Escherichia coli

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 together with measurements of enzyme activities and intracellular metabolite concentrations; Al Zaid Siddiquee K et al.; Metabolic flux analysis based on 13C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture . In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture . It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF- mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant . In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant . This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc . It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF- mutant . To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.

J Nippon Med Sch, 2003 Apr, 70(2), 151 - 6
Fetal plasma prostaglandin F(2alpha) and cortisol responses to high-dose endotoxin administration in fetal goats; Miura A et al.; Intrauterine inflammation/infection has been associated with prenatal mortality and morbidity . However, few studies have been performed to investigate how the fetus responds to intrauterine inflammation/infection in utero . In the present study, fetal plasma prostaglandin (PG) F(2alpha) and cortisol responses to high-dose fetal endotoxin administration were evaluated in late gestation goats (n=8) . After 160 microg/kg of fetal weight of endotoxin (Escherichia coli, O111:B4 lipopolysaccharide) administration via the fetal jugular vein over a 5-min period, fetal plasma PGF(2alpha) and cortisol levels, fetal blood gases and pH were measured periodically . After endotoxin administration, fetal plasma cortisol levels significantly increased to 9.5+/-0.8 ng/mL and 9.3+/-0.7 ng/mL after 1 and 3h, respectively (p<0.05) and plasma PGF(2alpha) levels did not change throughout the study . These results suggest that absent PGF(2alpha) and attenuated cortisol responses to high-dose fetal endotoxin administration, relative to the adult, may be a self-protective mechanism that diminishes premature delivery and fetal asphyxia.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7480 - 5 Epub 2003 Jun 11.
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice; de Boer E et al.; Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput . Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates . Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation . We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag . Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution . Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads . Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues . Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

J Biol Chem, 2003 Aug 8, 278(32), 29710 - 8 Epub 2003 Jun 11.
On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter; Minakhin L et al.; Bacterial promoters of the extended -10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity . Open promoter complexes can be formed on an extended -10 Escherichia coli galP1 promoter at temperatures as low as 6 degrees C, when complexes on most promoters are closed . Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended -10 motif and/or containing the -35 promoter consensus element . A panel of E . coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation . At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif . However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent . Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other . In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif . We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations.

J Biochem (Tokyo), 2003 May, 133(5), 651 - 7
Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon, Thermoplasma acidophilum; Nemoto N et al.; We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography . Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species . The gene encoding GGGP synthase in T . acidophilum was cloned after PCR amplification of the gene from the genomic DNA . The recombinant enzyme was expressed in Escherichia coli and purified . A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis . The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer . As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures . Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.

J Biochem (Tokyo), 2003 May, 133(5), 607 - 14
Molecular cloning of a cDNA for the human phospholysine phosphohistidine inorganic pyrophosphate phosphatase; Yokoi F et al.; We previously reported the isolation from bovine liver of a novel 56-kDa inorganic pyrophosphatase named phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase) . It is a unique enzyme that hydrolyzes not only oxygen-phosphorus bonds in inorganic pyrophosphate but also nitrogen-phosphorus bonds in phospholysine, phosphohistidine and imidodiphosphate in vitro . In this study, we determined the partial amino acid sequence of the purified bovine LHPPase . To investigate whether humans have the same enzyme, we isolated a cDNA clone from a HeLa cell cDNA library that encodes for the human homologue of LHPPase . Although its sequence does not include the consensus sequence of a typical inorganic pyrophosphatase, it does contain a similar sequence of the active site in other phosphatases such as protein-tyrosine phosphatase, dual-specific phosphatase and low molecular weight acid phosphatase . Human LHPPase was highly expressed in the liver and kidney, and moderately in the brain . The recombinant protein was produced in E . coli . Its ability to hydrolyze oxygen-phosphorus bonds and nitrogen-phosphorus bonds was confirmed . The enzymatic characteristics of this human protein were similar to those of purified bovine LHPPase . Thus, we concluded that the cDNA encoded the human counterpart of bovine LHPPase.

Arch Biochem Biophys, 2003 Jul 1, 415(1), 94 - 100
A protein isolated from Escherichia coli, identified as GroEL, reacts with anti-beta spectrin antibodies; Czogalla A et al.; We found that a protein of molecular weight close to 65kDa, present in Escherichia coli cells, reacts with anti-beta spectrin antibodies . A method of purification of this protein was designed . The method consists of the following: nonionic detergent extraction, gel filtration chromatography, ion-exchange chromatography using DEAE-Servacell, and two FPLC ion-exchange chromatography runs: the first without urea, the second in its presence . This method allowed us to obtain a highly purified protein . The results of mass spectrometry analysis suggest that the investigated protein is GroEL (Hsp60 Class) . Using computer programs, by sequence analysis of both proteins we tried to explain why GroEL isolated from E . coli reacts with anti-beta spectrin antibodies . Both proteins may share a single epitope for the antibodies on their surfaces . Additionally, such an assumption is supported by the results of experiments in which antibodies interacting with GroEL were obtained from anti-beta spectrin serum and were shown to react with both GroEL and beta spectrins.

Arch Biochem Biophys, 2003 Jul 1, 415(1), 80 - 6
Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain; Ma Z et al.; The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K . The two proteinase K fragments remain associated and retained enzymatic activity . Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies . A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis . The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide . However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM . Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.

J Food Prot, 2003 Jun, 66(6), 1007 - 12
Weibull distribution function based on an empirical mathematical model for inactivation of Escherichia coli by pulsed electric fields; Rodrigo D et al.; The pulsed electric field inactivation kinetics of Escherichia coli suspended in orange juices with three different concentrations of carrot juice (0, 20, and 60%) was studied . Electric field strengths ranged from 25 to 40 kV/cm, and treatment times ranged from 40 to 340 micros . Experimental data were fitted to Bigelow, Hulsheger, and Weibull distribution functions, and the Weibull function provided the best fit (with the lowest mean square error) . The dependency of each model's kinetic constant on electric field strength and carrot juice concentration was studied . A secondary model was developed to describe the relationship of Weibull parameters a and n to electric field strength and carrot juice concentration . An empirical mathematical model based on the Weibull distribution function, relating the natural logarithm of the survival fraction to treatment time, electric field strength, and carrot juice concentration, was developed . Parameters were estimated by a nonlinear regression . The results of this study indicate that the error rate for the model's predictions was 6.5% and that the model was suitable for describing E . coli inactivation.

Folia Microbiol (Praha), 2003, 48(2), 243 - 7
Occurrence of extended-spectrum beta-lactamases among Escherichia coli isolates from hospitalized and healthy children; Franiczek R et al.; The prevalence of extended-spectrum beta-lactamases (ESBL) was determined among isolates of Escherichia coli (n = 63) isolated from hospitalized (43) and healthy (20) children . Ten isolates (21%) were ESBL-positive for two screening tests, the double disk-synergy test and the Oxoid Combination Disk method . One ESBL-positive isolate came from a healthy child . The transfer frequency of oxyimino-beta-lactam resistance from ESBL-producing isolates to E . coli K12 C600 recipient strain ranged from 10(-8) to 10(-5) per donor cell . Donor strains and transconjugants displayed susceptibility patterns typical of ESBL producers . They were resistant to oxyimino-beta-lactams but susceptible to clavulanic acid and carbapenems . Seven out of the 10 ESBL-positive isolates were found to produce MR/MS fimbria, which may play an important role in the colonization of the human intestinal mucosa.

Folia Microbiol (Praha), 2003, 48(2), 162 - 7
Heterogeneity of Escherichia coli derived from artiodactyla animals analyzed with the use of rep-PCR fingerprinting; Baldy-Chudzik K et al.; Genetic polymorphism of 83 isolates of E . coli, derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Swierkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers . The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates . The arithmetic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E . coli isolates into three similarity groups containing various numbers of isolates . The groups comprised isolates derived from two, three and four species of the source animals . The isolates derived from each source segregated in the dendrogram in a different way, both within the similarity groups and among them, indicating an individual repertoire of E . coli in the examined species of animals . The similarity relations among E . coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (< or = 20%), indicated an essential interstrain differentiation in terms of the distribution of REP sequences . Our results confirmed the hypothesis of the oligoclonal characters of populations obtained from particular sources . The rep-PCR fingerprinting method with REP primers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.

