenesis induced in E . coli.

Mutat Res, 2001 May 10, 485(4), 319 - 29
The SOS-dependent upregulation of uvrD is not required for efficient nucleotide excision repair of ultraviolet light induced DNA photoproducts in Escherichia coli; Crowley DJ et al.; We have shown previously that induction of the SOS response is required for efficient nucleotide excision repair (NER) of the major ultraviolet light (UV) induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), but not for repair of 6-4 photoproducts (6-4PP) or for transcription-coupled repair of CPDs {1} . We have proposed that the upregulation of cellular NER capacity occurs in the early stages of the SOS response and enhances the rate of repair of the abundant yet poorly recognized genomic CPDs . The expression of three NER genes, uvrA, uvrB, and uvrD, is upregulated as part of the SOS response . UvrD differs from the others in that it is not involved in lesion recognition but rather in promoting the post-incision steps of NER, including turnover of the UvrBC incision complex . Since uvrC is not induced during the SOS response, its turnover would seem to be of great importance in promoting efficient NER . Here we show that the constitutive level of UvrD is adequate for carrying out efficient NER of both CPDs and 6-4PPs . Thus, the upregulation of uvrA and uvrB genes during the SOS response is sufficient for inducible NER of CPDs . We also show that cells with a limited NER capacity, in this case due to deletion of the uvrD gene, repair 6-4PPs but cannot perform transcription-coupled repair of CPDs, indicating that the 6-4PP is a better substrate for NER than is a CPD targeted for transcription-coupled repair.

Graefes Arch Clin Exp Ophthalmol, 2001 Aug, 239(8), 609 - 12
Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin; Becker MD et al.; BACKGROUND: Intravital microscopy allows imaging of specific cell populations in vivo . The value of this technique is well established, but would be enhanced if one could distinguish functional states of cells in vivo . Interleukin-2 (IL-2) is expressed upon stimulation of T-cells and is a commonly used marker for T-cell activation . This study tests the use of enhanced green fluorescent protein (GFP) as a reporter gene for interleukin-2 (IL-2) expression in vivo . METHODS: Characterization of mice that have the GFP gene under the control of IL-2 regulatory sequences has previously been published . Uveitis was induced by injection of E . coli endotoxin into the vitreous of these IL-2/GFPki transgenic mice . Four hours later, 3 microg of recombinant mouse IL-2 was injected into the anterior chambers of one group of mice . In vivo imaging of infiltrating cells in the iris stroma was performed with fluorescence microscopy at 6, 24, 48, and 72 h after endotoxin injection . The absolute number of fluorescent cells per mm2 was evaluated . RESULTS: Eyes with endotoxin-induced uveitis had cells that expressed GFP and were identifiable by intravital microscopy . The fluorescent cells were exclusively seen in the subset of cells that had infiltrated the iris stroma or arrested along the vascular endothelium . The number of GFP-positive infiltrating cells in the iris increased from undetectable at baseline to 0.5 cells/mm2 at 6 h and 1.3 cells/mm2 at 72 h . The animals that received endotoxin as well as IL-2 tended to have more GFP-positive cells at the 48-h and 72-h time points, but these differences were not statistically significant CONCLUSIONS: GFP is commonly used as a reporter gene for in vitro expression assays . The results presented here document that transgenic mice with GFP under the control of IL-2 regulatory elements can be used with intravital microscopy for in vivo expression assays that allow detection of activated T-cells at multiple time points within the same animal . This provides a novel method for temporal and spatial studies on the state of cell activation in inflammatory responses.

Onderstepoort J Vet Res, 2001 Jun, 68(2), 91 - 9
Changes in some factors of the innate immunity and serum zinc and iron concentrations in pigs following intravenous administration of Escherichia coli lipopolysaccharide; Andonova M et al.; The changes in some factors of the innate immunity (phagocytosis, complement, lysozyme); haematological parameters-leukocytes, erythrocytes, differential white blood cell counts, haemoglobin, haematocrit and the serum concentrations of the microelements zinc and iron in six 2- to 3-months-old female piglets after the intravenous administration of lipopolysaccharide from Escherichia coli 0111:B4 were determined . It was found out that 1 h after the administration of lipopolysaccharide at the dosage rate of 10 microg/kg body weight resulted in a decrease in the phagocytic parameters, i.e . the phagocytic number and the index of phagocytic activity, which was followed by an increase in their values between post treatment hours 2 and 4 . The leukocyte counts had decreased by hour 2 after the injection, but thereafter increased, and at post treatment hour 72, a leukocytosis was observed . The differential white blood cell counts were characterized by a shift to the left between hours 2 and 4 and a statistically significant increase in lymphocyte counts at hour 48 of the experiment . The serum zinc concentrations were increased an hour after the lipopolysaccharide application; after which their average values were lower . The haemolytic activities (CH50) of the classical and the alternative pathways of complement activation decreased . The haemolytic activity (CH50) for the classical pathway began to increase at hour 48 following the treatment . Significant changes were not observed in lysozyme activity, serum iron concentrations or the related haematological parameters (erythrocytes and haemoglobin).

EMBO J, 1983, 2(5), 799 - 804
Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy; Evstafieva AG et al.; Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives . The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes . Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens . The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome . They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform) . Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.

