Biochim Biophys Acta, 2003 Jun 26, 1649(1), 16 - 23 Naturally occurring Phe151Leu substitution near a conserved folding module lowers stability of glutathione transferase P1-1; Lin HJ et al.; Glutathione transferases (GSTs) are a family of enzymes that detoxify electrophilic compounds, such as carcinogens or drugs, by conjugating them to glutathione . The enzymes have contributed to the understanding of protein structure, due to large differences in amino acid sequence within the family, yet similar architecture and folding . Our objective was to conduct a systematic survey of GSTP1 polymorphisms and their function . Nearly all variants detected were known polymorphisms: IVS4+13C>A; Ile105Val; Ala114Val; and g.2596T>C (Ser185Ser) . However, we also found a novel Phe151Leu substitution in an African-American subject (1 out of 111) . Kinetic parameters for the conjugation reaction with 1-chloro-2,4-dinitrobenzene (CDNB) were determined for the novel variant enzyme purified via heterologous expression in Escherichia coli . Five substrates were used for measurement of specific activities, including isothiocyanate compounds that occur in cruciferous vegetables (benzylisothiocyanate, phenethylisothiocyanate, and sulforaphane) . Such isothiocyanate substrates are potential cancer chemopreventive agents that are conjugated by GSTs . No major change in kinetic parameters was observed . However, the half-life at 50 degrees C of the Leu 151 enzyme was reduced to 12 min, as compared to 28 min for the Phe 151 enzyme . Residue 151 is located at the N-terminus of helix alpha6 in GST motif II, surrounded by hydrophobic residues, and near the conserved "hydrophobic staple" and N-capping box motifs . These local structural elements aid in formation of helix alpha6 and promote proper folding and protein stability . Analysis of the three-dimensional structure showed that substitution of Phe 151 with Leu produces a hydrophobic cavity in the GSTP1 core, thereby destabilizing its structure . Phe151Leu represents one of the first-described allelic variations in a protein folding motif. Poult Sci, 2003 Jun, 82(6), 870 - 5 Molecular approaches to disease control; Babiuk LA et al.; Recent advances in molecular biology, genomics, and immunology are revolutionizing our approach to managing infectious diseases of humans, livestock, and poultry . One of the most interesting additions to the armamentarium of research focusing on controlling infectious diseases has been a better understanding of how the host's innate immune system recognizes "danger" signals . Additionally, there has been recognition of the relationship between the innate and the specific arms of the immune system . For example, the recent discovery that CpG motifs can modulate immune responses has been used both as an adjuvant to enhance the responses to vaccines, as well as a direct immunostimulant to prevent infections . Using an Escherichia coli chicken model, we have been able to prevent cellulitis in chickens with CpG alone . Thus, CpG can be used immunoprophylactically to reduce infectious diseases . In addition, we will describe how CpG formulations with various antigens; recombinant proteins, peptides, and conventional vaccines can enhance immune responses to each of these different vaccine combinations . What is even more interesting is that CpG incorporation in vaccines can shift the immune response from a predominant T helper 2 (Th2)-like immune response generally induced by killed or subunit proteins to a much more balanced Th1-Th2 response . These immunomodulatory effects have significant implications for management of infectious diseases of all vertebrates. Zhonghua Fu Chan Ke Za Zhi, 2003 Mar, 38(3), 154 - 7, 3-1 {Evaluation of construction and expression shiga toxin2 A-luteinizing hormone releasing hormone recombinant toxin}; Yue Y et al.; OBJECTIVE: The purpose is to construct shiga toxin2A-luteinizing hormone releasing hormone (Stx2A-LHRH) recombinant toxin which can kill cancer cells specifically and try to get a new target-binding drug . METHODS: The fragment of Stx2A-LHRH DNA amplified by polymerase chain reaction (PCR) were cloned into plasmid pET-20b(+) vector . The recombinant plasmid pET-Stx2A-LHRH was constructed successfully and identified by endonucleases digestion and sequencing analysis then it was transformed to Escherichia coli BL21 (DE3) and expressed under the induction of isopropyl-beta-D-thiogalactopyranoside . RESULTS: The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel thin layer scanning proved a special band of a molecular weight of about 38,000, accounting for 10.32% of total amount of the supernatant protein . Stx2A-LHRH recombinant toxin can kill HeLa cells clearly . CONCLUSION: Stx2A-LHRH recombinant toxin may become a choice of target-binding drug in the future. Org Lett, 2003 Jun 26, 5(13), 2223 - 6 Studies on the substrate specificity of Escherichia coli galactokinase; Yang J et al.; In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates . Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application . This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK . The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes . {reaction: see text} Hum Mutat, 2003 Jul, 22(1), 24 - 34 Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients; Gao HZ et al.; Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1 . ASS functions as a rate-limiting enzyme in the urea cycle . Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide . In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries . By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations . Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified . On the basis of primary sequence comparisons with the crystal structure of E . coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein . It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one) . Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14 . It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe . The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild . Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum . However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease . Hum Mutat, 2003 Jul, 22(1), 12 - 23 Clear relationship between ETF/ETFDH genotype and phenotype in patients with multiple acyl-CoA dehydrogenation deficiency; Olsen RK et al.; Mutations in electron transfer flavoprotein (ETF) and its dehydrogenase (ETFDH) are the molecular basis of multiple acyl-CoA dehydrogenation deficiency (MADD), an autosomal recessively inherited and clinically heterogeneous disease that has been divided into three clinical forms: a neonatal-onset form with congenital anomalies (type I), a neonatal-onset form without congenital anomalies (type II), and a late-onset form (type III) . To examine whether these different clinical forms could be explained by different ETF/ETFDH mutations that result in different levels of residual ETF/ETFDH enzyme activity, we have investigated the molecular genetic basis for disease development in nine patients representing the phenotypic spectrum of MADD . We report the genomic structures of the ETFA, ETFB, and ETFDH genes and the identification and characterization of seven novel and three previously reported disease-causing mutations . Our molecular genetic investigations of these nine patients are consistent with three clinical forms of MADD showing a clear relationship between the nature of the mutations and the severity of disease . Interestingly, our data suggest that homozygosity for two null mutations causes fetal development of congenital anomalies resulting in a type I disease phenotype . Even minute amounts of residual ETF/ETFDH activity seem to be sufficient to prevent embryonic development of congenital anomalies giving rise to type II disease . Overexpression studies of an ETFB-D128N missense mutation identified in a patient with type III disease showed that the residual activity of the mutant enzyme could be rescued up to 59% of that of wild-type activity when ETFB-D128N-transformed E . coli cells were grown at low temperature . This indicates that the effect of the ETF/ETFDH genotype in patients with milder forms of MADD, in whom residual enzyme activity allows modulation of the enzymatic phenotype, may be influenced by environmental factors like cellular temperature . Nature, 2003 Jun 19, 423(6942), 893 - 7 RecBCD enzyme is a bipolar DNA helicase; Dillingham MS et al.; Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination . For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme . Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity . We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation . These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex . This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function. Nature, 2003 Jun 19, 423(6942), 889 - 93 RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity; Taylor AF et al.; Helicases are molecular motors that move along and unwind double-stranded nucleic acids . RecBCD enzyme is a complex helicase and nuclease, essential for the major pathway of homologous recombination and DNA repair in Escherichia coli . It has sets of helicase motifs in both RecB and RecD, two of its three subunits . This rapid, highly processive enzyme unwinds DNA in an unusual manner: the 5'-ended strand forms a long single-stranded tail, whereas the 3'-ended strand forms an ever-growing single-stranded loop and short single-stranded tail . Here we show by electron microscopy of individual molecules that RecD is a fast helicase acting on the 5'-ended strand and RecB is a slow helicase acting on the 3'-ended strand on which the single-stranded loop accumulates . Mutational inactivation of the helicase domain in RecB or in RecD, or removal of the RecD subunit, altered the rates of unwinding or the types of structure produced, or both . This dual-helicase mechanism explains how the looped recombination intermediates are generated and may serve as a general model for highly processive travelling machines with two active motors, such as other helicases and kinesins. J Biol Chem, 2003 Sep 5, 278(36), 34717 - 24 Epub 2003 Jun 18. Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization; Tanaka M et al.; We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization . Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degrees C and that its formation was completed in approximately 3 h . A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to approximately 90 monomers and a bowl-like structure with long and short axes of 400 and 190 A, respectively . Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 A-1 corresponding to the 4.8-A spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine . These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril . The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine. J Biol Chem, 2003 Sep 19, 278(38), 36341 - 9 Epub 2003 Jun 18. Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1; Kirchhofer D et al.; Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases . In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form . By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1 . To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced . First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C . Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm) . Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2 . Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines . Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA . Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1. J Exp Bot, 2003 Aug, 54(389), 1833 - 9 Epub 2003 Jun 18. Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure; Heiss S et al.; Higher plants respond to cadmium exposure with the production of phytochelatins (PCn), small heavy metal binding peptides, which are synthesized from glutathione by phytochelatin synthase (PCS) . The isolation of a PCS cDNA clone from Brassica juncea L . cv . Vitasso, a candidate species for phytoremediation, is reported here . CLUSTAL analysis revealed a close relationship of BjPCS1 with PCS proteins from Arabidopsis thaliana and Thlaspi caerulescens . BjPCS1 expressed as recombinant protein in E . coli had PCS activity in vitro that was activated by 50 microM Cu and 200 microM Cd to a similar extent . Immunoblot analysis with an antiserum directed against recombinant BjPCS1 showed constitutive PCS expression during plant development . As a percentage of the total protein, the expression was higher in the roots, internodes and petioles in comparison with the leaf tissue . When B . juncea plants were treated with 25 microM cadmium, PCn accumulated increasingly over a 6 d period . Levels in shoots were about 3-fold higher than in roots . Prolonged cadmium exposure caused a significant increase of PCS protein in leaves, whereas in roots PCS protein levels were not affected. J Exp Bot, 2003 Aug, 54(389), 1997 - 9 Epub 2003 Jun 18. Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice; Suzuki K et al.; A cDNA fragment was cloned from rice immature seeds by the RT-PCR method . The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis . The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS . The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period . This is the first report of the cloning of the uxs gene from monocots. Drug Metab Dispos, 2003 Jul, 31(7), 955 - 66 Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation; Weaver R et al.; Detailed cytochrome P450 (P450) inhibition profiles are now required for the registration of novel molecular entities . This method uses combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reductases . Kinetic analysis of single substrate with single P450 (sP450) yielded apparent Km values of 25, 2, 20, 9, and 3 microM, for CYP1A2, 2C9, 2C19, 2D6, and 3A4, respectively . Combined substrates with combined P450s (cP450) yielded apparent Km values of 65, 4, 19, 7, and 2 microM . Selectivity of the substrates for each P450 isoform was checked . Phenacetin proved to be the least selective substrate . However, the ratio of the various P450s was modified in the final assay such that metabolism of phenacetin by other enzymes was approximately 20% of the metabolism by CYP1A2 . IC50 determinations with alpha-naphthoflavone (0.04 microM), sulfaphenazole (0.26 microM), tranylcypromine (9 microM), quinidine (0.02 microM), and ketoconazole (0.01 microM) were similar for sP450 and cP450 enzymes . The assay was further evaluated with 11 literature compounds and 52 in-house new chemical entities, and the data compared with radiometric/fluorescent values . The overall protein level of the assay was reduced from the original starting point, as this led to some artificially high IC50 measurements when compared with existing lower protein assays (radiometric/fluorometric) . This method offers high throughput P450 inhibition profiling with potential advantages over current radiometric or fluorometric methods. J Biotechnol, 2003 Jun 26, 103(2), 113 - 7 Production and characterization of bioactive recombinant resistin in Escherichia coli; Juan CC et al.; Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity . The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance . Here, we report the production of bioactive recombinant resistin in Escherichia coli . cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells . The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E . coli JM109 . After IPTG induction, the rec . resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol . The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity . After a quasi-static-like refolding process, the secondary structure of the rec . resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure . No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions . The rec . resistin showed a dose-dependent antagonizing action against insulin in {3H}-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin . A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1) . This result may indicate that the rec . resistin does not need to form homodimers to establish its bioactivity . The rec . resistin will be useful for exploring the biological functions of this newly discovered hormone. Int J Parasitol, 2003 Jul, 33(7), 721 - 31 Characterisation of a Rho homologue of Schistosoma mansoni; Vermeire JJ et al.; The development and survival of the helminth parasite, Schistosoma mansoni, is dependent on its ability to interpret signals from its environment . Currently, little is known about signal transduction in schistosomes . Rho is a member of a super-family of small GTP-binding proteins . Rho is involved in a number of cell signalling pathways with effects on actin cytoskeleton organisation, gene transcription, cell cycle progression, and membrane trafficking . We have cloned an S . mansoni protein (Rho1) that has 71-75% identity and approximately 85% similarity with human Rho A, B, and C proteins . We have optimised expression of recombinant S . mansoni Rho1 protein in Escherichia coli by co-expression with rare tRNAs . Western blot analysis results showed expression of Rho1 protein in adult worm stages especially female worms . In vitro prenylation of recombinant S . mansoni Rho1 determined that, similar to Rho from other organisms, Rho1 is geranynlgeranylated but not farnesylated . A search of the gene database indicates that Rho GTPases exist as a small family in S . mansoni including orthologues of Rho, Cdc42, and Rac . These data suggest that S . mansoni Rho1 plays a role in signalling in adult worms, especially females. J Theor Biol, 2003 Jul 21, 223(2), 199 - 203 In-phase implies large likelihood for independent codon model: distinguishing coding from non-coding sequences; Zheng WM et al.; It is proven that under the independent codon model, the likelihood of a DNA coding sequence read according to the correct frame is asymptotically larger than that read with an incorrect frame . Based on this proposition, a single set of probabilities of the codon usage is enough for discriminating the six frames of coding sequences under the independent codon model . The direct coding sequence of Escherichia coli genome is taken as an example to examine the codon independency by using the mutual information and chi2 analysis . The contrast between the coding frame and the two offset frames is evident . A self-learning approach for generating training set is proposed to estimate probability parameters. BMC Mol Biol . 2003 Jun 18;4(1):8. Functional interaction between RNase III and the Escherichia coli ribosome; Allas U et al.; BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria . Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity . RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA . It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end . When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3) . We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes . Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific . CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site. J Proteome Res, 2003 May-Jun, 2(3), 239 - 42 Carbamylation of proteins in 2-D electrophoresis--myth or reality? McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, Herbert B. Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps . This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point . Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS . We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification . The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing . Therefore, it is the temperature during these post-extraction procedures that is the most critical factor . Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times . However, under poorly controlled sample preparation and storage conditions, it can become a major event. Biotechniques, 2003 Jun, 34(6), 1272 - 9 Microarray-based method for combinatorial library sequence mapping and characterization; Abecassis V et al.; Here we describe a DNA-chip-based method for high-throughput sequence mapping . This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products . Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations . The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family . We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution. Shock, 2003 Jul, 20(1), 41 - 5 Hypothermia induces interleukin-10 and attenuates injury in the lungs of endotoxemic rats; Sarcia PJ et al.; We recently reported that hypothermia protects against intrapulmonary nitric oxide overproduction and nitric oxide-mediated lung injury in endotoxemic rats . Few studies have been performed to investigate whether hypothermia reduces inflammation by affecting favorable changes in chemokine and pro- and anti-inflammatory cytokine profiles . In this study, we tested the hypothesis that hypothermia decreases concentrations of growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1), interleukin (IL)-1beta, IL-6, and myeloperoxidase and increases concentration of IL-10 in the lungs endotoxemic rats . Twelve rats were anesthetized and randomized to treatment with either hypothermia (T = 18-24 degrees C; n = 6) or normothermia (T = 36-38 degrees C, n = 6) . Endotoxin (15 mg/kg of Escherichia coli lipopolysaccharide) was administered intravascularly and lung tissue was harvested 150 min later . Three additional rats were sham instrumented and maintained as normothermic but not given endotoxin . Hematoxylin & eosin staining was performed for qualitative inspection of tissues . Quantitative analyses of lung homogenates were performed using enzyme-linked immunosorbent assays for IL-1beta, IL-6, IL-10, and GRO/CINC-1 . Myeloperoxidase concentrations were determined using a colorimetric assay . Hypothermia attenuated the induction of intrapulmonary IL-1beta (P < 0.05), IL-6 (P < 0.05), GRO/CINC-1 (P < 0.05), and myeloperoxidase (P < 0.05) caused by endotoxin . Inspection of the lungs revealed that hypothermia similarly attenuated histological signs of injury, such as interstitial edema and neutrophil accumulation . Hypothermia increased the intrapulmonary concentration of IL-10 more than 3-fold over that measured in the normothermia (endotoxin-exposed) group (P < 0.05) . Hypothermia inhibits neutrophil recruitment in the lungs of endotoxemic rats in part by decreasing proinflammatory cytokine expression . Additionally, hypothermia induces intrapulmonary IL-10 expression . Further studies are needed to investigate whether IL-10 mediates the anti-inflammatory effects of hypothermia. Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8188 - 92 Epub 2003 Jun 17. A mechanism of covalent substrate binding in the x-ray structure of subunit K of the Escherichia coli dihydroxyacetone kinase; Siebold C et al.; Dihydroxyacetone (Dha) kinases are homologous proteins that use different phosphoryl donors, a multiphosphoryl protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system in bacteria, ATP in animals, plants, and some bacteria . The Dha kinase of Escherichia coli consists of three subunits, DhaK and DhaL, which are colinear to the ATP-dependent Dha kinases of eukaryotes, and the multiphosphoryl protein DhaM . Here we show the crystal structure of the DhaK subunit in complex with Dha at 1.75 A resolution . DhaK is a homodimer with a fold consisting of two six-stranded mixed beta-sheets surrounded by nine alpha-helices and a beta-ribbon covering the exposed edge strand of one sheet . The core of the N-terminal domain has an alpha/beta fold common to subunits of carbohydrate transporters and transcription regulators of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system . The core of the C-terminal domain has a fold similar to the C-terminal domain of the cell-division protein FtsZ . A molecule of Dha is covalently bound in hemiaminal linkage to the N epsilon 2 of His-230 . The hemiaminal does not participate in covalent catalysis but is the chemical basis for discrimination between short-chain carbonyl compounds and polyols . Paralogs of Dha kinases occur in association with transcription regulators of the TetR/QacR and the SorC families, pointing to their biological role as sensors in signaling. J Bacteriol, 2003 Jul, 185(13), 3972 - 7 The dinB operon and spontaneous mutation in Escherichia coli; McKenzie GJ et al.; Apparently conflicting data regarding the role of SOS-inducible, error-prone DNA polymerase IV (DinB) in spontaneous mutation are resolved by the finding that mutation is reduced by a polar allele with which dinB and neighboring yafN are deleted but not by two nonpolar dinB alleles . We demonstrate the existence of a dinB operon that contains four genes, dinB-yafN-yafO-yafP . The results imply a role for yafN, yafO, and/or yafP in spontaneous mutation. J Bacteriol, 2003 Jul, 185(13), 3948 - 57 Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus; Bartolucci S et al.; LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation . One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS) . Fine mapping performed in comparison to the genomic sequence of S . solfataricus P2 indicated an extended deletion of approximately 13 kb . The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058 . In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid . Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily . Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside). J Bacteriol, 2003 Jul, 185(13), 3871 - 7 NikAB- or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors; Furuya N et al.; The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer . We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer . In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18 . Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64 . This recombination was found to be independent of both the recA gene and conjugative DNA transfer . The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination . Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site . Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E . coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene . These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E . coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB. J Bacteriol, 2003 Jul, 185(13), 3863 - 70 Functional characterization of cysteine residues in GlpT, the glycerol 3-phosphate transporter of Escherichia coli; Fann MC et al.; In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate . Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration approximately 35 microM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and {2-(trimethylammonium)ethyl}methanethiosulfonate (MTSET), have no effect . Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS . The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution . To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop . In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes . Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway. J Bacteriol, 2003 Jul, 185(13), 3821 - 7 The C-terminal flexible domain of the heme chaperone CcmE is important but not essential for its function; Enggist E et al.; CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli . It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c . The recently solved structure of soluble CcmE revealed a compact core consisting of a beta-barrel and a flexible C-terminal domain with a short alpha-helical turn . In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus . Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely . For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence (131)DENYTPP(137) was required . When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE . Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE. J Bacteriol, 2003 Jul, 185(13), 3804 - 12 Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli; Franke S et al.; The cus determinant of Escherichia coli encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux . CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance . Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance . Substitutions for several other methionine residues of this protein did not have any effect . The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein . CusF bound one copper per polypeptide . The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm . Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis . CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay . In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF . These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex. J Bacteriol, 2003 Jul, 185(13), 3773 - 9 Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin; van den Brink-van der Laan E et al.; MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis . The crystal structure of Escherichia coli MurG (S . Ha, D . Walker, Y . Shi, and S . Walker, Protein Sci . 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site . To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E . coli in the absence of detergent . Surprisingly, we found that lipid vesicles copurify with MurG . Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression . This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell . The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells . The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%) . MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol . These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane . In addition, the results show a special role for cardiolipin in the MurG-membrane interaction. J Bacteriol, 2003 Jul, 185(13), 3745 - 52 Sites of interaction between the FecA and FecR signal transduction proteins of ferric citrate transport in Escherichia coli K-12; Enz S et al.; Transcription of the fecABCDE ferric citrate transport genes of Escherichia coli K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm . FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor . In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR(237-317) fragment interacts with the N-terminal FecA(1-79) fragment . In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis . They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins . The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium . Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E) . One FecR mutant, FecR (D138E, V197A), induced fecA promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA . The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the fec transport genes and identified sites in FecR and FecA that are important for signal transduction. J Bacteriol, 2003 Jul, 185(13), 3696 - 702 Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli; Minagawa S et al.; Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner . The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2) . The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes . Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon . Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously . In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP . Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E . coli. J Bacteriol, 2003 Jul, 185(13), 3690 - 5 Mutants suppressing novobiocin hypersensitivity of a mukB null mutation; Adachi S et al.; The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli . A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin . In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation . All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit . We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain. J Biol Chem, 2003 Sep 12, 278(37), 35265 - 71 Epub 2003 Jun 17. Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex; Samanta S et al.; Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis . MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain . The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W . (2000) EMBO J . 19, 5951-5961) . Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct . Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface . Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter . Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle . Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected . These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation. J Biol Chem, 2003 Aug 29, 278(35), 32501 - 6 Epub 2003 Jun 17. The twin-arginine leader-binding protein, DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase; Papish AL et al.; The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria . Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits . Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase . In this work, the targeting of DmsD within Escherichia coli was investigated . Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E . coli and a dmsABC mutant E . coli under anaerobic conditions . In contrast, DmsD is predominantly found in the cytoplasmic fraction of a Delta tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex . Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wild-type E . coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane . Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E . coli inner membrane . Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E . coli inner membrane. J Biol Chem, 2003 Sep 12, 278(37), 35597 - 608 Epub 2003 Jun 16. Downstream DNA sequence effects on transcription elongation . Allosteric binding of nucleoside triphosphates facilitates translocation via a ratchet motion; Holmes SF et al.; The ability of RNA polymerase (RNAP) to adopt multiple conformations is central to transcriptional regulation . In previous work, we demonstrated that RNAP can exist in an unactivated state that catalyzes synthesis slowly and an activated state that catalyzes synthesis rapidly, with the transition from the unactivated to the activated state being induced by the templated NTP binding to an allosteric site on the RNAP . In this work, we investigate the effects of downstream DNA sequences on the kinetics of single nucleotide incorporation . We demonstrate that changing the identity of the DNA base 1 bp downstream (+2) from the site of incorporation (+1) can regulate the catalytic activity of RNAP . Combining these data with sequence and structural analyses and molecular modeling, we identify the streptolydigin-binding region (Escherichia coli beta residues 543-546), which lies across from the downstream DNA, as the putative allosteric NTP binding site . We present a structural model in which the NTP binds to the streptolydigin loop and upon pairing with the +1 DNA base in the unactivated state or the +2 DNA base in the activated state facilitates translocation via a ratchet motion . This model provides an alternative mechanism for pausing as well as a structural explanation not only for our kinetic data but also for data from elongation studies on yeast RNAP II. Yi Chuan Xue Bao, 2003 Mar, 30(3), 209 - 14 Expression of biologically active human tumor necrosis factor beta by Streptomyces lividans; Hong B et al.; The production of human tumor necrosis factor beta (hTNF beta) in Streptomyces lividans was studied . The expression of hTNF beta uses the transcription, translation and secretion signals of subtilisin inhibitor VSI which is naturally produced by Streptomyces venezuelae CBS762.70 . In direct secretory expression cassette, hTNF beta cDNA was fused 2 amino acids after the signal peptidase cleavage site . In fusion expression cassette, hTNF beta cDNA was fused after the total vsi gene . In intracellular expression cassette, hTNF beta cDNA was fused after initiation codon ATG . The expression cassettes were subcloned into Streptomyces multi-copy plasmid pIJ486 respectively and transformed into S . lividans TK24 . The recombinant strains were designated as S . lividans (pIJ486-hTNF beta), S . lividans (pIJ486-vsi-hTNF beta) and S . lividans (pIVPA-hTNF beta) . The analysis of expressed proteins by Western blotting and biological activity measurements revealed hTNF beta was expressed by the recombinant strains with bioactivity . The molecular weight of directly secreted product is around 16 kDa, and the expression level at 48 hours in NB medium was 0.7 mg/L . Intact product could be obtained when hTNF beta was expressed intracellularly, although it may be degraded to lower molecular weight product when cultivation was prolonged . The intracellular expression level at 48 hours in NB medium was 25.1 mg/L. RNA, 2003 Jul, 9(7), 787 - 93 DnaK-facilitated ribosome assembly in Escherichia coli revisited; Alix JH et al.; Assembly helpers exist for the formation of ribosomal subunits . Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE) . Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes . Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits . On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits . It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process. J Exp Bot, 2003 Jul, 54(388), 1655 - 64 Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit; Aluru MR et al.; The biosynthesis of capsaicinoids in the placenta of chilli fruit is modelled to require components of the fatty acid synthase (FAS) complex . Three candidate genes for subunits in this complex, Kas, Acl, and Fat, isolated based on differential expression, were characterized . Transcription of these three genes was placental-specific and RNA abundance was positively correlated with degree of pungency . Kas and Acl were mapped to linkage group 1 and Fat to linkage group 6 . None of the genes is linked to the pungency locus, C, on linkage group 2 . KAS accumulation was positively correlated with pungency . Western blots of placental extracts and histological sections both demonstrated that the accumulation of this enzyme was correlated with fruit pungency and KAS was immunolocalized to the expected cell layer, the placental epidermis . Enzyme activity of the recombinant form of the placental-specific KAS was confirmed using crude cell extracts . These FAS components are fruit-specific members of their respective gene families . These genes are predicted to be associated with Capsicum fruit traits, for example, capsaicinoid biosynthesis or fatty acid biosynthesis necessary for placental development. J Histochem Cytochem, 2003 Jul, 51(7), 941 - 9 Analysis of the mouse CSF-1 gene promoter in a transgenic mouse model; Abboud SL et al.; CSF-1 stimulates monocyte and osteoclast populations . However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear . To identify regulatory regions that control normal CSF-1 gene expression, a -774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo . Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression . Transient transfection of osteoblasts with promoter deletion constructs showed that the -774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences . To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the -774/+183-bp region driving the E . coli beta-galactosidase (lacZ) reporter gene were generated . beta-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney . At the cellular level, the pattern of beta-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization . This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney . These findings indicate that the -774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues . CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development. Hypertension, 2003 Jul, 42(1), 103 - 9 Epub 2003 Jun 16. Downregulation of angiotensin subtype 1 receptor in rostral ventrolateral medulla during endotoxemia; Chan JY et al.; We reported recently that an upregulation of the inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a crucial determinant for the elicitation of cardiovascular depression during experimental endotoxemia . The current study evaluated the hypothesis that a downregulation of the molecular synthesis and functional expression of angiotensin subtype 1 receptor (AT1R) in the RVLM is consequential to this upregulated iNOS . In adult Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (15 mg/kg) elicited a reduction, followed by an augmentation and a secondary decrease in sympathetic vasomotor outflow, together