World J Gastroenterol, 2003 Jun, 9(6), 1352 - 5
Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis; Jiang XH et al.; AIM: To construct and express a humanized M(2) autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC) . METHODS: cDNA fragments encoding M(2)-reactive epitopes of pyruvate dehydrogenase complex E(2) (PDC-E(2)), branched chain 2-oxo-acid dehydrogenase complex E(2) (BCOADC-E(2)) and 2-oxo-glutarate dehydrogenase complex E(2) (OGDC-E(2)) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells . The fragments were cloned into the plasmid vector pQE-30 and then transferred into E . coli M15 (pREP4) for expression, which was induced by isopropylthio-beta-D-galactoside . The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M(2)-positive sera confirmed at Euroimmun Research Center (Germany) . Using the purified BPO, M(2) antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA . RESULTS: The expressed BPO was observed with both antigenic reactivity and specificity of M(2) autoantigens . The determination of M(2) antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay . With the newly established method, M(2) antibodies were found in 100 % (20/20) of patients with PBC . Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M(2) antibodies . There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M(2)-antibody positive . CONCLUSION: Compared with the routine immunofluorescence assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.

World J Gastroenterol, 2003 Jun, 9(6), 1282 - 6
Isolation and analysis of a novel gene over-expressed during liver regeneration; Li YC et al.; AIM: To isolate and analyze a novel gene over-expressed during liver regeneration . METHODS: Total RNA of regenerating liver was extracted from liver tissue after 0-4-36-36-36 hr short interval successive partial hepatectomy (SISPH) . Reverse transcription-polymerase chain reaction was used to synthesize double strand cDNA, after the tissue was digested by proteinase K and Sfi A/B . The double-strand cDNA was ligated to lambdaTriplEx2 . lambdaphage packaging reaction was performed and E . coli XL1-Blue was infected for titering and amplifying . One expressed sequence tag was probed by Dig and phage in situ hybridization was carried out to isolate positive clones . Positive recombinant lambdaTriplEx2 was converted to the corresponding pTriplEx2, and bioinformatics was used to analyze full-length cDNA . RESULTS: We isolated a novel full-length cDNA during liver regeneration following SISPH . CONCLUSION: We have succeeded in cloning a novel gene, based on bioinformatics . We postulate that this gene may function in complicated network in liver regeneration . On the one hand, it may exert initiation of liver regeneration via regulating nitric oxide synthesis . On the other hand, it may protect damaged residue lobus following SISPH.

World J Gastroenterol, 2003 Jun, 9(6), 1256 - 60
Cross-reactivity of hypervariable region 1 chimera of hepatitis C virus; Xiu BS et al.; AIM: To analyze the amino acid sequences of hypervariable region 1 (HVR1) of HCV isolates in China and to construct a combinatorial chimeric HVR1 protein having a very broad high cross-reactivity . METHODS: All of the published HVR1 sequences from China were collected and processed with a computer program . Several representative HVR1's sequences were formulated based on a consensus profile and homology within certain subdivision . A few reported HVR1 mimotope sequences were also included for a broader representation . All of them were cloned and expressed in E.coli . The cross-reactivity of the purified recombinant HVR1 antigens was tested by ELISA with a panel of sera from HCV infected patients in China . Some of them were further ligated together to form a combinatorial HVR1 chimera . RESULTS: Altogether 12 HVR1(s) were selected and expressed in E.coli and purified to homogeneity . All of these purified antigens showed some cross-reactivity with sera in a 27 HCV positive panel . Recombinant HVR1s of No . 1, 2, 4, and 8# showing broad cross-reactivities and complementarity with each other, were selected for the ligation elements . The chimera containing these 4 HVR1s was highly expressed in E.coli . The purified chimeric antigen could react not only with all the HCV antibody positive sera in the panel but also with 90/91 sera of HCV -infected patients . CONCLUSION: The chimeric antigen was shown to have a broad cross-reactivity . It may be helpful for solving the problem caused by high variability of HCV, and in the efforts for a novel vaccine against the virus.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3227 - 35
Peptidyl-tRNA hydrolase from Sulfolobus solfataricus; Fromant M et al.; An enzyme capable of liberating functional tRNA(Lys) from Escherichia coli diacetyl-lysyl-tRNA(Lys) was purified from the archae Sulfolobus solfataricus . Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA(fMet) as a substrate . N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom . Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity . The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes . Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type . Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH . In vitro assays confirm that the two enzymes ensure the recycling of tRNA(Lys) from diacetyl-lysyl-tRNA(Lys) . Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions . Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3092 - 100
Cc RNase: the Ceratitis capitata ortholog of a novel highly conserved protein family in metazoans; Rampias TN et al.; Complementary DNA encoding a protein, designated Cc RNase, was isolated from the insect Ceratitis capitata . Deduced amino acid sequence analysis demonstrates that the Cc RNase has strong sequence homology with other uncharacterized proteins predicted from EST sequences belonging to different animal species, therefore defining a new protein family, which is conserved from Caenorhabditis elegans to humans . Phylogenetic analysis data in addition to extensive homolog searches in all available complete genomes suggested that all family members are true orthologs . Proteins belonging to this family are composed of 95-101 amino acids . The C.capitata orthologous protein was expressed in Escherichia coli . Despite the fact that the amino acid sequence of Cc RNase does not share any significant similarities with other known ribonucleases, our data give strong evidence in support of the assignment of enzymatic activity to the recombinant protein . The expressed molecule exhibits ribonucleolytic activity against poly(C) and poly(U) synthetic substrates, as well as rRNA . It is also demonstrated that expression of Cc RNase in E.coli inhibits growth of the host cells.

Nucleic Acids Res, 2003 Jun 15, 31(12), 3050 - 6
Single-chain Tet transregulators; Krueger C et al.; We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker . TetR-based transregulators are widely used to regulate gene expression in eukaryotes . They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain . Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines . In particular, the reverse 'tet-on' phenotype of rtTA variants is also present in the sc variants . Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction . The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.

Nucleic Acids Res, 2003 Jun 15, 31(12), 2990 - 4
Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations; Sohail A et al.; Activation-induced cytidine deaminase (AID) is required for the maturation of antibodies in higher vertebrates, where it promotes somatic hypermutation (SHM), class switch recombination and gene conversion . While it is known that SHM requires high levels of transcription of the target genes, it is unclear whether this is because AID targets transcribed genes . We show here that the human AID promotes C to T mutations in Escherichia coli which are stimulated by transcription . The mutations are strand-biased and occur preferentially in the non-transcribed strand of the target gene . Human AID purified from E.coli is active without prior treatment with a ribonuclease and deaminates cytosines in plasmid DNA in vitro . Further, the action of this enzyme is greatly stimulated by the transcription of the target gene in a strand-dependent fashion . These results confirm the prediction that AID may act directly on DNA and show that it can act on transcribing DNA in the absence of specialized DNA structures such as R-loops . It suggests that AID may be recruited to variable genes through transcription without the assistance of other proteins and that the strand bias in SHM may be caused by the preference of AID for the non-transcribed strand.

J Biol Chem, 2003 Aug 22, 278(34), 32413 - 22 Epub 2003 Jun 10.
Molecular and biochemical characterization of an enzyme responsible for the formation of hypericin in St . John's wort (Hypericum perforatum L.); Bais HP et al.; A major gene termed Hyp-1 encoding for hypericin (HyH) biosynthesis was cloned and characterized from Hypericum perforatum (St . John's wort) cell cultures . H . perforatum leaves are widely used as an herbal remedy in the treatment of mild to moderate depression . Hypericin, a photosensitive and red-colored naphthodianthrone, has been reported as the bioactive compound responsible for reversing the depression symptoms . In this study a novel red-color-based colony screening method for examining a cDNA library (lambda-TriplEX2) derived from H . perforatum cell cultures revealed the gene responsible for hypericin biosynthesis after the administration of emodin, a precursor of hypericin . The selected clones were expressed in Escherichia coli (BM 25.8 line) and were further screened for biosynthesis of emodin to hypericin, which resulted in an 84.6% conversion . The full-length cDNA sequence of Hyp-1 is 782 nucleotides in length with an open reading frame of 477 nucleotides coding for a protein of 159 amino acids, with a 45.1% homology to Bet.v.1 class allergens . Reverse transcriptase-PCR analysis showed high levels of Hyp-1 transcripts in dark-grown cell cultures compared with the levels in light-grown cell cultures and leaves . Southern blot analysis showed the presence of a single Hyp-1 gene in H . perforatum . Furthermore, Hyp-1 was expressed with a His6 affinity tag linked to its N terminal region using the expression vector pET-28a, and the recombinant Hyp-1 protein was able to convert HyH from emodin under in vitro conditions . HyH product inhibition was observed with emodin analogues, rhein, rhein methyl ester, and DNA3-55-1 . Our results demonstrate a direct and complex conversion of emodin to HyH that is solely catalyzed by Hyp-1, a Bet.v.1 class allergen from H . perforatum.