J Biol Chem, 2001 Nov 30, 276(48), 45470 - 5 Epub 2001 Oct 02.
Intra- and intermolecular beta-pleated sheet formation in glutamine-repeat inserted myoglobin as a model for polyglutamine diseases; Tanaka M et al.; An aberrant structure of the expanded polyglutamine might be involved in the formation of aggregates in CAG repeat diseases . To elucidate structural properties of the expanded polyglutamine, we prepared sperm whale myoglobin (Mb) mutants, in which 12, 28, 35, and 50 repeats of glutamine were inserted at the corner between the C and D helices (Gln(12), Gln(28), Gln(35), and Gln(50), respectively) . Circular dichroism and IR spectroscopies showed that the expanded polyglutamine, which was recognized by the monoclonal antibody 1C2 in Gln(28), Gln(35), and Gln(50) Mb forms an antiparallel beta-pleated sheet structure . Gln(50) Mb aggregates were found to comprise an intermolecular antiparallel beta-pleated sheet . Fluorescence together with (1)H NMR spectra revealed partial unfolding of the protein surface in Gln(35) and Gln(50) Mb, although the structural changes in the protein core were rather small . The present results indicate that the fluctuating beta-pleated sheet of the expanded polyglutamine exposed on the protein surface facilitates the formation of aggregates through intermolecular interactions . The present study has first established and characterized structural properties of a molecular model for polyglutamine diseases in which various lengths of polyglutamine including a pathologically expanded glutamine repeat were inserted into a structurally known protein.

J Biol Chem, 2001 Dec 14, 276(50), 47615 - 22 Epub 2001 Oct 02.
Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery; Smisterova J et al.; Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells . Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes . Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency . The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure . In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl . If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures . The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology . We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.

J Biol Chem, 2001 Dec 7, 276(49), 45969 - 78 Epub 2001 Oct 02.
Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates . Evidence for reversible tight-binding inhibition; Nemeria N et al.; Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created . Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme . The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm . According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions . The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants . This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism . Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177) . Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region . In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment . In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Insect Biochem Mol Biol, 2001 Nov 1, 31(12), 1145 - 53
Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica; Wei SH et al.; Strains of the housefly, Musca domestica, highly resistant to organophosphate (OP) and other insecticides are known because they overproduce glutathione S-transferases (GSTs) . Previous work has shown that overproduction in these strains involved numerous isozymes with glutathione conjugating activities (Pesticide Biochem . Physiol., 25 (1986) 169; Mol . General Genetics, 227 (1991) 355; J . Biol . Chem., 267 (1992) 1840; Mol . General Genetics, 245 (1994) 236; J . Mol . Evol., 43 (1996) 236) . The current work describes the purification and identification of a M . domestica GST isozyme (pI 7.1) broadly specific for substrates from a housefly strain, Cornell-HR, that is highly resistant against OP-insecticides, and the isolation of two new MdGST genes using the antibody made against it . This isozyme, which was identified from amongst more than 20 isoelectric forms of GSTs of the same subunit size, was highly active for conjugating GSH to the model substrate 3,4-dichloronitrobenzne (DCNB) . When expressed in Escherichia coli, one of the cloned GSTs, MdGST-6A, produces an enzyme that conjugates glutathione to the insecticides methyl parathion and lindane . On indication that it was the most active isozyme toward several xenobiotics among several MdGSTs tested, we advance the notion that MdGST-6A probably plays an important role in M . domestica Cornell-HR's resistance towards OP-insecticides . MdGST-6A and a second closely related one found in this work, MdGST-6B, are members of the traditional insect class I family (theta-class) and share the greatest homologies with a cluster of Drosophila GSTs on locus 55 . In addition to having the unusually broad substrate specificity, the sequence of the new group of enzymes reveals that it has a highly diverged hydrophobic motif in its active site as compared to other class I GSTs from insects.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 199 - 205
An enteroaggregative Escherichia coli strain of serotype O111:H12 damages and invades cultured T84 cells and human colonic mucosa; Abe CM et al.; The pathogenic mechanisms of enteroaggregative Escherichia coli (EAEC) are not well defined . We investigated the interaction of EAEC strain 236 (serotype O111:H12) with polarised Caco-2 and T84 human intestinal epithelial cells lines, and with human jejunal and colonic mucosa . Strain 236 adhered to both polarised cell lines and to both intestinal tissue types, but caused severe damage and was invasive only in T84 cells and colonic mucosa . In contrast, prototype EAEC strain 042, which also adhered to the cultured intestinal cell lines, did not adhere to or invade jejunal or colonic tissue . These observations suggest a heterogeneity of virulence properties within the EAEC category of diarrhoea-causing E . coli.

Mol Cell, 2001 Sep, 8(3), 486 - 7
To cleave or not to cleave? Insights from the LexA crystal structure; Walker GC; In the September 7, 2001 issue of Cell, Luo et al . describe the structure of LexA protein in two states, cleavable and noncleavable . This structure offers new insights into how LexA and other structurally related proteins, such as lambda and UmuD, undergo autocatalytic cleavage using a Ser-Lys dyad.

Biochem J, 2001 Oct 15, 359(Pt 2), 427 - 34
Study of the interaction of the medium chain mu 2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28; Follows ER et al.; The medium chain mu 2 subunit (AP50) of the clathrin-associated adapter protein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXXPhi, where X can be any residue and Phi is a large hydrophobic residue . Using surface plasmon resonance combined with structural information, we have analysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4) . The crystal structure of AP50 in complex with a CTLA-4-derived peptide was determined to 3.6 A (1 A=0.1 nm) resolution . The binding domain of AP50 (residues 164-435) was expressed in Escherichia coli and purified . In agreement with previous reports, the AP50 domain bound to residues 152-174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine residue (position 165) . The interaction exhibited fast kinetics with rapid on and off rates and a K(d) of 0.7 microM . In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of binding of AP50 with five peptides with single changes in and around the YXXPhi motif to the equivalent residues of CD28 was made . T162H greatly reduced binding, whereas T161L had little effect . Mutations G163S, V164D and K167N all exhibited reduced binding . Modelling of the single amino acid changes using structural information, was in broad agreement with the binding data, demonstrating that residues outside of the YXXPhi motif are also important in the interaction of membrane proteins with AP50.