with progressive hypotension and bradycardia . There was also a progressive increase in iNOS mRNA and protein level in the ventrolateral medulla . This was followed by a significant downregulation of both mRNA and protein levels of AT1R in the ventrolateral medulla, alongside reduced efficacy of angiotensin II (50 pmol) to induce an increase in systemic arterial pressure, heart rate, or sympathetic vasomotor outflow on unilateral microinjection into the RVLM . Pretreatment with microinjection of a selective iNOS inhibitor, S-methylisothiourea (250 pmol) bilaterally into the RVLM significantly reversed the reduction in both synthesis and activity of AT1R . We conclude that a downregulation of molecular synthesis and functional expression of AT1R in the ventrolateral medulla is consequential to the overproduction of NO through upregulation of iNOS in the RVLM and may underlie the cardiovascular depression that takes place during experimental endotoxemia. J Biol Chem, 2003 Sep 5, 278(36), 33839 - 47 Epub 2003 Jun 16. A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal; Argentaro A et al.; During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad . The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9 . By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro . The formation of a SOX9.CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9 . A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells . The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/autosomal sex reversal (CD/SRA) . This mutant binds importin beta normally despite defective nuclear import . Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes . Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9 . These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes. J Biol Chem, 2003 Aug 22, 278(34), 32300 - 6 Epub 2003 Jun 16. Characterization of the interactions within the mazEF addiction module of Escherichia coli; Zhang J et al.; In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin . The mazEF system is known as an addiction module located on the Escherichia coli chromosome . MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex . MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression . Here we show that the binding of purified (His)6MazE to the mazEF promoter DNA was enhanced by MazF . The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF-(His)6 complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain . The ratio of MazE to MazF(His)6 in the MazE-MazF(His)6 complex is about 1:2 . Because both MazE and MazF-(His)6 exist as dimers by themselves, the MazE-MazF-(His)6 complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)6 dimers . The interaction between MazE and MazF was also characterized with the yeast two-hybrid system . It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF . Site-directed mutagenesis at this region revealed that Leu55 and Leu58 play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA . The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF. Di Yi Jun Yi Da Xue Xue Bao, 2003 Jun, 23(6), 549 - 52 {Recombinant Helicobacter pylori urease B subunit and its biological properties}; Lu DS et al.; OBJECTIVE: To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein . METHODS: The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain . RESULTS: The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62,000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP . The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp . CONCLUSION: The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp. Biochem Biophys Res Commun, 2003 Jul 4, 306(3), 786 - 91 Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli; Pradel N et al.; Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system . The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate . Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion . In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes . Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions . These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system. Biochem Biophys Res Commun, 2003 Jul 4, 306(3), 666 - 73 Reconstitution of archaeal ribonuclease P from RNA and four protein components; Kouzuma Y et al.; Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5(') end of matured tRNA molecules . A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively . These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies . The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P . horikoshii . All four proteins exhibited the binding activity to the RNase P RNA . In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P . horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity . However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity . The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P. Infect Genet Evol, 2003 Jul, 3(2), 111 - 7 Evidence for the presence of a type III secretion system in diffusely adhering Escherichia coli (DAEC); Kyaw CM et al.; Diffusely adhering Escherichia coli (E . coli) strains (DAEC) represent a potential cause of diarrhoea in infants, and the detection of type three secretion system (TTSS) genes in DAEC would substantiate their pathogenic nature . In this work, four isolates of DAEC, recovered from stools of diarrhoeic children, were analysed by PCR, in order to detect the presence of TTSS genes . Primers targeted to the escC, escJ, escN and escV, some of the most conserved TTSS genes in enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), were used in order to verify the occurrence of homologous genes in our DAEC isolates . By this approach, we were able to characterise DNA fragments corresponding to putative escJ and escN genes in all DAEC isolates . Furthermore, DNA fragments homologous to the escC and escV genes were also amplified from all isolates . Besides the similarity found among the DAEC esc homologues with EPEC and EHEC esc genes, the nucleotide sequence analysis of the flanking regions of the amplified DNA fragments suggests that the putative DAEC esc genes are organised in the same manner as observed in EPEC and in EHEC strains . The results described here provide strong evidence for the presence of a TTSS in the DAEC strains analysed, implicating a pathogenic nature of these isolates. Mol Genet Metab, 2003 Jun, 79(2), 142 - 5 A novel mutation (G233D) in the glycogen phosphorylase gene in a patient with hepatic glycogen storage disease and residual enzyme activity; Tang NL et al.; We identified a novel mutation in the glycogen phosphorylase gene (PGYL) in a Chinese patient with glycogen storage disease (GSD) type VI . The patient presented with gross hepatomegaly since the age of two without history of any hypoglycemic attack . Otherwise, he was largely asymptomatic . Liver tissue enzyme assays revealed a mild deficiency of total glycogen phosphorylase . Both PGYL and PHKA2 genes were sequenced . The patient was homozygous of a missense mutation G233D in PGYL . This location forms a hairpin turn secondary structure and the small glycine residue is completely conserved in all the orthologous proteins from Escherichia coli to mammals . This is the sixth reported mutation of this form of GSD. Biochemistry, 2003 Jun 24, 42(24), 7587 - 93 High-precision, high-throughput stability determinations facilitated by robotics and a semiautomated titrating fluorometer; Edgell MH et al.; The use of statistical modeling to test hypotheses concerning the determinants of protein structure requires stability data (e.g., the free energy of denaturation in H(2)O, DeltaG(HOH)) from hundreds of protein mutants . Fluorescence-monitored chemical denaturation provides a convenient method for high-precision, high-throughput DeltaG(HOH) determination . For eglin c we find that a throughput of about 20 min per protein can be attained in a two-channel semiautomated titrating fluorometer . We find also that the use of robotics for protein purification and preparation of the solutions for chemical denaturation gives highly precise DeltaG(HOH) values in which the standard deviation of values from multiple preparations (+/-0.051 kcal/mol) differs very little from multiple measurements from a single preparation (+/-0.040 kcal/mol) . Since the variance introduced into model fitting by DeltaG(HOH) increases as the square of measurement error, there is a premium on precision . In fact, the fraction of stability behavior explicable by otherwise perfect models goes from 98% to only 50% over the error range commonly reported for chemical denaturation measurements (0.1-0.6 kcal/mol) . We have found that the precision of chemical denaturation DeltaG(HOH) measurements depends most heavily on the precision of the instrument used, followed by protein purity and the capacity to precisely prepare the solutions used for titrations. Biochemistry, 2003 Jun 24, 42(24), 7509 - 17 Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase; Louie TM et al.; 4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH(2)) utilizing monooxygenase . Its cosubstrate, FADH(2), is supplied by HpaC, an NAD(P)H-flavin oxidoreductase . Because HpaB is the first enzyme for 4HPA metabolism, FADH(2) production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA . An important question is how FADH(2) is produced and used, as FADH(2) is unstable in the presence of free O(2) . One solution is metabolic channeling by forming a transitory HpaB-HpaC complex . However, our in vivo and in vitro data failed to support the interaction . Further investigation pointed to an alternative scheme for HpaB to sequester FADH(2) . The intracellular HpaB concentration was about 122 microM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH(2) (K(d) of 70 nM), suggesting that most FADH(2) is bound to HpaB in vivo . The HpaB-bound FADH(2) was either used to rapidly oxidize 4HPA or slowly oxidized by O(2) to FAD and H(2)O(2) in the absence of 4HPA . Thus, HpaB's high intracellular concentration, its high affinity for FADH(2), its property of protecting bound FADH(2) in the absence