J Immunol Methods, 2003 Jun 1, 277(1-2), 117 - 25
A fast Western blot procedure improved for quantitative analysis by direct fluorescence labeling of primary antibodies; Bergendahl V et al.; The procedures for Western blots have been around for a long time and recent developments have increased the sensitivity for luminescent techniques so that the need for radioactive probes has been limited to only a few applications . Nevertheless, most protocols require more than 6 h and are often performed over more than a day . The majority of techniques require a secondary antibody conjugated to an enzyme that catalyzes a color reaction in order to amplify a detectable signal . However, both processes, the binding of a secondary antibody and the catalyzed reaction with the dye, are sources for errors and the latter is disadvantageous for a signal that is linear over a larger range of detected antigen . In order to improve the procedure most commonly used for quantitative analysis and convenience, we investigated the use of fluorescence labeling of primary monoclonal antibodies against Escherichia coli RNA polymerase subunits (beta', sigmaE and sigmaFecI) and their use in Western blots . We achieved a sensitivity (<1 ng detectable protein) comparable to most luminescent techniques . Additionally, we reduced the procedure time significantly to less than 1 h after SDS-PAGE and transfer to a membrane . Above all, we obtained a linear signal over the range of 30 ng to 1 microg of protein (dependent on protein size) making quantitative analysis of Western blots easier and more reliable.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 67 - 72
Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli; Zhang H et al.; The expressions of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli were compared under a variety of growth conditions and in several genetic backgrounds . LacZ operon fusions were constructed and were inserted in single copies into strain MC4100 and into its fnr, arcA or hemA carrying derivatives . During aerobic growth the expressions of both ubiD and ubiX depended on the carbon source: succinate>glycerol>glucose . Mutations in fnr, arcA or hemA increased the expressions of both genes . During anaerobic growth in LB medium glucose strongly inhibited the expression of ubiD but not of ubiX.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 33 - 40
Regulation of glnB gene promoter expression in Azospirillum brasilense by the NtrC protein; Huergo LF et al.; In Azospirillum brasilense the glnB and glnA genes are clustered in an operon regulated by three different promoters: two located upstream of glnB (glnBp1-sigma(70), and glnBp2-sigma(N)) and one as yet unidentified promoter, in the glnBA intergenic region . We have investigated the expression of the glnB gene promoter using glnB-lacZ gene fusions, mutation analysis, heterologous expression and DNA band-shift assays . Deletion of the glnB promoter region showed that NtrC-binding sequences were essential for glnB expression under nitrogen limitation . The A . brasilense NtrC protein activated transcription of glnB-lacZ fusions in the heterologous genetic background of Escherichia coli . Expression of glnB-lacZ fusions in two A . brasilense ntrC mutants differed from that in the wild-type strain . In vitro studies also indicated that the purified NtrC protein from E . coli was able to bind to the glnB promoter region of A . brasilense . Our results show that the NtrC protein activates glnBglnA expression under nitrogen limitation in A . brasilense.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 25 - 31
Membrane-derived oligosaccharides (MDOs) are essential for sodium dodecyl sulfate resistance in Escherichia coli; Rajagopal S et al.; We studied the role of membrane-derived oligosaccharides (MDOs) in sodium dodecyl sulfate (SDS) resistance by Escherichia coli . MDOs are also known as osmoregulated periplasmic glucans . Wild-type E . coli MC4100 grew in the presence of 10% SDS whereas isogenic mdoA and mdoB mutants could not grow above 0.5% SDS . Similarly, E . coli DF214, a mutant (pgi, zwf) unable to grow on glucose, exhibited conditional sensitivity to SDS in that it grew in gluconate and glucose or galactose but not in gluconate and mannose or sorbose . DF214 requires both gluconate and glucose/galactose because the gluconate is used for energy production, while glucose/galactose is used for MDO synthesis . Finally, the fate of E . coli cells subjected to SDS shock either during growth or when used as an inoculum is dependent on the presence or absence of sufficient MDOs . In both cases, cells grown under high-osmolarity (low-MDO) conditions were rapidly lysed by 5% SDS . Based on findings from a wild-type E . coli (MC4100), two mdo mutants and strain DF214 we conclude that MDOs are required for SDS resistance.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 15 - 20
Isolation of high molecular weight DNA from soil for cloning into BAC vectors; Berry AE et al.; Isolation of high molecular weight DNA fragments from soil, in excess of 1 Mb, and of sufficient quality for cloning into an Escherichia coli-streptomycete artificial chromosome vector is described . The combination of indirect extraction of cells, using a nycodenz extraction technique, followed by lysis of biomass immobilised in agarose plugs, allowed fragments in excess of 1 Mb to be purified.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jun, 135(2), 197 - 218
Pyruvate kinase: current status of regulatory and functional properties; Munoz ME et al.; Pyruvate kinase (PK) is a key enzyme for the glycolytic pathway and carbon metabolism in general . On the basis of the relevance and enormous diverse properties of this enzyme, this paper describes the results of a current and extensive review that determines the sites of conservation and/or difference in PK sequences, and the differences in the functional and regulatory properties of the enzymes . An alignment and analysis of 50 PK sequences from different sources and a phylogenetic tree are presented . This analysis was performed with reference to crystallographically characterized PK principally from E . coli, cat and rabbit muscle . A number of attributes of the enzyme that make it of particular interest in biomedicine and industry are also discussed.

J Mol Biol, 2003 Jun 20, 329(5), 941 - 8
Open complex formation in vitro by sigma38 (rpoS) RNA polymerase: roles for region 2 amino acids; Lee SJ et al.; Non-functional mutants of sigma(38)(sigma(S)) were studied in vitro to identify the nature of their defects . Mutations in four amino acids led to severe defects in DNA binding and enzyme isomerization with promoter fork junction probes containing single-stranded non-template DNA . The same properties were previously seen with DNA mutations at the fork junction, implying that sigma:DNA interactions at the fork junction are used both for DNA binding and enzyme isomerization . An overlapping set of four mutants had defects that appear to be associated with DNA melting to create the fork junction . When mapped onto the sigma(70) structure, these groups of mutants suggest motifs used by sigma factors to melt DNA and isomerize RNA polymerase to form functional open promoter complexes.

J Mol Biol, 2003 Jun 20, 329(5), 875 - 89
Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA; Ravin NV et al.; Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC . A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon . We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression . Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure . SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues . Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif . Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB . Yeast two-hybrid analysis indicated that these regions contact SopB directly . Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.

J Mol Biol, 2003 Jun 20, 329(5), 853 - 7
Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria; Piwowarski J et al.; The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation . Here, we demonstrate that the hPNPase is localized in mitochondria . Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.

Vaccine, 2003 Jun 20, 21(21-22), 2844 - 51
A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine; Sugino M et al.; Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins . We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1) . Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts . Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits . Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding . rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.

Vaccine, 2003 Jun 20, 21(21-22), 2761 - 6
A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein; Fingerut E et al.; In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested . The fiber protein is responsible for binding the virus to the target cell . The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein . The hexon, fiber capsid protein and knob-s were produced in E . coli and injected into chickens . Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity . Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine . We assume that production of only part of the fiber enables the protein produced in E . coli to fold correctly . Antibodies produced against the hexon protein showed no SN activity . In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber . The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

Mol Biochem Parasitol, 2003 Jun, 129(1), 61 - 8
Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue; Higazi TB et al.; Angiogenesis is an important step in the development of ocular onchocercaisis . In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity . The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism . These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis . Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone . Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation . The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis . Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns . Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O . volvulus revealed that some polymorphism existed among the various isolates tested . However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

Cardiovasc Res, 2003 Jun 1, 58(3), 602 - 10
Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity; Sichelschmidt OJ et al.; OBJECTIVE: The cardioprotective effects of trapidil on ischemic reperfused (I/R) rabbit hearts were studied . Recently, we had shown that trapidil might activate protein kinase A (PKA) . In this study, we examined the exact mode of PKA stimulating activity of trapidil . Finally, we investigated the effect of trapidil on the phosphorylation state of phospholamban (PLB), a major PKA target in the heart and key regulator of Ca(2+) sequestration via the sarcoplasmic reticulum Ca(2+)-ATPase . METHODS: Langendorff-hearts of New Zealand White rabbits were perfused at constant volume and subjected to global low-flow ischemia for 2 h, followed by 1 h of reperfusion . Subsequently, hearts were used for Western blot analysis of PLB phosphorylation . Furthermore, three different regulatory subunits and one catalytic subunit of PKA were overexpressed in E . coli . These PKA subunits were purified and used in an in vitro assay system to test the impact of trapidil on PKA activities in the absence and presence of cAMP . RESULTS: I/R resulted in a significant increase in left ventricular end-diastolic pressure and creatine kinase efflux in the hearts . Trapidil (10 microM) prevented these alterations . Using recombinant cAMP-free PKA isoforms, it was found that trapidil specifically stimulated PKAII but only did so in the presence of small amounts of added cAMP . Furthermore, the PKA-dependent 16Ser phosphorylation of PLB was markedly reduced in I/R . Trapidil largely normalized the 16Ser phosphorylation of PLB . CONCLUSIONS: The data demonstrate cardioprotective actions of trapidil in I/R and show a PKAII-dependent cAMP sensitizing effect of the compound . They also indicate PKA-dependent PLB phosphorylation as a target, suggesting an improved Ca(2+) uptake by the sarcoplasmic reticulum . This action might be involved in the cardioprotective effects of trapidil.