Biochem J, 2001 Oct 15, 359(Pt 2), 369 - 74
Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency; Ren X et al.; Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species . In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation {Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem . Biophys . Res . Commun . 198, 1240-1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem . J . 332, 251-255} . Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH . The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation . To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains . The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%, 15-20% and 25-30% of total bacterial proteins respectively . The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co(2+)-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration . The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46 x 10(5) M(-1) and 1.03 x 10(5) M(-1) respectively, which were less by one order of magnitude than that of the intact 2F3 antibody . The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394 units/micromol, which approaches the activity of rabbit liver GPX.

J Am Chem Soc, 2001 Oct 10, 123(40), 9749 - 59
Studies on the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA: elucidation of the inactivation mechanism and identification of cysteine-114 as the entrapped nucleophile; Dakoji S et al.; The inhibitory properties of (methylenecyclopropyl)formyl-CoA (MCPF-CoA), a metabolite derived from a natural amino acid, (methylenecyclopropyl)glycine, against bovine liver enoyl-CoA hydratase (ECH) were characterized . We have previously demonstrated that MCPF-CoA specifically targets ECHs, which catalyze the reversible hydration of alpha,beta-unsaturated enoyl-CoA substrates to the corresponding beta-hydroxyacyl-CoA products . Here, we synthesized (R)- and (S)-diastereomers of MCPF-CoA to examine the stereoselectivity of this inactivation . Both compounds were shown to be competent inhibitors for bovine liver ECH with nearly identical second-order inactivation rate constants (k(inact)/K(I)) and partition ratios (k(cat)/k(inact)), indicating that the inactivation is nonstereospecific with respect to ring cleavage . The inhibitor, upon incubation with bovine liver ECH, labels a tryptic peptide, ALGGGXEL, near the active site of the protein, where X is the amino acid that is covalently modified . Cloning and sequence analysis of bovine liver ECH gene revealed the identity of the amino acid residue entrapped by MCPF-CoA as Cys-114 (mature sequence numbering) . On the basis of gHMQC (gradient heteronuclear multiple quantum coherence) analysis with {3-(13)C}-labeled MCPF-CoA, the ring cleavage is most likely induced by the nucleophilic attack at the terminal carbon of the exomethylene group (C(2)') . We propose a plausible inactivation mechanism that involves relief of ring strain and is consistent with examples found in the literature . In addition, these studies provide important clues for future design of more efficient and selective inhibitors to control and/or regulate fatty acid metabolism.

Biochemistry, 2001 Aug 14, 40(32), 9677 - 84
Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase; van den Brink-van der Laan E et al.; Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases . In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins . Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding . In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC . For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites . On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein . We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC . The size of the insertion sites decreases with an increase in headgroup size . These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface . It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.

Biochemistry, 2001 Aug 14, 40(32), 9631 - 7
Kinetics in the pre-steady state of the formation of cystines in ribonucleoside diphosphate reductase: evidence for an asymmetric complex; Erickson HK; Two folded polypeptides, designated R1 and R2, respectively, combine in an as yet undefined stoichiometry to form ribonucleoside diphosphate reductase (ribonucleotide reductase) from Escherichia coli . Two pairs of cysteines in each R1 protomer have been implicated in the enzymatic mechanism . One pair, cysteines 225 and 462, is located in the active site of the enzyme and forms a cystine concomitant with the reduction of the ribonucleotide . The other pair, cysteines 754 and 759, is located near the carboxy terminus and is thought to reduce the cystine in the active site by disulfide interchange; either thioredoxin or glutaredoxin is then thought to reduce the cystine that results . Rapid quenching and site-directed immunochemistry have been used to follow the formation of the cystine in the active site and the peripheral cystine simultaneously during the pre-steady state . Prereduced R1 dimer of ribonucleoside diphosphate reductase, in the presence of ATP and CDP, was mixed with R2 dimer in an apparatus for quench flow . The reaction was quenched with a solution of acetic acid and N-ethylmaleimide, the protein was then precipitated with trichloroacetic acid, and the precipitate was separated into two portions . The percent of the cystine in the active site in one of the portions was determined as described previously {Erickson, H . K . (2000) Biochemistry 39, 9241-9250} . A similar method was employed to determine the percent of the peripheral cystine in the other portion of the precipitate . It was found that while the formation of both of these cystines was initiated by the addition of R2 dimer, presumably as products of the reduction of CDP, the peripheral cystine appeared to form more rapidly and in a higher yield than the cystine in the active site . These results demonstrate that the formation of the cystine between cysteines 754 and 759 of ribonucleotide reductase from E . coli is kinetically competent . A mechanism consistent with the prior formation of the cystine between cysteine 225 and cystene 462 as well as the kinetics for the formation of each cystine with time is presented . Because twice as much of the peripheral cystine than cystine in the active site had formed during the pre-steady state, it follows that the enzymatically competent complex between R1 dimers and R2 dimers cannot be symmetric.

Biochemistry, 2001 Aug 14, 40(32), 9587 - 95
Three-dimensional folding of the tRNA-like domain of Escherichia coli tmRNA; Zwieb C et al.; UV irradiation of Escherichia coli tmRNA both on and off the ribosome induced covalent cross-links between its 3'- and its 5'-terminal segments . Cross-linking was unaffected in a molecule that lacked the tag-peptide codon region and pseudoknots 2, 3, and 4 . Intact and truncated cross-linked tmRNAs were aminoacylated as efficiently as the respective nonirradiated molecules, suggesting that the added UV-induced bonds did not disturb tmRNA conformation . Using RNase H digestion followed by primer extension with reverse transcriptase, two cross-linked sites were identified within the tRNA-like region of tmRNA . The first was formed between nucleotides U9/U10 near the 5' end and nucleotides C346/U347 in the T loop . The second cross-link involved residues at positions 25-28 and 326-329 within helix 2a . Together with comparative sequence analysis, these findings yielded a three-dimensional model of the tRNA-like domain of E . coli tmRNA . Despite significant reduction of the D domain and the proximity of U9/U10 and C346/U347, the model closely resembles the L-shaped structure of canonical tRNA.