of 4HPA, and its ability to rapidly use FADH(2) to oxidize 4HPA when 4HPA is available can coordinate FADH(2) production and utilization by HpaB and HpaC in vivo . This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date. Biochemistry, 2003 Jun 24, 42(24), 7467 - 76 Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study; Blanca G et al.; The recently discovered human DNA polymerase lambda (DNA pol lambda) has been implicated in translesion DNA synthesis across abasic sites . One remarkable feature of this enzyme is its preference for Mn(2+) over Mg(2+) as the activating metal ion, but the molecular basis for this preference is not known . Here, we present a kinetic and thermodynamic analysis of the DNA polymerase reaction catalyzed by full length human DNA pol lambda, showing that Mn(2+) favors specifically the catalytic step of nucleotide incorporation . Besides acting as a poor coactivator for catalysis, Mg(2+) appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol lambda and in an increased sensitivity to end product (pyrophosphate) inhibition . Comparison with the closely related enzyme human DNA pol beta, as well as with other DNA synthesising enzymes (mammalian DNA pol alpha and DNA pol delta, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol lambda . A deletion mutant of DNA pol lambda, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochemical properties comparable to the full length enzyme but clearly different from the close homologue DNA pol beta, highlighting the existence of important differences between DNA pol lambda and DNA pol beta, despite a high degree of sequence similarity. Biochemistry, 2003 Jun 24, 42(24), 7442 - 7 Nearest neighbor analysis of the SecYEG complex . 2 . Identification of a SecY-SecE cytosolic interface; Satoh Y et al.; Although the importance of interactions involving both the cytosolic and transmembrane regions of SecY and SecE has been documented, no information has been available for the physical contact sites of these translocase subunits in their cytosolic domains . We now carried out site-specific cross-linking experiments to identify SecY and SecE regions that are physically close . Cysteines introduced into SecY residue 244 in the fourth cytosolic domain (C4) as well as into residues 354-356 and 362 in the C5 domain could be cross-linked with natural or engineered residues at positions 79 and 81 in the central part of the cytosolic loop of SecE . These cross-linkages were abolished by the Gly240 mutation in the SecY C4 region as well as by prlG alterations in SecE transmembrane segment 3, known to compromise SecY-SecE interaction . We suggest that the cytosolic and intramembrane interactions bring these two subunits together, forming a functionally crucial SecYE interface involving the SecY C5 region and the conserved cytosolic segment of SecE. Biochemistry, 2003 Jun 24, 42(24), 7434 - 41 Nearest neighbor analysis of the SecYEG complex . 1 . Identification of a SecY-SecG interface; Satoh Y et al.; Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane . To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity . Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG . These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG . Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop . Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG . In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY . These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG. Biochemistry, 2003 Jun 24, 42(24), 7427 - 33 Recombinant water-soluble chlorophyll protein from Brassica oleracea var . Botrys binds various chlorophyll derivatives; Schmidt K et al.; A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var . Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli . The protein has been refolded in vitro to study its pigment binding behavior . Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein {Satoh, H., Nakayama, K., Okada, M . (1998) J . Biol . Chem . 273, 30568-30575} . WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding . WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein . We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes . If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited . We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl . This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives. Biochemistry, 2003 Jun 24, 42(24), 7294 - 302 Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity; Schenk G et al.; Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids . Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively) . Addition of substrate leads to a purely six-coordinate form . A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography {Tomchick et al . (2001) Biochemistry 40, 7509-7517} . While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed . A recent study using density-functional theory {Lehnert and Solomon (2002) J . Biol . Inorg . Chem . 8, 294-305} indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions . In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems . Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active . We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity. Methods Find Exp Clin Pharmacol, 2003 May, 25(4), 253 - 7 cDNA cloning and high-level expression of a thrombin-like enzyme from Agkistrodon acutus venom; Zha XD et al.; Agkistrodon acutus (Guenther), a poisonous snake species of the family of Crotalidae, is mainly found south of the Yellow River in China . The main symptom of this poison is massive hemorrhage, in which thrombin-like enzymes (TLEs) in the venom play an important role . TLEs are abundant, especially in the venom of A . acutus . We isolated the total RNA from the venom gland tissue of A . acutus and amplified the cDNAs of the TLEs using reverse transcription-polymerase chain reaction (RT-PCR) . The cDNAs were cloned into vector pThioHis B and were expressed as fusion proteins in the form of inclusion bodies, which accounted for nearly 50% of the total cell proteins . The inclusion bodies were washed, dissolved, refolded and purified by affinity chromatography . The purity was higher than 97%, as indicated by capillary zone electrophoresis . The renatured recombinant enzyme exhibited arginine esterase activity, as tested by the BAEE method, and also showed a fibrinogen cleavage effect, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . This method provides a fast and convenient system for studying the structure-function relationships in TLE isoenzymes, and also a practical way for mass production of TLEs in the pharmaceutical industry. J Biol Chem, 2003 Sep 26, 278(39), 37664 - 71 Epub 2003 Jun 14. Essential cell division protein FtsZ assembles into one monomer-thick ribbons under conditions resembling the crowded intracellular environment; Gonzalez JM et al.; Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli . In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion . Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick . When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer . We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers . In vivo these ribbons would fit into the Z-ring even in the absence of other interactions . Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked. J Biol Chem, 2003 Sep 5, 278(36), 34505 - 16 Epub 2003 Jun 13. Biosynthesis of phytosterols . Kinetic mechanism for the enzymatic C-methylation of sterols; Nes WD et al.; Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity . From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively . Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism . The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet . The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet . 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet . Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy . 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center . Comparison of the initial velocity data using AdoMet versus {2H3-methyl}AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity . {25-2H}24(28)-Methylenecycloartanol, {28E-2H}24 (28)-methylenelanosterol, and {28Z-2H}24(28)-methylene lanosterol were prepared and paired with AdoMet or {methyl-3H3}AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols . The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond. Genetics, 2003 Jun, 164(2), 501 - 9 COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster; Desset S et al.; ZAM and Idefix are two endogenous retroviruses whose expression is tightly controlled in Drosophila melanogaster . However, a line exists in which this control has been perturbed, resulting in a high mobilization rate for both retroviruses . This line is called the U (unstable) line as opposed to the other S (stable) lines . In the process of analyzing this control and tracing the genetic determinant involved, we found that ZAM and Idefix expression responded to two types of controls: one restricting their expression to specific somatic cells in the ovaries and the other silencing their expression in S lines but permitting it in U lines . While studying this second control in the U or S backgrounds, we found that the heterochromatic locus 20A2-3 on the X chromosome, previously implicated in the regulation of a third retroelement, gypsy, also controlled both ZAM and Idefix . We report here that genetic determinants necessary for endogenous retrovirus silencing occur at the 20A2-3 locus, which we call COM, for centre organisateur de mobilisation . We propose that if this point of control becomes mutated during the life of the fly, it may trigger processes reactivating dormant endogenous retroviruses and thus bring about sudden bursts of mobilization. Chem Res Toxicol, 2003 Jun, 16(6), 721 - 32 Predicting the genotoxicity of thiophene derivatives from molecular structure; Mosier PD et al.; We report several binary classification models that directly link the genetic toxicity of a series of 140 thiophene derivatives with information derived from the compounds' molecular