Res Microbiol, 2003 May, 154(4), 283 - 7
Channeling phage DNA through membranes: from in vivo to in vitro; Letellier L et al.; We discuss current models of phage DNA transport through membranes . We present results that attempt to answer the following questions: is there a single mechanism of transport for all types of phage? is DNA transported as a free molecule or in association with proteins? what is the driving force for transport?

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jul 5, 791(1-2), 323 - 36
Guidelines for analytical method development and validation of biotechnological synthesis of drugs . Production of a hydroxyprogesterone as model; Lindholm J et al.; In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients . In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field . Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E . coli using progesterone as substrate . The process liquid was extracted and analyzed using an HPLC-DAD system . The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107% . The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions . The tolerances (C.V . values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process) . In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jul 5, 791(1-2), 127 - 35
Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization; Song J et al.; A protocol utilizing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) using a simplified trimethylsilyl (TMS) derivatization protocol was developed and validated for the determination of hydroxylated metabolites of 3-keto-4-ene steroids such as testosterone, progesterone and androstenedione . Hydroxylated metabolites catalyzed by human CYP1B1 were extracted with methylene chloride and derivatized with BSTFA-10% TMCS . To get an optimal derivatizing condition, the effect of various incubation times and temperatures was evaluated . When the incubation temperature and time in the presence of the TMS derivatizing agent were increased, the 3-keto group became derivatized with TMS to form a 3-TMS derivative . To minimize the formation of the TMS ether on the 3-keto group, a reaction condition of 56 degrees C for 10 min was used for the routine measurement of the steroids and their hydroxylated metabolite . Performance studies including linearity of calibration curves, extraction efficiency and precision were performed . Linearity of the calibration curves was satisfactory from 0.125 to 5 microM for most compounds except 21-hydroxyprogesterone and 16alpha-hydroxyandrostenedione which deviated from linearity at the lower concentrations . Mean percentage extraction recoveries were greater than 80% for all compounds . Most compounds showed good precisions with C.V.s of within-day precision of less than 5% and C.V.s of between-day precision of less than 10% . The selected ion chromatograms from the recombinant human CYP1B1 incubations with testosterone, progesterone and androstenedione showed evidence of 6beta-, 16alpha-, 2alpha-, and 15alpha-hydroxytestosterone, 6alpha- and 16alpha-hydroxyprogesterone and 6alpha- and 16alpha-hydroxyandrostenedione, respectively . There was no significant interference associated with Escherichia coli membrane extracts in detecting hydroxylated metabolites . This procedure provides a rapid and sensitive method for the evaluation of steroid hydroxylation by CYP isoenzymes.

Bioresour Technol, 2003 Sep, 89(3), 249 - 53
Characterization of an Escherichia coli lysA insertion targeted mutant using phenotype arrays; Li X et al.; The objective of this study was to investigate the effect of a lysine biosynthesis insertion mutation on the growth response and phenotype of Escherichia coli . The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria . This E . coli insertion mutant exhibited altered growth physiology and phenotype of the recipient E . coli . The constructed mutant could grow in the absence of lysine supplementation although the extent of growth after 7 h incubation in the presence of most lysine concentration was significantly (p<0.05) decreased compared to that observed with the parent E . coli strain . The mutant was also less able to utilize carbon and nitrogen substrates than the parent E . coli strain as determined by using phenotype arrays . These results suggest that the carbon and nitrogen phenotype profiles of E . coli when measured on phenotype arrays are altered after targeted insertion mutagenesis in the lysA gene . Creation of altered phenotypes may have potential for pharmaceutical and biotechnological applications of lysine E . coli metabolism.

J Orthop Res, 2003 Jul, 21(4), 573 - 83
Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7; Hidaka C et al.; BACKGROUND: Cartilage has a limited capacity to heal . Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy . Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro . In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair . METHODS: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses . Biopsies were performed to evaluate early healing at 4 weeks . At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells . RESULTS: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control . Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted . By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups . CONCLUSIONS: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects . CLINICAL RELEVANCE: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life . This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing.

Int J Immunopathol Pharmacol, 2003 May-Aug, 16(2), 157 - 66
Interleukin-4 and interferon-gamma production during HIV-1 infection and changes induced by antiretroviral therapy; Vecchiet J et al.; Several lines of evidence indicate that a switch of the cytokine pattern from a predominant type 1 (antiviral and cell mediated response) to type 2 (polyclonal humoral immune response) occurs during the course of Human Immunodeficiency Virus-1 (HIV-1) infection, and represents a key event in the progression of immunodeficiency and dysregulated immune activation . We proposed to further investigate this immunological aspect of HIV-1 disease, in naive and in patients treated with Highly Active Antiretroviral Therapy (HAART) . The prototypic cytokines chosen were Interleukin (IL)-4 and Interferon-gamma (IFN-gamma), whose in vitro production was determined in mononuclear cell cultures stimulated with different T lymphocyte mitogenic agents (anti-CD3, Phytohaemoagglutin-P -PHA-, E . coli B04/035 Lipopolysaccharide -LPS-) . We classified all the patients on the basis of the number of CD4+ lymphocytes and we found a progressive, even if not significant decrease in the baseline production of IFN-gamma with the progression of the immunodeficiency . The mean value of baseline IFN-gamma in the group of patients with CD4+>500 cells/microL was 7.79 +/- 3.1 pg/mL while in the group with CD4+<200 cells/microL it was 4.66 +/- 2.22 . We didn't find significant differences in the baseline production of IL-4 in these groups and in IFN-gamma and IL-4 production in LPS-stimulated cultures . We also re-assessed 12 patients after one year's follow-up . They presented a significant increase in IFN-gamma production compared to the first assessment in the LPS-stimulated cultures (baseline IFN-gamma 2.87 +/- 1.17 pg/mL, after 12 months 19.15 +/- 5.19 pg/mL; p= 0.03) . In the 12 patients in follow-up IL-4 production showed a decreased in PHA-stimulated cultures with mean values of 16.65 +/- 14.32 pg/mL at baseline and 6.54 +/- 6.54 pg/mL after follow-up . These results highlight the immunorestoring effects of HAART . IL-4 production was lower in the treated subjects compared to the naive ones in PHA-stimulated cultures (mean values: IL-4=13.42 +/- 11.08 pg/mL in the naive patients and 9.75 +/- 65 pg/mL in the treated patients) . The IFN-gamma values in anti-CD3 stimulated cultures were also higher in the treated patients, but this increase was not significant.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jun, 35(6), 503 - 10
A new model of trispecific antibody resulting the cytotoxicity directed against tumor cells; Song LP et al.; Bispecific antibodies (BsAb) with specificity to both tumor cells and CD3 molecule were believed to be promising immunological tools for the therapy with minimal residual diseases by activating cytotoxic T cells . However, without costimulatory molecule CD28, the activated T cells tended to apoptosis . In order to kill tumor cells more efficiently, a recombinant multifunctional single-chain trispecific antibody (scTsAb), which contains anti-ovarian carcinoma (OC) scFv, anti-CD3 scFv and VH domain of anti-CD28 antibody, was constructed and expressed in E . coli BL21 Star strain . The scTsAb showed strong binding avidities to membrane antigen of SK-OV-3 cell, CD3 molecule on Jurkat cell, and recombinant CD28 antigen . It was further demonstrated that this scTsAb could activate peripheral blood T cells to elicit strong cytotoxicity against SK-OV-3 cells . This new type of recombinant scFv antibody set up a new technological platform for T cells based immunotherapy against cancer, especially with the failure on MHC antigen presentation or absence of costimulating signal.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7471 - 5 Epub 2003 Jun 09.
RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase; Frugier M et al.; The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs . The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3' end of bound tRNA . Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain . Here we take the domain for synthesis of alanyl adenylate and fuse it to "artificial" peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla . Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem . Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity . Aminoacylation efficiency and specificity also depended on the specific peptide sequence . The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions . They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

J Biol Chem, 2003 Aug 29, 278(35), 33256 - 67 Epub 2003 Jun 09.
Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei; Klunker D et al.; Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner . Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol . Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol . These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria . In Methanosarcina mazei Go1, both chaperonins are similarly abundant and are moderately induced under heat stress . The M . mazei GroEL/GroES proteins have the structural features of their bacterial counterparts . The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1 . As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit . The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.