FEBS Lett, 2001 Aug 3, 502(3), 73 - 8
Molecular cloning and disruption of a novel gene encoding UDP-glucose: tetrahydrobiopterin alpha-glucosyltransferase in the cyanobacterium Synechococcus sp . PCC 7942; Choi YK et al.; The gene encoding UDP-glucose:tetrahydrobiopterin alpha-glucosyltransferase (BGluT) was cloned from the genomic DNA of Synechococcus sp . PCC 7942 . The encoded protein consisting of 359 amino acid residues was verified in vitro and in vivo to be responsible for the synthesis of tetrahydrobiopterin (BH4)-glucoside produced in the organism . The BGluT gene is the first cloned in pteridine glycosyltransferases and also a novel one cloned so far in UDP-glycosyltransferases . The mutant cells disrupted in the BGluT gene produced only aglycosidic BH4 at a level of 8.3% of the BH4-glucoside in wild type cells and exhibited half of the wild type growth in normal photoautotrophic conditions . These results suggest that the glucosylation of BH4 is required for the maintenance of the high cellular concentration of the compound, thereby supporting the normal growth of Synechococcus sp . PCC 7942.

J Endotoxin Res, 2001, 7(3), 237 - 41
Signal integration in lipopolysaccharide (LPS)-stimulated murine macrophages; Vogel S et al.; Using a panel of LPS-inducible genes, selected for the capacity of their products to contribute to endotoxicity, normal macrophages were compared to macrophages deficient in CD14, CD11b/CD18, or TLR4 to elicit gene expression in response to Escherichia coli LPS or the LPS mimetic, Taxol . All genes were TLR4-dependent . At low doses of LPS or Taxol, all genes were also CD14-dependent; however, IP-10 and ICSBP remained poorly inducible even at much higher concentrations . A distinct subset of genes (COX-2, IL-12 p40, and IL-12 p35) was CD11b/CD18-dependent . NF-B translocation and MAPK phosphorylation were dysregulated in receptor-deficient macrophages . In contrast to E . coli LPS, a Porphyromonas gingivalis LPS preparation was found to be TLR2-, rather than TLR4-dependent, and resulted in differential expression of genes within the panel . These data suggest that: (i) TLR4 is necessary, but not sufficient, to induce the full repertoire of genes examined; (ii) CD14 and CD11b/CD18 facilitate signaling for induction of select subsets of genes that are also TLR4-dependent; and (iii) signaling through TLR2 versus TLR4 differs quantitatively/qualitatively . These data support an LPS signaling complex on murine macrophages that minimally includes CD14, CD11b/CD18, and TLR4 to respond to E . coli LPS to elicit the full spectrum of gene expression.

J Biol Chem, 2001 Dec 7, 276(49), 45762 - 71 Epub 2001 Oct 01.
Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors; Leung D et al.; Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences . The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker . The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol . A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding . We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease . Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

J Biol Chem, 2001 Dec 14, 276(50), 47394 - 401 Epub 2001 Oct 01.
Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C; Gruz P et al.; DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins . The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA) . In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III . Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC) . Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC . The length of the synthesized DNA product reached at least 200 nucleotides . Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E . coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis . The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.

J Biol Chem, 2001 Dec 14, 276(50), 47474 - 82 Epub 2001 Oct 01.
Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis; Ko TP et al.; The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method . The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role . In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta-strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103 . The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs . Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C(70) polymer rather than the C(55) polymer . Ala-69 and Ala-143 are located near the top of the tunnel . In contrast to the A143V reaction, the C(30) intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant . These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C(55) product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C(55) product . The roles of residues located on a flexible loop were investigated . The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values . W75A showed an 8-fold increase of the FPP K(m) value, and 22-33-fold increases in the IPP K(m) values were observed for E81A and S71A . The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.

J Biol Chem, 2001 Dec 7, 276(49), 45744 - 50 Epub 2001 Oct 01.
Characterization of the dimerization domain in the FNR transcription factor; Moore LJ et al.; The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its {4Fe-4S} cluster and conversion to a monomeric form . As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain . Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo . Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization . Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface . This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face . In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face . These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.

J Biol Chem, 2001 Dec 14, 276(50), 47178 - 84 Epub 2001 Oct 01.
Crystal structure of MtaN, a global multidrug transporter gene activator; Godsey MH et al.; MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta . The 2.75 A resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (alpha5) that is involved in dimerization by the formation of an antiparallel coiled-coil . The hydrophobic core and helices alpha1 through alpha4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted . Differences between the orientation of alpha5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers . These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary.

J Biol Chem, 2001 Nov 30, 276(48), 44770 - 6 Epub 2001 Oct 01.
Olfaction in the gypsy moth, Lymantria dispar: effect of pH, ionic strength, and reductants on pheromone transport by pheromone-binding proteins; Kowcun A et al.; The pheromone-binding proteins (PBPs) are 16-kDa abundant proteins in specialized olfactory hairs in insects . The mechanism by which the PBPs remove the pheromone from the inner surface of sensory hairs and deliver it to the sensory cell remains unclear . Existing qualitative models postulate that pheromone is released near the dendrite by a decrease in pH or by a reduced form of the PBP . This study focuses on the two PBPs from the gypsy moth and the enantiomers of the pheromone cis-2-methyl-7,8-epoxyoctadecane . The pH dependence of pheromone binding has revealed three ionizations that are important . The type of ligand influences two of these ionizations . We propose that the (-)-enantiomer of the pheromone interacts with one of the ionizable residues on the protein while the (+)-enantiomer does not . Simultaneous variation of pH and KCl concentration in the physiological range or reduction of disulfide bridges does not change the affinity of PBP for pheromone . We propose a revised model of pheromone transport from the inner surface of the sensory hair to the sensory neuron.