structure . Genetic toxicity was measured using an SOS Chromotest . IMAX (maximal SOS induction factor) values were recorded for each of the 140 compounds both in the presence and in the absence of S9 rat liver homogenate . Compounds were classified as genotoxic if IMAX >or= 1.5 in either test or nongenotoxic if IMAX < 1.5 for both tests . The molecular structures were represented by numerical descriptors that encoded the topological, geometric, electronic, and polar surface area properties of the thiophene derivatives . The classification models used were linear discriminant analysis (LDA), k-nearest neighbor classification (k-NN), and the probabilistic neural network (PNN) . These were used in conjunction with either a genetic algorithm or a generalized simulated annealing to find optimal subsets of descriptors for each classifier . The quality of the resulting models was determined by the number of misclassified compounds, with preference given to models that produced fewer false negative classifications . Model sizes ranged from seven descriptors for LDA to three descriptors for k-NN and PNN . Very good classification results were obtained with all three classifiers . Classification rates for the LDA, k-NN, and PNN models were 80, 85, and 85%, respectively, for the prediction set compounds . Additionally, a consensus model was generated that incorporated all three of the basic model types . This consensus model correctly predicted the genotoxicity of 95% of the prediction set compounds. Plant Physiol, 2003 Jun, 132(2), 1097 - 106 Epub 2003 May 15. A conserved 19-amino acid synthetic peptide from the carboxy terminus of phosphoenolpyruvate carboxylase inhibits the in vitro phosphorylation of the enzyme by the calcium-independent phosphoenolpyruvate carboxylase kinase; Alvarez R et al.; Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein . Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain . However, no further information was available as to where PEPCk-C4 PEPC interactions take place . In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk . In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 microm) . This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A . In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column . Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect . Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states . The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize {Zea mays}) enzymes. Plant Physiol, 2003 Jun, 132(2), 1065 - 76 Arabidopsis contains a large superfamily of acyl-activating enzymes . Phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme a synthetases; Shockey JM et al.; Acyl-activating enzymes are a diverse group of proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of a thioester bond . This group of enzymes is found in all living organisms and includes the acyl-coenzyme A synthetases, 4-coumarate:coenzyme A ligases, luciferases, and non-ribosomal peptide synthetases . The members of this superfamily share little overall sequence identity, but do contain a 12-amino acid motif common to all enzymes that activate their acid substrates using ATP via an enzyme-bound adenylate intermediate . Arabidopsis possesses an acyl-activating enzyme superfamily containing 63 different genes . In addition to the genes that had been characterized previously, 14 new cDNA clones were isolated as part of this work . The protein sequences were compared phylogenetically and grouped into seven distinct categories . At least four of these categories are plant specific . The tissue-specific expression profiles of some of the genes of unknown function were analyzed and shown to be complex, with a high degree of overlap . Most of the plant-specific genes represent uncharacterized aspects of carboxylic acid metabolism . One such group contains members whose enzymes activate short- and medium-chain fatty acids . Altogether, the results presented here describe the largest acyl-activating enzyme family present in any organism thus far studied at the genomic level and clearly indicate that carboxylic acid activation metabolism in plants is much more complex than previously thought. Plant Physiol, 2003 Jun, 132(2), 883 - 92 Epub 2003 May 15. The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns; Bonin CP et al.; l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins . The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes . To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope . GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed . Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant . These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types . We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme. Plant Physiol, 2003 Jun, 132(2), 453 - 60 AraCyc: a biochemical pathway database for Arabidopsis; Mueller LA et al.; AraCyc is a database containing biochemical pathways of Arabidopsis, developed at The Arabidopsis Information Resource . The aim of AraCyc is to represent Arabidopsis metabolism as completely as possible with a user-friendly Web-based interface . It presently features more than 170 pathways that include information on compounds, intermediates, cofactors, reactions, genes, proteins, and protein subcellular locations . The database uses Pathway Tools software, which allows the users to visualize a bird's eye view of all pathways in the database down to the individual chemical structures of the compounds . The database was built using Pathway Tools' Pathologic module with MetaCyc, a collection of pathways from more than 150 species, as a reference database . This initial build was manually refined and annotated . More than 20 plant-specific pathways, including carotenoid, brassinosteroid, and gibberellin biosyntheses have been added from the literature . A list of more than 40 plant pathways will be added in the coming months . The quality of the initial, automatic build of the database was compared with the manually improved version, and with EcoCyc, an Escherichia coli database using the same software system that has been manually annotated for many years . In addition, a Perl interface, PerlCyc, was developed that allows programmers to access Pathway Tools databases from the popular Perl language . AraCyc is available at the tools section of The Arabidopsis Information Resource Web site . Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7702 - 7 Epub 2003 Jun 12. Detailed map of a cis-regulatory input function; Setty Y et al.; Most genes are regulated by multiple transcription factors that bind specific sites in DNA regulatory regions . These cis-regulatory regions perform a computation: the rate of transcription is a function of the active concentrations of each of the input transcription factors . Here, we used accurate gene expression measurements from living cell cultures, bearing GFP reporters, to map in detail the input function of the classic lacZYA operon of Escherichia coli, as a function of about a hundred combinations of its two inducers, cAMP and isopropyl beta-d-thiogalactoside (IPTG) . We found an unexpectedly intricate function with four plateau levels and four thresholds . This result compares well with a mathematical model of the binding of the regulatory proteins cAMP receptor protein (CRP) and LacI to the lac regulatory region . The model is also used to demonstrate that with few mutations, the same region could encode much purer AND-like or even OR-like functions . This possibility means that the wild-type region is selected to perform an elaborate computation in setting the transcription rate . The present approach can be generally used to map the input functions of other genes. Science, 2003 Jun 13, 300(5626), 1718 - 22 Selective charging of tRNA isoacceptors explains patterns of codon usage; Elf J et al.; We modeled how the charged levels of different transfer RNAs (tRNAs) that carry the same amino acid (isoacceptors) respond when this amino acid becomes growth-limiting . The charged levels will approach zero for some isoacceptors (such as tRNA2Leu) and remain high for others (such as tRNA4Leu), as determined by the concentrations of isoacceptors and how often their codons occur in protein synthesis . The theory accounts for (synonymous) codons for the same amino acid that are used in ribosome-mediated transcriptional attenuation, the choices of synonymous codons in trans-translating transfermessenger RNA, and the overrepresentation of rare codons in messenger RNAs for amino acid biosynthetic enzymes. J Biol Chem, 2003 Sep 12, 278(37), 34925 - 33 Epub 2003 Jun 12. The Escherichia coli RecQ helicase functions as a monomer; Xu HQ et al.; The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans . In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques . The results obtained clearly indicate that E . coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C . Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate . Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration . The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein . The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes . Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action. J Biol Chem, 2003 Aug 29, 278(35), 32608 - 17 Epub 2003 Jun 12. Characterization of a trap mutant of the AAA+ chaperone ClpB; Weibezahn J et al.; The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE) . The order of action of ClpB and KJE on aggregated proteins is unknown . We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP . This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time . Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB . The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions . ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates. Vaccine, 2003 Jul 4, 21(23), 3228 - 35 A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encoding Plasmodium falciparum sexual-stage antigen, Pfs230; Fanning SL et al.; Mammalian expression vectors encoding region C of malaria transmission-blocking vaccine candidate Pfs230 (aa 443-1132) with and without a 3' glycosylphosphatidylinositol (GPI) anchor signal sequence were tested for their immunogenicity in mice . The plasmid containing the GPI anchor signal sequence consistently induced higher titers of anti-Pfs230 antibodies using three delivery systems: intramuscular (i.m.), intradermal (i.d.), and gene gun (g.g.) . In contrast, the isotype profile elicited varied depending on the delivery system and was not effected by the presence of the GPI anchor sequence . Both gene gun and intradermal administration induced primarily an IgG1 response, while intramuscular injection induced both IgG1 and IgG2a antibodies . Regardless of the mode of delivery, all the plasmids encoding Pfs230 region C primed for a mixed IgG1/IgG2a response to an intraperitoneal (i.p.) injection of E . coli-produced recombinant Pfs230 region C . None of these vaccination strategies were more effective than r230/MBP.C alone in generating malaria transmission-blocking immunity. FEBS Lett, 2003 Jun 19, 545(2-3), 127 - 32 OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity; Law CJ et al.; The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli . Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive . We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin . While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature. Biochem Biophys Res Commun, 2003 Jun 27, 306(2), 590 - 7 Characterization of Drosophila nitric oxide synthase: a biochemical study; Sengupta R et al.; The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW . The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa . The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity . The trypsin digestion patterns were different from nNOS . The full-length DNOS protein had high degree of stability against trypsin . The activity assay of trypsin-digested protein confirmed the same result . Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity . Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea . Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN . The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant. Biochem Biophys Res Commun, 2003 Jun 27, 306(2), 570 - 6 Hydrogen peroxide-induced microsatellite instability in the Escherichia coli K-12 endogenous tonB gene; Yamamura E et al.; Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms . Using an endogenous tonB gene as a mutation selective marker in Escherichia coli, we have examined whether endogenous oxidative mutagenesis can contribute to genetic instability . We have also used oxyR(+) and oxyR(-) strains to evaluate how hydrogen peroxide scavenging system can contribute to genetic instability . The highest mutation frequency induced by hydrogen peroxide was 3.8x10(-6) at 600 microM and 5.3 x 10(-6) at 40 microM in oxyR(+) and oxyR(-), respectively . Hydrogen peroxide induced minus frameshift mutations predominantly followed by transversions (G:C-->T:A, G:C-->C:G, and A:T-->T:A) . The types and the nature of the mutations did not differ between strains . Frameshift mutations occurred at G:C and A:T sites equally, and in repeated and non-repeated sequences equally . It is evident that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication and contribute to genomic instability . Our results further indicate that oxyR regulon does not take part in the DNA-repair pathway against oxidative damage induced by hydrogen peroxide. J Endotoxin Res, 2003, 9(2), 108 - 12 Differences in the mechanism of nitric oxide production between mouse vascular endothelial cells and macrophages; Sugiyama T et al.; The detailed mechanism of NO production in mouse vascular endothelial cells, END-D, was studied . The NO production in END-D cells was triggered by gamma interferon (IFN-gamma), but not LPS . However, LPS augmented the NO production in IFN-gamma-stimulated END-D cells . A high level of NO production was due to the expression of an inducible type of NO synthase (iNOS) in those cells . A significant amount of NO was detected 18 h after IFN-gamma stimulation, accompanied by the delayed iNOS expression . The JAK/STAT signal pathway mediated IFN-gamma-induced NO production, but did not participate in the LPS-induced augmentation . Further, no activation of nuclear factor (NF)-kappaB was involved in the NO production in END-D cells stimulated with either IFN-gamma and/or LPS . The mechanism of NO production in END-D cells was suggested to be different from that in mouse macrophages . The differential regulation of NO production in mouse vascular endothelial cells and macrophages is discussed. J Endotoxin Res, 2003, 9(2), 97 - 100 Endotoxin boosts the vascular endothelial growth factor (VEGF) in rabbits; Hahn RG; Vascular endothelial growth factor (VEGF) is a cytokine that greatly increases vascular permeability and thereby promotes hypovolemia . The present study examines whether the plasma VEGF concentration is increased by a bolus injection of endotoxin 20 microg/kg in 30 rabbits, and whether the response is modified by vitamin A, which doubles the endotoxin clearance . The results show that endotoxin stimulates a gradual increase in the VEGF concentration to a peak 5 h later which is approximately 100 times higher than the baseline concentration . No statistically significant difference was found between the rabbits that received no further treatment (n = 10) and the ones that were given 1000 IE/kg of vitamin A intravenously 1 h before (n = 10) or after (n = 10) the endotoxin . The rise in VEGF correlated with the development of fever, and the VEGF concentrations were higher in animals with a severely affected physical status as judged by the breathing pattern and changes in posture or reactivity . In conclusion, release of VEGF is part of the cytokine response to endotoxin with a peak occurring 5 h after a bolus injection, and the rise is pronounced also in the presence of a high endotoxin clearance. J Endotoxin Res, 2003, 9(2), 85 - 90 C2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt; Koide N et al.; The effect of C(2)-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied . The non-toxic concentration of C(2)-ceramide inhibited LPS-induced NO production . It was due to the attenuated expression of the inducible type of NO synthase (iNOS) . C(2)-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS . On the other hand, C(2)-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-kappaB activation . Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production . C(2)-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt. J Pharm Pharmacol, 2003 Apr, 55(4), 527 - 31 Involvement of inwardly rectifying K+ channels in secretory responses of human ileal mucosa; Burleigh DE; In acute secretory diarrhoea the primary event driving fluid secretion is a transcellular, electrogenic, serosal to mucosal transport of chloride ions . Such transport requires the maintenance of an electrically negative cell membrane voltage, which is achieved through a basolateral outward leakage of potassium ions . The aim of this study was to investigate the nature of K(+) channel involvement in facilitating secretory processes in the human ileum . Muscle-stripped mucosal preparations of human ileal mucosa were set up in Ussing chambers for recording short-circuit current and transmucosal conductance . Escherichia coli heat-stable toxin and vasoactive intestinal peptide (VIP) produced concentration-dependent increases in short-circuit current . Responses to the heat-stable toxin were unaffected by basolateral application of 4-aminopyridine (5 mM), glibenclamide (10 microM) or a combination of charybdotoxin (0.3 microM) plus apamin (0.3 microM) . However, basolateral barium (0.2-5 mM) caused a concentration-dependent inhibition . Responses to VIP were similarly affected by barium (0.05-1 mM) . These results suggested that electrogenic chloride transport by human ileal mucosa required the presence of basolateral K(+) channels . The use of selective K(+)-channel inhibitors and low concentrations of barium suggested that the channels involved might be of the inwardly rectifying type. Parasitol Res, 2003 Aug, 90(6), 467 - 72 Epub 2003 Jun 12. Recombinant Duffy binding-like-alpha domains of Plasmodium falciparum erythrocyte membrane protein 1 elicit antibodies in rats that recognise conserved epitopes; Oguariri RM et al.; Plasmodium falciparum parasites remodel the surface of human erythrocytes on invasion by the insertion of parasite-derived proteins in knob-like protrusions . P . falciparum erythrocyte membrane protein 1 (PfEMP-1), a variant surface antigen, has been shown to be anchored in these knobs and mediates adhesion to various host endothelial receptors . These proteins also undergo clonal antigenic variation as a means of immune evasion . Duffy binding-like-alpha(DBL-alpha) domain together with the cysteine-rich interdomain region form the head structure of the PfEMP1 molecule . In this report, we used ten different recombinant DBL-alpha fusion proteins expressed in Escherichia coli to generate antibodies in experimental animals . Five out of ten recombinant DBL-alpha fusion proteins were immunogenic and induced antibodies that reacted with conserved peptides derived from PfEMP1 . Indirect immunofluorescence assay was used to localise PfEMP-1-DBL-alpha expressed in parasitised erythrocytes . Positive fluorescence reactivity was observed within the cytoplasm and with membrane structures but not on the surface of intact P . falciparum-infected erythrocytes. Histochem Cell Biol, 2003 Jun, 119(6), 469 - 75 Epub 2003 Jun 11. Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide; Abdelwahab SA et al.; Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry . The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus . After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection . The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP. Appl Microbiol Biotechnol, 2004 Jan, 63(4), 407 - 17 Epub 2003 Jun 12. Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments toget