Clin Cancer Res, 2003 Jun, 9(6), 2374 - 83
Truncated galectin-3 inhibits tumor growth and metastasis in orthotopic nude mouse model of human breast cancer; John CM et al.; PURPOSE: The goal of this research was to evaluate a potential therapeutic agent for breast cancer based on galectin-3 that has been implicated in tumorigenicity and metastasis of breast cancer . The hypothesis was that therapy with NH(2)-terminally truncated form of galectin-3 (galectin-3C) will be efficacious for reduction in tumor growth and for inhibition of metastases . EXPERIMENTAL DESIGN: Recombinant human galectin-3 was produced in Escherichia coli from which galectin-3C was derived by collagenase enzyme digestion . Toxicity, pharmacokinetic, and organ biodistribution studies were performed in nude mice . For efficacy studies, nude mice bearing orthotopically implanted tumors derived from breast cancer cell line MDA-MB-435 were treated with galectin-3C or a vehicle control i.m . twice daily for 90 days . RESULTS: The maximum tolerated dose of galectin-3C in nude mice was determined to be >125 mg/kg without overt adverse effects . The elimination half-life when administered i.m . was found to be 3.0 h in the serum and 4.3 h in the cellular fraction of the blood . Organ biodistribution studies revealed that galectin-3C localized in the liver, kidneys, and spleen but not in the heart or lungs . We found that the mean tumor volumes and weights were statistically significantly less in mice treated with galectin-3C compared with control mice, and that fewer numbers of mice exhibited lymph node metastases in the treated group compared with the control group . CONCLUSIONS: Galectin-3C is not overtly toxic, and is efficacious in reducing metastases and tumor volumes and weights in primary tumors in an orthotopic nude mouse model of human breast cancer.

Eukaryot Cell, 2003 Jun, 2(3), 560 - 8
RBP38, a novel RNA-binding protein from trypanosomatid mitochondria, modulates RNA stability; Sbicego S et al.; We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria . This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms . Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis . Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA . In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38 . The half-life of metabolically labeled RNA decreased from approximately 160 to approximately 60 min after depletion . In contrast, there was no change in transcriptional activity . These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.

Biochemistry, 2003 Jun 17, 42(23), 7044 - 9
Contribution to stability and folding of a buried polar residue at the CARM1 methylation site of the KIX domain of CBP; Wei Y et al.; The transcriptional coactivator and acetyltransferase CREB Binding Protein (CBP) is comprised of several autonomously folded and functionally independent domains . The KIX domain mediates interactions between CBP and numerous transcriptional activators . The folded region of KIX has all the structural features of a globular protein, including three alpha-helices, two short 3(10) helices, and a well-packed hydrophobic core . KIX contains a buried cation-pi interaction between the positively charged guanidinium group of Arg 600 and the aromatic ring of Tyr 640 . Arg 600 is a site for regulatory methylation by CARM1/PRMT4, which negates the CREB-binding function of the KIX domain . The role of the Arg 600-Tyr 640 buried polar interaction in specifying and stabilizing the structure of KIX was investigated by comparing the folding of wild-type KIX with the single point mutants Y640F and R600M . The Y640F mutant disrupts a hydrogen bond involving the Tyr 640 OH and the backbone of V595 but still allows for the cation-pi interaction while the R600M mutant disrupts the cation-pi interaction . Both wild type KIX and Y640F exhibit properties expected of native like, globular proteins such as a single oligomerization state (monomer), cooperative thermal and urea-induced unfolding transitions, and a well-packed core . In contrast, the R600M mutant has properties reminiscent of a molten globule state, including a tendency to aggregate, noncooperative thermal unfolding transition, and a loosely packed core . Thus, the buried cation-pi interaction is critical for specifying the unique cooperatively folded structure of KIX.

Can J Microbiol, 2003 Mar, 49(3), 171 - 80
Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85; Jun HS et al.; Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized . Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences . The amino acid sequences exhibited similarities of between 53 and 60% . The xyn10D, xyn10E, and truncated xyn10deltaACBM were expressed in Escherichia coli and purified to homogeneity . The purified Xyn10D, Xyn10E, and Xyn10BdeltaCBM exhibited the same temperature optimum (40 degrees C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively . Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DdeltaCBM did not bind to the substrates . The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose . RT-PCR analysis showed that the three genes were co-transcribed as a single transcript . Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F . succinogenes grown on either glucose or cellulose as the source of carbohydrate.

J Biotechnol, 2003 May 8, 102(3), 223 - 231
Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations; Stepanov VG et al.; A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases . The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants . No expensive reagents or equipment were required . Plasmid quality was evaluated by gel electrophoresis and restriction analysis . The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.

Nat Biotechnol, 2003 Jul, 21(7), 790 - 5 Epub 2003 Jun 08.
Systematic discovery of analogous enzymes in thiamin biosynthesis; Morett E et al.; In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species . In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity . Here we fill such gaps by analyzing anticorrelating occurrences of genes across species . Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins . So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway . For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity . The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity.

Crit Care Med, 2003 Jun, 31(6), 1802 - 7
Role of ubiquitin-proteasome pathway in skeletal muscle wasting in rats with endotoxemia; Chai J et al.; OBJECTIVE: To investigate the mechanism of muscle protein breakdown under endotoxemia condition . DESIGN: Randomized, controlled, animal experiment in a hospital institute . SETTING: Experimental laboratory . INTERVENTION: Either saline or endotoxin (Escherichia coli O(55)B(5), 10 mg/kg) were administered into the peritoneal cavity in rats . MEASUREMENTS AND MAIN RESULTS: The rate of total protein breakdown was increased by 29% and 61% in extensor digitorum longus muscle at 2 hrs and 6 hrs, whereas the myofibrillar proteolytic rate was increased by 155%, 222%, and 40% at 2 hrs, 6 hrs, and 12 hrs, respectively, in the endotoxin treatment group compared with that of the pair-fed normal control group . Meanwhile, compared with the normal control group, the level of 2.4-kilobase (kb) messenger RNA (mRNA) for ubiquitin in extensor digitorum longus muscle in rats was increased by 153% and 470% at 2 hrs and 6 hrs . There were 87% and 117% increases in 1.2-kb mRNA for E2-14K, and 89% and 168% increase in RC2 mRNA expression in extensor digitorum longus muscle in endotoxemic rats than normal control rats at 2 hrs and 6 hrs after injection of endotoxin peritoneally . The tumor necrosis factor-alpha and interleukin-6 concentrations in rat plasma progressively increased after endotoxin treatment, but tumor necrosis factor-alpha peaked at the 2-hr time point, whereas interleukin-6 peaked at 12 hrs . Endotoxin administration resulted in a marked increase in endotoxin level at 2 hrs and 6 hrs . No significant change was observed in soleus muscle after endotoxin injection . A significantly positive correlation was found between the net release of 3-methylhistidine and respective values of endotoxin, intensity of mRNA expression of 2.-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 in extensor digitorum longus muscle (r =.9882, .9731, .9653, .9814, p <.05) . However, no significant correlation was seen between tumor necrosis factor-alpha or interleukin-6 and respective values of 3-methylhistidine, mRNA expression of 2.4-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 (r =.3580, .4521, .5277, .4931, p >.05; r =.3950, .1767, .2136, .2519, p >.05, respectively.) in soleus muscle . CONCLUSIONS: Endotoxemia can induce enhancement of skeletal muscle protein breakdown, mainly involving myofibrillar protein and white, fast-twitch extensor digitorum longus muscle . Ubiquitin-proteasome proteolytic pathway plays an important and major role in skeletal muscle proteolysis . Endotoxin, tumor necrosis factor-alpha, and interleukin-6 can directly or indirectly regulate muscle protein breakdown.

J Biol Chem, 2003 Sep 5, 278(36), 34555 - 67 Epub 2003 Jun 06.
Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants; Telmer PG et al.; The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft . The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM . Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose . Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity . Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose . The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP . These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand . Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.