Invest Ophthalmol Vis Sci, 2001 Oct, 42(11), 2563 - 6
Inhibition of leukocyte sticking and infiltration, but not rolling, by antibodies to ICAM-1 and LFA-1 in murine endotoxin-induced uveitis; Becker MD et al.; PURPOSE: Cell-adhesion molecules are critical elements in intravascular rolling and sticking of leukocytes during acute inflammation . In this process, selectins are thought to be involved in initial adhesion and rolling, and integrin-Ig superfamily interactions are believed primarily to mediate stronger adhesion and transendothelial migration . This study clarifies the role of two adhesion molecules, intercellular adhesion molecule (ICAM)-1 and leukocyte functional antigen (LFA)-1, in endotoxin-induced uveitis (EIU) . METHODS: Intravital microscopy was used to record the movement and location of leukocytes in the irises of mice with uveitis induced by intravitreal injection of 250 ng Escherichia coli endotoxin . Each mouse concurrently received an intraperitoneal injection of monoclonal neutralizing antibodies for ICAM-1, LFA-1, or both or control irrelevant antibodies . RESULTS: Mice treated with endotoxin and control antibodies had an inflammatory response that was clearly present at the 6- and 24-hour time points and was mostly resolved by 48 hours . Mice that received anti-ICAM-1 or anti-LFA-1 had significantly fewer cells infiltrating their irises at 6 and 24 hours . Detailed analysis of the 6-hour time point recordings revealed that neither anti-ICAM-1 nor anti-LFA-1 significantly reduced the number of leukocytes rolling on venule endothelial surfaces, but the treatments reduced the number of firmly adherent cells . CONCLUSIONS: These data confirm previous reports that ICAM-1 and LFA-1 are important mediators of EIU . The dynamic in vivo images clearly support the hypothesis that integrin-mediated cell adhesion is more critical for the firm adhesion of sticking cells than for leukocyte rolling.

Int Immunol, 2001 Oct, 13(10), 1233 - 42
Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70; Udono H et al.; Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL) . In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses . Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli . Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response . The point of injection was crucial for CTL induction . CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses . Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.

Eur J Endocrinol, 2001 Oct, 145(4), 497 - 503
In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion; Bolognani F et al.; OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro . Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E . coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas) . DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts . In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo . METHODS: GH and PRL release was measured by specific RIAs . In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) . Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures . RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively . In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting . In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals . Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas . CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.

Mol Microbiol, 2001 Sep, 41(6), 1459 - 67
Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision; Hinerfeld D et al.; The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays . Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends . Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon . Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916 . Mutations in these residues reduced binding of Xis . In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision . A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage . This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision . Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.

Mol Microbiol, 2001 Sep, 41(6), 1445 - 58
Enterohaemorrhagic and enteropathogenic Escherichia coli use a different Tir-based mechanism for pedestal formation; DeVinney R et al.; Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria . EHEC and a related pathogen, enteropathogenic E . coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation . An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host . In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC . In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir . The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC . In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC . EHEC forms pedestals with both wild-type EPEC Tir and the non-tyrosine-phosphorylatable EPEC Tir Y474F . Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell . These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.

Mol Microbiol, 2001 Sep, 41(6), 1237 - 47
Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin; Pei S et al.; The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT) . As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues . In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol . To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine . Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity . These single or double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins . Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.

Cell Microbiol, 2001 Oct, 3(10), 669 - 79
Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli; Ide T et al.; Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells . The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane . Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell . In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes . Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far . Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane . Osmoprotection revealed a minimal pore size of 3-5 nm . The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques . Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55-65 nm and to be raised 15-20 nm above the membrane plane . We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro . These results, together with the homologies of EspB and EspD to proposed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

Biochemistry, 2001 Oct 9, 40(40), 12220 - 9
A face on transmembrane segment 8 of the lactose permease is important for transport activity; Green AL et al.; Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 2 (TMS-2) play a critical role in conformational changes associated with lactose transport {Green, A . L., Anderson, E . J., and Brooker, R . J . (2000) J . Biol . Chem . 275, 23240-23246} . In the current study, mutagenesis was conducted along the side of predicted TMS-8 that contains the first amino acid in the conserved loop 8/9 motif . Several substitutions at positions 261, 265, 272, and 276 were markedly defective for downhill lactose transport although these mutants were well expressed . Substitutions along the entire side of TMS-8 containing the first amino acid in the loop 8/9 motif displayed defects in uphill lactose transport . Again, substitutions at positions 261, 265, 268, 272, and 276 were the most defective, with several of these mutants showing no lactose accumulation against a gradient . According to helical wheel plots, Phe-261, Thr-265, Gly-268, Asn-272, and Met-276 form a continuous stripe along one face of TMS-8 . These results are discussed according to our hypothetical model, in which the two halves of the protein form a rotationally symmetrical dimer . In support of this model, alignment of predicted TMS-2 and TMS-8 shows an agreement between the amino acid residues in these transmembrane segments that are critical for lactose transport activities.

Biochemistry, 2001 Oct 9, 40(40), 12207 - 14
Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC; Marmor S et al.; The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala) . The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue . The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states . Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI) . Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site . The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.

Biochemistry, 2001 Oct 9, 40(40), 12150 - 6
Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals; Dizdaroglu M et al.; Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA . We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals . Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases . Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases . Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics . The kinetic parameters also depended on the identity of the individual modified base being removed . Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study . This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates . The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.

J Struct Biol, 2001 Aug, 135(2), 139 - 46
ATP-induced structural change of the thermosome is temperature-dependent; Gutsche I et al.; Protein folding by chaperonins is powered by ATP binding and hydrolysis . ATPase activity drives the folding machine through a series of conformational rearrangements, extensively described for the group I chaperonin GroEL from Escherichia coli but still poorly understood for the group II chaperonins . The latter--archaeal thermosome and eukaryotic TRiC/CCT--function independently of a GroES-like cochaperonin and are proposed to rely on protrusions of their own apical domains for opening and closure in an ATP-controlled fashion . Here we use small-angle neutron scattering to analyze structural changes of the recombinant alpha-only and the native alphabeta-thermosome from Thermoplasma acidophilum upon their ATPase cycling in solution . We show that specific high-salt conditions, but not the presence of MgATP alone, induce formation of higher order thermosome aggregates . The mechanism of the open-closed transition of the thermosome is strongly temperature-dependent . ATP binding to the chaperonin appears to be a two-step process: at lower temperatures an open state of the ATP-thermosome is predominant, whereas heating to physiological temperatures induces its switching to a closed state . Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action .

J Struct Biol, 2001 Aug, 135(2), 115 - 25
Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy; Roseman AM et al.; We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms . This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure . Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP . Seven ATP molecules bind cooperatively to one heptameric ring of GroEL . This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism . The conformation of GroEL-ATP was determined at approximately 15-A resolution . In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state . Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map .

J Struct Biol, 2001 Aug, 135(2), 95 - 103
Review: a structural view of the GroE chaperone cycle; Grallert H et al.; The GroE chaperone system consists of two ring-shaped oligomeric components whose association creates different functional states . The most remarkable property of the GroE system is the ability to fold proteins under conditions where spontaneous folding cannot occur . To achieve this, a fully functional system consisting of GroEL, the cochaperone GroES, and ATP is necessary . Driven by ATP binding and hydrolysis, this system cycles through different conformational stages, which allow binding, folding, and release of substrate proteins . Some aspects of the ATP-driven reaction cycle are still under debate . One of these open questions is the importance of so-called "football" complexes consisting of GroEL and two bound GroES rings . Here, we summarize the evidence for the functional relevance of these complexes and their involvement in the efficient folding of substrate proteins .

J Struct Biol, 2001 Aug, 135(2), 84 - 93
Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones; Ben-Zvi AP et al.; Molecular chaperones are essential for the correct folding of proteins in the cell under physiological and stress conditions . Two activities have been traditionally attributed to molecular chaperones: (1) preventing aggregation of unfolded polypeptides and (2) assisting in the correct refolding of chaperone-bound denatured polypeptides . We discuss here a novel function of molecular chaperones: catalytic solubilization and refolding of stable protein aggregates . In Escherichia coli, disaggregation is carried out by a network of ATPase chaperones consisting of a DnaK core, assisted by the cochaperones DnaJ, GrpE, ClpB, and GroEL-GroES . We suggest a sequential mechanism in which (a) ClpB exposes new DnaK-binding sites on the surface of the stable protein aggregates; (b) DnaK binds the aggregate surfaces and, by doing so, melts the incorrect hydrophobic associations between aggregated polypeptides; (c) ATP hydrolysis and DnaK release allow local intramolecular refolding of native domains, leading to a gradual weakening of improper intermolecular links; (d) DnaK and GroEL complete refolding of solubilized polypeptide chains into native proteins . Thus, active disaggregation by the chaperone network can serve as a central cellular tool for the recovery of native proteins from stress-induced aggregates and actively remove disease-causing toxic aggregates, such as polyglutamine-rich proteins, amyloid plaques, and prions .

J Mol Biol, 2001 Oct 5, 312(5), 949 - 62
Quantitative analysis of DNA binding by the Escherichia coli arginine repressor; Szwajkajzer D et al.; Allosteric activation of the hexameric arginine repressor (ArgR) for specific operator DNA binding appears to involve alteration in its quaternary structure . Current models for activation include subunit assembly and/or domain rearrangements in response to binding of the coeffector l-arginine . To investigate the molecular basis for ArgR operator interactions, we have carried out a series of quantitative analyses of ArgR subunit assembly and of the affinity, stoichiometry, cooperativity, and l-arginine- and DNA sequence-dependence of ArgR-DNA binding . The results indicate that subunit assembly plays no role in activation, although communication among subunits of the ArgR hexamer is required for specific DNA binding . The data suggest that DNA is also an allosteric effector of ArgR .

J Mol Biol, 2001 Oct 5, 312(5), 935 - 47
Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks; Jones JM et al.; Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicase activity whose apparent function is to promote resumption of DNA synthesis following replication-fork arrest . Here, we describe how initiation of helicase activity on DNA forks is influenced by both fork structure and by single-strand DNA-binding protein . PriA could recognize and unwind forked substrates where one or both arms were primarily duplex, and PriA required a small (two bases or larger) single-stranded gap at the fork in order to initiate unwinding . The helicase was most active on substrates with a duplex lagging-strand arm and a single-stranded leading-strand arm . On this substrate, PriA was capable of translocating on either the leading or lagging strands to unwind the duplex ahead of the fork or the lagging-strand duplex, respectively . Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex . Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lagging-strand template . While single-strand-binding protein could inhibit binding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates . In the cell, single-strand-binding protein and fork structure may direct PriA helicase to translocate along the lagging-strand template of forked structures such that the primosome is specifically assembled on that DNA strand .

J Mol Biol, 2001 Oct 5, 312(5), 927 - 34
Energetics, stability, and prediction of transmembrane helices; Jayasinghe S et al.; We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability . Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences . We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 % . Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli . In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation . An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins . We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used .