J Biol Chem, 2003 Aug 15, 278(33), 30806 - 12 Epub 2003 Jun 06.
The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase; Choi-Rhee E et al.; Escherichia coli acetyl-CoA carboxylase (ACC) is composed of four different protein molecules . These proteins form a large but very unstable complex . Hints of a sub-complex between the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) subunits have been reported in the literature, but the complex was not isolated and thus the protein stoichiometry could not be determined . We report isolation of the BC.BCCP complex . By use of affinity chromatography using two different affinity tags it was shown that the complex consists of a two BCCP molecules per BC molecule . The molar ratio in the complex is the same as the ratio of the subunit proteins synthesized in vivo . We conclude that the complex consists of a dimer of BC plus four BCCP molecules instead of the 2BC.2BCCP complex previously assumed . This subunit ratio allows two conflicting models of the ACC mechanism to be rectified . We also report that the N-terminal 30 or so residues of BCCP are responsible for the interaction of BCCP with BC and that the BC.BCCP complex is a substrate for biotinylation in vitro.

J Biol Chem, 2003 Aug 22, 278(34), 31958 - 63 Epub 2003 Jun 06.
A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO; Roberts SA et al.; CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present . The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues . The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule . Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper . This arrangement may mediate electron transfer from substrates to the T1 copper . Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site . The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding . The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.

J Biol Chem, 2003 Aug 22, 278(34), 31701 - 8 Epub 2003 Jun 06.
RNA polymerase subunit requirements for activation by the enhancer-binding protein Rhodobacter capsulatus NtrC; Richard CL et al.; Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R . capsulatus sigma 70 RNA polymerase, but does not activate the Escherichia coli sigma 70-RNA polymerase at the nifA1 promoter . We utilized R . capsulatus:E . coli hybrid RNA polymerases assembled in vitro to investigate the subunits required for protein-protein interaction with RcNtrC at the nifA1mut1 promoter . Assembly of core Rc alpha beta beta' or hybrid RNA polymerases containing the Rc beta beta' subunits absolutely require the inclusion of an omega subunit, with the Ec omega subunit only partially promoting RNA polymerase assembly . The Rc alpha Ec beta beta' RNA polymerase is not activated by RcNtrC . Moreover, a mutant form of the Rc alpha lacking the alpha C-terminal domain, when assembled with the Rc beta beta'omega and sigma 70 subunits, is activated by RcNtrC . These results suggest that the R . capsulatus alpha subunit is not important for RcNtrC interaction . All hybrid RNA polymerases that contained the Rc beta' were activated by RcNtrC, suggesting that the Rc beta' subunit plays an important role . It is proposed that RcNtrC recruits R . capsulatus sigma 70-RNA polymerase to the promoter through interaction with Rc beta' . RcNtrC interacts with RNA polymerase from a unique position, with dimers centered at -118 bp from the start site . Placing the RcNtrC tandem binding sites on the opposite face of the helix (-113 bp) completely abolished transcription activation . Moving the RcNtrC tandem binding sites 20 bp closer to or further from the promoter significantly reduced activation, again suggesting unique spatial constraints on how RcNtrC interacts with the R . capsulatus RNA polymerase.

J Biol Chem, 2003 Aug 22, 278(34), 32014 - 9 Epub 2003 Jun 06.
POLN, a nuclear PolA family DNA polymerase homologous to the DNA cross-link sensitivity protein Mus308; Marini F et al.; The Drosophila Mus308 gene is unusual in encoding both a family A DNA polymerase domain and a DNA/RNA helicase domain . A mus308 mutation was shown to result in increased sensitivity to DNA cross-linking agents, leading to the hypothesis that Mus308 functions in the repair of DNA interstrand cross-links . Recently a mammalian ortholog of Mus308, POLQ, has been identified . We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase also related to Mus308 . The human cDNA encodes a protein of 900 amino acid residues . The region starting from residue 419 shares 33% identity (48% similarity) with the equivalent region of Escherichia coli DNA polymerase I . POLN is expressed in human cell lines with numerous alternatively spliced transcripts, and a full-length human coding region that comprises 24 exons within 160 kilobases of genomic DNA . Expression analysis by northern blotting and in situ hybridization showed highest expression of full-length POLN in human and mouse testis . POLN localized to the nucleus when expressed as a enhanced green fluorescent protein (GFP)-tagged protein in human fibroblasts . GFP-tagged recombinant POLN had DNA polymerase activity on activated calf thymus DNA and on a singly primed template.

Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 169 - 74
Nucleoside diphosphate kinase-like activity in adenylate kinase of Mycobacterium tuberculosis; Meena LS et al.; Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high-energy phosphoryl-transfer reaction between ATP and AMP to form ADP . In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S-transferase fusion protein . Purified Ak converted AMP into ADP in the presence of {gamma-32P}ATP or {gamma-32P}GTP . Replacement of arginine-88 of adk with glycine resulted in the loss of enzymic activity . The purified protein also showed Ndk (nucleoside diphosphate kinase)-like activity as it transferred terminal phosphate from {gamma-32P}ATP to all nucleoside diphosphates, converting them into corresponding triphosphates . However, Ndk-like activity of Ak was not observed with {gamma-32P}GTP . Immunoblot analysis of various cellular fractions of M . tuberculosis H37Rv revealed that Ak is a cytoplasmic protein . The dual activity of Ak as both nucleoside mono- and di-phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.

J Radiat Res (Tokyo), 2002 Dec, 43 Suppl, S137 - 40
Mutation frequency of plasmid DNA and Escherichia coli following long-term space flight on Mir; Takahashi A et al.; To elucidate the biological influence of space radiation, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) . We prepared dried samples of plasmid DNA and repair-deficient and wild type cells of Escherichia (E.) coli . After a 40-day space flight on board the Russian space station Mir, the mutation frequencies of the rpsL gene were estimated by transformation of E . coli and by assessment of conversion of rpsL wild type phenotype (SmS) to its mutant phenotype (SmR) . The experimental findings indicate that mutation frequencies of space samples were not significantly different from those of ground control samples in plasmid DNA and both E . coli strains . It may suggest that space radiation did not influence mutation frequency.

In Vivo, 2003 Mar-Apr, 17(2), 169 - 72
Effect of vitamin C, E-acetate and beta-carotene on the cytostatic activity of nicotinamide (vitamin B3); Getoff N; From experiments in vitro (E . coli bacteria AB 1157) it was established that the cytostatic activity of nicotinamide (vitamin B3) in aqueous, aerated media (pH = 7.4) under irradiation with gamma-rays can be strongly enhanced in the presence of antioxidant vitamins . Whereas the addition of beta-carotene (beta-car) increases nicotinamide activity by a factor of 1.7, vitamin E (vit.E) of 2.16 and vitamin C (vit.C) of 2.3, in the presence of all three vitamins (beta-car, vit.E and C) a four times enhancement is observed . This ability of the vitamins to affect so strongly the cytostatic properties of nicotinamide is of interest for the radiation therapy of patients.

J Clin Microbiol, 2003 Jun, 41(6), 2669 - 71
Multiplex PCR assay for identification of human diarrheagenic Escherichia coli; Toma C et al.; A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed . The targets selected for each category were eae for enteropathogenic E . coli, stx for Shiga toxin-producing E . coli, elt and est for enterotoxigenic E . coli, ipaH for enteroinvasive E . coli, and aggR for enteroaggregative E . coli . This assay allowed the categorization of a diarrheagenic E . coli strain in a single reaction tube.

J Clin Microbiol, 2003 Jun, 41(6), 2448 - 53
Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease; Friedrich AW et al.; The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown . In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P < 0.000001) . The 76 stx(1c)-harboring E . coli isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination . Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing E . coli isolates . Moreover, all but three of these strains contained saa, the gene encoding an STEC autoagglutinating adhesin . The virulence profiles of Stx1c-producing E . coli isolates were mostly serotype independent and heterogeneous . This enabled us to subtype the isolates within the same serotype . The individuals infected with Stx1c-producing E . coli strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic . We conclude that Stx1c-producing E . coli strains represent a significant subset of eae-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and saa as their putative virulence factors . The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.

J Clin Microbiol, 2003 Jun, 41(6), 2367 - 71
Precise characterization of norovirus (Norwalk-like virus)-specific monoclonal antibodies with broad reactivity; Yoda T et al.; We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus) . In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins . In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus . Similar reactivity was observed with the equivalent region of genogroup I Norovirus . In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide . To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short {80-amino-acid} protein fragment) . A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study . Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.

J Clin Microbiol, 2003 Jun, 41(6), 2300 - 5
Studies of epidemiology and seroprevalence of bovine noroviruses in Germany; Deng Y et al.; Jena virus (JV) is a bovine enteric calicivirus that causes diarrhea in calves . The virus is approximately 30 nm in diameter and has a surface morphology similar to the human Norwalk virus . The genome sequence of JV was recently described, and the virus has been assigned to the genus Norovirus of the family CALICIVIRIDAE: In the present study, the JV capsid gene encoded by open reading frame 2 was cloned into the baculovirus transfer vector pFastBac 1, and this was used to transform Escherichia coli to generate a recombinant bacmid . Transfection of insect cells with the recombinant baculovirus DNA resulted in expression of the JV capsid protein . The recombinant JV capsid protein undergoes self-assembly into virus-like particles (VLPs) similar to JV virions in size and appearance . JV VLPs were released into the cell culture supernatant, concentrated, and then purified by CsCl equilibrium gradient centrifugation . Purified JV VLPs were used to hyperimmunize laboratory animals . An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and characterized initially with clinical specimens containing defined human noroviruses and bovine diarrheal samples from calves experimentally infected with JV; the ELISA was specific only for JV . The ELISA was used to screen 381 diarrheal samples collected from dairy herds in Thuringia, Hesse, and Bavaria, Germany, from 1999 to 2002; 34 of these samples (8.9%) were positive for JV infection . The unexpectedly high prevalence of JV was confirmed in a seroepidemiological study using 824 serum or plasma samples screened using an anti-JV ELISA, which showed that 99.1% of cattle from Thuringia have antibodies to JV.

J Clin Microbiol, 2003 Jun, 41(6), 2294 - 9
Diagnostic potential of parechovirus capsid proteins; Alho A et al.; To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione S-transferase (GST)-tagged recombinant proteins . The fusion proteins were used to raise antisera in rabbits . VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence . Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection . When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive . Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay . The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test . Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults . Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.

Carbohydr Res, 2003 Jun 16, 338(12), 1339 - 43
Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase; Koh S et al.; This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24 . The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1) . The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.

Carbohydr Res, 2003 Jun 16, 338(12), 1333 - 7
Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima; Parisot J et al.; A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli . The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da . The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography . The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h . Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.

Acta Pharmacol Sin, 2003 Jun, 24(6), 505 - 11
Small envelope protein E of SARS: cloning, expression, purification, CD determination, and bioinformatics analysis; Shen X et al.; AIM: To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions . METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain . The secondary structure feature of the protein was determined by circular dichroism (CD) technique . The possible functions of this protein were annotated by bioinformatics methods, and its possible three-dimensional model was constructed by molecular modeling . RESULTS: The pure sample of SARS E protein was obtained . The secondary structure feature derived from CD determination is similar to that from the secondary structure prediction . Bioinformatics analysis indicated that the key residues of SARS E protein were much conserved compared to the E proteins of other coronaviruses . In particular, the primary amino acid sequence of SARS E protein is much more similar to that of murine hepatitis virus (MHV) and other mammal coronaviruses . The transmembrane (TM) segment of the SARS E protein is relatively more conserved in the whole protein than other regions . CONCLUSION: The success of expressing the SARS E protein is a good starting point for investigating the structure and functions of this protein and SARS coronavirus itself as well . The SARS E protein may fold in water solution in a similar way as it in membrane-water mixed environment . It is possible that beta-sheet I of the SARS E protein interacts with the membrane surface via hydrogen bonding, this beta-sheet may uncoil to a random structure in water solution.

Mol Microbiol, 2003 Jun, 48(6), 1711 - 25
RdgB acts to avoid chromosome fragmentation in Escherichia coli; Bradshaw JS et al.; Bacterial RecA protein is required for repair of two-strand DNA lesions that disable whole chromosomes . recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosome-disabling lesions . recA-dependent mutants reveal chromosomal lesion avoidance pathways . Here we characterize one such mutant, rdgB/yggV, deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life . The rdgB recA lethality is suppressed by inactivation of endonuclease V (gpnfi) specific for DNA-hypoxanthines/xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines . We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB . rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks . Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants . Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.

Mol Microbiol, 2003 Jun, 48(6), 1679 - 92
Ribosome-DnaK interactions in relation to protein folding; Ghosh J et al.; Bacterial ribosomes or their 50S subunit can refold many unfolded proteins . The folding activity resides in domain V of 23S RNA of the 50S subunit . Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo . The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro . The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact . The DnaK binding influenced the protein folding activity of domain V modestly . Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit . DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.

Mol Microbiol, 2003 Jun, 48(6), 1481 - 9
Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803; Spence E et al.; The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system . Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations . We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP) . Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place . Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network . These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium . The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.

Biotechnol Prog, 2003 May-Jun, 19(3), 1076 - 80
Applicability of new expression vectors for both engineering uses and biological studies; Chao YP et al.; To be applicable for both engineering and biological uses, the plasmid with the features of tight regulation, high-level expression, and subtle modulation (or homogeneous induction) is required . IPTG-inducible promoters are of particular interest since they acquire the latter two merits but usually lack stringency . To this end, two plasmids have been developed to contain the T7 A1 promoter along with either lacI(q) or lacI gene . As a production system, the cells harboring the plasmids with the lacZ gene clone enabled production of the maximal protein accounting for 35% total cell content upon induction by a saturating IPTG level . This protein yield is amplified over 700-fold relative to that at the uninduced state . As a system for biological study, the ppc negative strain bearing the plasmid with the ppc gene clone failed to grow on glucose without IPTG induction but immediately resumed its growth in the presence of IPTG . Moreover, the level of the ppc gene product in the cell was varied by various IPTG, and the result revealed that the wild-type ppc level was sufficient to support the saturated growth of the cell on glucose . Overall, it illustrates the applicability of these plasmids to needs in the post-genome era.

Biotechnol Prog, 2003 May-Jun, 19(3), 982 - 6
Nucleic acid separations utilizing immobilized metal affinity chromatography; Murphy JC et al.; Immobilized metal affinity chromatography (IMAC) is widely used for protein purification, e.g., in the isolation of proteins bearing the well-known hexahistidine affinity tag . We report that IMAC matrixes can also adsorb single-stranded nucleic acids through metal ion interactions with aromatic base nitrogens and propose that metal affinity technologies may find widespread application in nucleic acid technology . Oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose . The affinity of yeast RNA for IDA-chelated metal ions decreases in the following order: Cu(II), Ni(II), Zn(II), and Co(II) . Adsorption isotherms for 20-mer oligonucleotide homopolymers show that purines are strongly favored over pyrimidines and that double-stranded duplexes are not bound . IMAC columns have been used to purify plasmid DNA from E . coli alkaline lysates, to purify a ribozyme, to remove primers and imperfect products from PCR reactions, and to separate 20-mer oligonucleotide duplexes containing centered single-base mismatches . Potential further applications include SNP scoring, hybridization assays, and the isolation of polyadenylated messenger RNA.

Biotechnol Prog, 2003 May-Jun, 19(3), 915 - 20
On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment; Gao YG et al.; The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag . The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies . In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C . Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery . Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised . The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability . A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.

Biotechnol Prog, 2003 May-Jun, 19(3), 887 - 98
Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing; Heeboll-Nielsen A et al.; In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving (i) chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields . The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described . In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K(d) approximately 0.08 microM) was exhibited by Cu(2+)-charged type II support materials . Equilibrium adsorption of L1 to these nonporous supports was achieved very rapidly (<300 s), and approximately 90% of the tightly bound L1 could be desorbed in just one elution step by including >100 mM imidazole in the equilibration buffer . The influence of feedstock complexity on L1 adsorption to the Cu(2+)-charged type II magnetic chelators was studied using various dilutions of four crude chemical E . coli cell extracts containing denatured L1 protein . Undiminished L1 adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L(-1)) and spermine-treated chemical cell extract (feedstock B) . Efficient recovery of L1 from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu(2+)-chelator particles following batch adsorption of L1 . Over 70% of the initial L1 present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of approximately 10.