J Mol Biol, 2001 Oct 5, 312(5), 921 - 6
Crystallographic analysis of Lac repressor bound to natural operator O1; Bell CE et al.; Previous structures of Lac repressor bound to DNA used a fully symmetric "ideal" operator sequence that is missing the central G-C base-pair present in the three natural operator sequences . Here we have determined the X-ray crystal structure of a dimeric Lac repressor bound to a 22 base-pair DNA with the natural operator O1 sequence and the anti-inducer ONPF, at 4.0 A resolution . The natural operator is bent in the same way as the symmetric sequence, due to the binding of the hinge helices of the repressor to the minor groove at the central GCGG sequence of O1 . Comparison of the structures of the repressor bound to the natural and symmetric operators shows very similar overall structures, with only slight rearrangements of the headpiece domains of the repressor . Analysis of crystals with iodinated DNA shows that the operator is uniquely positioned and allows for the sequence registration of the DNA relative to the repressor to be determined . The kink in the operator is centered between the left half-site and the central G-C base-pair of O1 . Our results are most consistent with a previously proposed model in which, relative to the complex with the symmetric operator, the repressor accommodates binding to the natural operator sequence by shifting the position of the right headpiece by one base-pair step towards the center of O1 .

J Mol Biol, 2001 Oct 5, 312(5), 915 - 20
A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart; Xue H et al.; The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin . Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory . In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli . This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor . The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain . Results from laser scattering studies suggest that the fragment forms trimers in solution . In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent . A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers . One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across . This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier . The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo . Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily .

J Mol Biol, 2001 Oct 5, 312(5), 899 - 906
The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form; Ravin NV et al.; The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends . Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends . A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres) . We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage . We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates . Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers . N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends . Our results suggest the following model of N15 prophage DNA replication . Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally . After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL . After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids . Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends .

Biologicals, 2001 Jun, 29(2), 123 - 32
Residual DNA quantification in clinical batches of BBG2Na, a recombinant subunit vaccine against human respiratory syncytial virus; Lokteff M et al.; BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate . This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials . Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold System assay . These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process . The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 microg BBG2Na, the highest tested clinical dose of antigen . This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical . This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold(trade mark)assay .

J Vet Diagn Invest, 2001 Sep, 13(5), 421 - 4
In vitro detection of Shiga toxin using porcine alveolar macrophages; Mengeling WL et al.; Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin {Stx}) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals . In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time . Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability . They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question . These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx.

Genes Chromosomes Cancer, 2001 Nov, 32(3), 212 - 21
GPHN, a novel partner gene fused to MLL in a leukemia with t(11;14)(q23;q24); Eguchi M et al.; We report a novel MLL-associated chromosome translocation t(11;14)(q23;q24) in a child who showed signs of acute undifferentiated leukemia 3 years after intensive chemotherapy that included the topoisomerase-II inhibitor VP 16 . Screening of a cDNA library of the patient's leukemic cells showed a novel fusion transcript between MLL and the Gephyrin (GPHN) gene on 14q24 . The resulting MLL-GPHN fusion gene encodes MLL AT hook motifs and a DNA methyltransferase homology domain fused to the C-terminal half of Gephyrin, including a presumed tubulin binding site and a domain homologous to the Escherichia coli molybdenum cofactor biosynthesis protein MoeA . Genomic breakpoint analysis showed potential in vitro topoisomerase-II DNA-binding sites spanning the breakpoints in both MLL and GPHN but no flanking sequences that might mediate homologous recombination . This suggests that MLL-GPHN may have been generated by VP 16/topoisomerase-II-induced DNA double-strand breaks, followed by error-prone DNA repair via non-homologous end joining . Gephyrin was originally identified as a submembraneous scaffold protein that anchors and immobilizes postsynaptic membrane neurotransmitter receptors to underlying cytoskeletal elements . It also is reported to bind to phosphatidylinositol 3,4,5-triphosphate binding proteins involved in actin dynamics and downstream signaling and interacts with ATM-related family member RAFT1 . Gephyrin domains in the chimeric protein therefore could contribute novel signal sequences or might modify MLL activity by oligomerization or intracellular redistribution .

J Biol Chem, 2001 Nov 30, 276(48), 44953 - 62 Epub 2001 Sep 28.
RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex; Levesque L et al.; Nuclear export of ribonucleoprotein complexes requires cis-acting signals and recognition by receptors that mediate translocation through the nuclear pore complex . Translocation is likely to involve a series of physical interactions between the ribonucleoprotein complex and nucleoporins within the nuclear pore complex . Here, we have characterized the function of NXT1 in the context of the Tap-dependent RNA export pathway . Tap has been implicated in the nuclear export of RNA transcripts derived from Mason-Pfizer monkey virus that contain the constitutive transport element . We demonstrate that NXT1 stimulates binding of a Tap-RNA complex to nucleoporins in vitro, and we provide mutational analysis that shows these interactions are necessary for nuclear export of an intron-containing viral mRNA in vivo . Tap contains separate domains for binding to nucleoporins and NXT1, both of which are critical for its export function . RNA export is mediated by a heterodimer of Tap and NXT1, and the function of NXT1 on this pathway is to regulate the affinity of the Tap-RNA complex for nucleoporins within the nuclear pore complex . We propose that NXT1-dependent binding of the Tap-RNA complex to the nucleoporin p62, which we have reconstituted in vitro using recombinant proteins, represents a single step of the translocation reaction.

Toxicology, 2001 Oct 30, 167(3), 199 - 205
Nitric oxide and iron overload . Limitations of ESR detection by DETC; Galleano M et al.; The ability of the ESR technique based on diethyldithiocarbamate (DETC) administration was studied as a suitable method to assess NO generation in vivo . The technique was successfully employed to measure NO generation after LPS treatment . DETC2-Fe-NO adducts were detected in liver homogenates of iron overloaded animals . When iron was administered to the animals simultaneously with LPS, NO-dependent signal increased 122%, but the content of NO2- and NO3- in sera was significantly lower (44%) as compared to LPS-treated rats . Iron dextran administration was responsible for a three-fold increase in the DETC2-Fe-NO content in non-LPS treated rats, while NOS activity and sera NO2- and NO3- levels remained unaffected . The adduct generation rate by a chemical NO-source was recorded in the presence of either control or iron overloaded homogenates supplemented with DETC in vivo . The exposure of liver homogenates to NO was performed either by the addition of 1 mM SNAP as NO donor or infusing an aqueous NO solution . In the presence of iron overloaded samples the adduct generation rate was 3.8-4.4-fold higher than in the presence of control samples . This effect restricts the applicability of the method to experimental conditions where iron levels remain constant, therefore it is not suitable for NO generation studies in experimental models where animals were subjected to iron overload.

Drugs, 2001, 61(11), 1599 - 624
Insulin glargine: a review of its therapeutic use as a long-acting agent for the management of type 1 and 2 diabetes mellitus; McKeage K et al.; Insulin glargine is a recombinant human insulin analogue produced by DNA technology using a nonpathogenic strain of Escherichia coli . Two modifications of human insulin result in a stable molecule which is soluble in slightly acidic conditions (pH 4.0) and precipitates in the neutral pH of subcutaneous tissue . Because of these properties, absorption of insulin glargine is delayed and the analogue provides a fairly constant, basal insulin supply without peaks in plasma insulin levels for approximately 24 hours, similar to that achieved by a continuous subcutaneous insulin infusion . Insulin glargine is indicated as a once daily subcutaneous injection to provide basal glycaemic control in adults and children aged >6 years with type 1 diabetes mellitus and in adults with type 2 diabetes mellitus . Fasting plasma glucose and fasting blood glucose levels generally improved to a greater extent in patients with type 1 diabetes mellitus receiving insulin glargine than patients who administered Neutral Protamine Hagedorn (NPH) insulin . In patients with type 1 or 2 disease, glycosylated haemoglobin levels were slightly reduced and to a similar extent with insulin glargine and NPH insulin . Most clinical trials in patients with type 1 or 2 diabetes mellitus demonstrated a lower incidence of hypoglycaemia, especially nocturnal hypoglycaemia, compared with NPH insulin . One of the most common adverse events with insulin glargine treatment was injection site pain which, in some studies, occurred more frequently than in patients receiving NPH insulin . In all cases the symptoms were mild and treatment discontinuation was not required . Otherwise, the drug is well tolerated and does not appear to be immunogenic . CONCLUSIONS: Insulin glargine once a day provides basal control of glycaemia for approximately 24 hours without inducing peaks in plasma insulin levels in patients with type 1 or 2 diabetes mellitus . In long term, well designed trials insulin glargine once daily improved glycaemic control at least as effectively as NPH insulin given once or twice daily . The drug was well tolerated and in most studies the incidence of nocturnal hypoglycaemia was significantly less in patients treated with insulin glargine compared with patients receiving NPH insulin . Therefore, insulin glargine is likely to be a useful addition to the armamentarium of insulin therapy by establishing basal glycaemic control with once daily administration and a reduced risk of nocturnal hypoglycaemia.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1928 - 31
A transformation system for an ectomycorrhizal basidiomycete, Lyophyllum shimeji; Saito T et al.; A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraidehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli . This vector was introduced into protoplasts of L . shimeji and 3.4 transformants per microg plasmid DNA were obtained . In most of the transformants, multiple copies of the vector were integrated into the genomic DNA . The results indicate that pLS-hph is a useful vector for L . shimeji.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1832 - 9
A recombinant molt-inhibiting hormone of the kuruma prawn has a similar secondary structure to a native hormone: determination of disulfide bond arrangement and measurements of circular dichroism spectra; Katayama H et al.; In crustaceans, molt-inhibiting hormone (MIH) is presumed to regulate molting through suppressing synthesis and/or secretion of ecdysteroids by the Y-organ . Recently, a recombinant MIH of the kuruma prawn Penaeus japonicus was produced in E . coli . To approximate the secondary structure of native and recombinant MIH of P . japonicus containing six cysteine residues, the arrangements of disulfide bridges in both MIHs were determined by characterizing their enzymatic digests, and their circular dichroism spectra were measured . The arrangements of disulfide bonds in both MIHs were determined to be identical, and they were linked between Cys7 and Cys44, Cys24 and Cys40, and Cys27 and Cys53 . The circular dichroism spectra of both MIHs were very close, and demonstrated that they were rich in a-helix . a-Helix contents in native and recombinant MIHs were calculated to be 49.3% and 46.0%, respectively . All these results strongly suggested that the recombinant MIH was folded in the same manner as the native MIH.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1824 - 31
Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520; Tsujibo H et al.; The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides . The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed . The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli . The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids . The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases . The bxlA gene was expressed in E . coli, and the recombinant protein was purified to homogeneity . The enzyme was a monomer with a molecular mass of 82 kDa . The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested . Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan . High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1713 - 23
Influence of alpha-helices on the emulsifying properties of proteins; Poon S et al.; A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier . This molecule, peptide 1-55, has two potential amphipathic alpha-helices and a hydrophilic C-terminal domain . The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains . The emulsifying activity of the peptides lacking either of the alpha-helices was correlated with the hydrophobic moments of their respective helices . The hydrophobic moment is a measure of the amphipathicity of alpha-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains alpha-helices.

J Biol Chem, 2001 Nov 30, 276(48), 44419 - 26 Epub 2001 Sep 27.
Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases; Arakaki AK et al.; In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket . To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis . Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin . Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value . FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly . Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein . Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)

J Biol Chem, 2001 Nov 30, 276(48), 44521 - 6 Epub 2001 Sep 27.
Transfer of sulfur from IscS to IscU during Fe/S cluster assembly; Urbina HD et al.; The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins . NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated . We have employed {(35)S}cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins . IscS catalyzed transfer of (35)S from {(35)S}cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier . Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism . Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU . A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU . The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

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