J Allergy Clin Immunol, 2003 Jun, 111(6), 1328 - 36
Native Art v 1 and recombinant Art v 1 are able to induce humoral and T cell-mediated in vitro and in vivo responses in mugwort allergy; Schmid-Grendelmeier P et al.; BACKGROUND: Mugwort pollen is an important allergen source in hay fever and pollen-related food allergy . Little is known about the clinical relevance of the major mugwort allergen Art v 1 and its importance in allergy . OBJECTIVE: In this study we aimed to investigate the allergenicity of mugwort extract compared with the allergenicity of native (n)Art v 1 and recombinant (r)Art v 1, one major allergen of mugwort, in vivo and in vitro . METHODS: Thirty-two patients allergic to mugwort and 10 control subjects were investigated by means of skin prick and nasal provocation testing with different concentrations of mugwort extract, nArt v 1, and rArt v 1 . nArt v 1 was purified from aqueous mugwort extract, and rArt v 1 was cloned, expressed in Escherichia coli, and then purified . The in vitro allergenicity was measured by means of ImmunoCAP, ELISA, ELISA-inhibition experiments, and T-cell proliferation assays . RESULTS: nArt v 1 and rArt v 1 were able to elicit positive in vivo and in vitro reactions . The IgE-binding capacity, as determined by means of ELISA, was slightly higher for nArt v 1 than for rArt v 1, and both allergens were able to induce T-cell proliferation in sensitized patients . However, rArt v 1 elicited a reduced response in skin and nasal provocation tests compared with nArt v 1 . Compared with mugwort extract, both nArt v 1 and rArt v 1 showed lower sensitivity in patients with mugwort allergy in vivo . CONCLUSIONS: Art v 1, either in its native or recombinant form, is able to induce allergic reactions in patients with mugwort allergy . rArt v 1 induced comparable humoral and cell-mediated responses in vitro but showed reduced in vivo allergenicity compared with biochemically purified nArt v 1.

Afr Health Sci, 2001 Aug, 1(1), 3 - 8
Evaluation of the adjuvant effect of Escherichia coli heat-labile enterotoxin mutant (LTK63) on the systemic immune responses to intranasally co-administered measles virus nucleoprotein . Part I: antibody responses; Erume J et al.; The adjuvanticity and immunogenicity of the heat-labile enterotoxin (LT) of Escherichia coli and of its non-toxic mutant, LTK63, was evaluated after intranasal administration of CBA mice with recombinant measles virus nucleoprotein (rMVNP) with or without LT or LTK63 . Both LT and LTK63 were shown to be highly immunogenic with higher responses observed 4 weeks after the booster immunization . Although the nucleoprotein was immunogenic on its own, mice immunized with the nucleoprotein plus wild type LT produced significantly high antibody responses (p< 0.01) . Mice that received the rMVNP with LTK63 also generated strong antibody responses to rMVNP . These antibodies were also significantly higher than those of rMVNP alone (p< 0.05) . No significant differences were observed between groups of mice immunized intranasally with rMVNP plus LT or LTK63 (p> 0.05) . Data on IgG antibody isotype profiles showed that IgG 1 and IgG 2a were predominant in mice immunized with rMVNP + LT or LTK63 whereas IgG 1 predominated when rMVNP was given on its own implying that LT and LTK63 induce both Th1 and Th2-type immune responses . These results highlight the great potential of this non-toxic mutant of LT as a safe vaccine adjuvant.

J Biol Chem, 2003 Aug 22, 278(34), 31930 - 40 Epub 2003 Jun 03.
Kinetic mechanism for formation of the active, dimeric UvrD helicase-DNA complex; Maluf NK et al.; Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids . We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro . Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex . Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps . The results show that the active dimeric complex can form via two pathways . The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase . The rate-limiting step within the monomer pathway involves dimer assembly on the DNA . These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.

J Biol Chem, 2003 Aug 15, 278(33), 31033 - 42 Epub 2003 Jun 04.
Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK; Mogk A et al.; Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones . The mechanistic basis of this refolding process is poorly defined . We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously . Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK . While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps . Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress . We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.

J Biol Chem, 2003 Aug 15, 278(33), 30705 - 10 Epub 2003 Jun 04.
Direct interaction between Escherichia coli RNA polymerase and the zinc ribbon domains of DNA topoisomerase I; Cheng B et al.; Escherichia coli DNA topoisomerase I (encoded by the topA gene) is important for maintaining steady-state DNA supercoiling and has been shown to influence vital cellular processes including transcription . Topoisomerase I activity is also needed to remove hypernegative supercoiling generated on the DNA template by the progressing RNA polymerase complex during transcription elongation . The accumulation of hypernegative supercoiling in the absence of topoisomerase I can lead to R-loop formation by the nascent transcript and template strand, leading to suppression of transcription elongation . Here we show by affinity chromatography and overlay blotting that E . coli DNA topoisomerase I interacts directly with the RNA polymerase complex . The protein-protein interaction involves the beta' subunit of RNA polymerase and the C-terminal domains of E . coli DNA topoisomerase I, which are homologous to the zinc ribbon domains in a number of transcription factors . This direct interaction can bring the topoisomerase I relaxing activity to the site of transcription where its activity is needed . The zinc ribbon C-terminal domains of other type IA topoisomerases, including mammalian topoisomerase III, may also help link the enzyme activities to their physiological functions, potentially including replication, transcription, recombination, and repair.

J Biol Chem, 2003 Aug 22, 278(34), 31647 - 56 Epub 2003 Jun 04.
Proanthocyanidin biosynthesis in plants . Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA; Tanner GJ et al.; Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis . The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned . The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover . The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of approximately 10 micromol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin . LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins . Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known . Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome . This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.

Appl Environ Microbiol, 2003 Jun, 69(6), 3448 - 55
Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases; Rau J et al.; Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species . These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells . Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds . In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E . coli and thus for the lawsone-dependent anaerobic azo reductase activity . An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells . The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined . The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism . Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB . In addition it was proven that also a second oxygen-insensitive nitroreductase of E . coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.

Appl Environ Microbiol, 2003 Jun, 69(6), 3421 - 6
Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids; Lee SY et al.; A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R . eutropha intracellular PHA depolymerase gene . By with this metabolically engineered E . coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose . By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose . By integration of the PHA biosynthesis genes into the chromosome of E . coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics . This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.

Appl Environ Microbiol, 2003 Jun, 69(6), 3344 - 9
Interactions of insecticidal toxin gene products from Xenorhabdus nematophilus PMFI296; Sergeant M et al.; Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species . Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined . The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E . coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens . The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P . rapae and P . brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H . virescens . When each of these three genes was expressed individually in E . coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level . If the genes xptB1 and xptC1 were expressed in the same E . coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P . rapae and P . brassicae . Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H . virescens . Individual gene disruptions in X . nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E . coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes . The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene . Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.

Appl Environ Microbiol, 2003 Jun, 69(6), 3231 - 7
Escherichia coli heat shock protein DnaK: production and consequences in terms of monitoring cooking; Seyer K et al.; Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens . For a given process lethality (F(70)(10) of 1, 3, and 5 min), the intracellular concentration of DnaK in E . coli varied with the heating temperature (50 or 55 degrees C) . In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50 degrees C) applied for a longer time . Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process . Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress . Indeed, E . coli cells that had undergone a sublethal heat shock (105 min at 55 degrees C, F(70)(10) = 3 min) accompanied by a 12-h recovery (containing 76,786 +/- 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 +/- 6,056 molecules/cell) when later heated to 60 degrees C for 50 min (F(70)(10) = 5 min) . Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.

Appl Environ Microbiol, 2003 Jun, 69(6), 3029 - 35
Isolation and characterization of NaCl-sensitive mutants of Caulobacter crescentus; Zuleta LF et al.; An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl . A transposon Tn5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated . Three of the mutants had the Tn5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn5 in the nhaA gene, which encodes a Na(+)/H(+) antiporter, and one had the Tn5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase . All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions . All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock . The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes . Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the sigma(32) regulon, as opposed to what was observed for its Escherichia coli homolog.

J Surg Res, 2003 Apr, 110(2), 413 - 8
Endotoxin impairs endothelium-dependent vasodilation more in the coronary and renal arteries than in other arteries of the rat; Piepot HA et al.; Endotoxemia may result in endothelial dysfunction, and some vascular beds may be affected more than others . To test this hypothesis, we studied, in vitro, the reactivity of isolated rat coronary, renal, superior mesenteric, and hepatic arteries exposed to endotoxin (E . coli, 50 microg . mL(-1)) or saline for 2 h at 37 degrees C . Vascular smooth muscle function was tested using 125 mM KCl, the vasoconstrictors norepinephrine (NE), and the thromboxane analog U46619 (coronary artery) . Endothelium-dependent vasorelaxation was tested with acetylcholine (ACh) in preconstricted vessels . Although differing between vessel types, the smooth muscle contractile responses were not affected by endotoxin, either in the presence or absence of L-arginine . Endotoxin impaired the response to ACh in rat coronary arteries (92.7 +/- 4.6% vasodilation in control and 41.3 +/- 11.6% in endotoxin-exposed segments) and in renal arteries (66.7 +/- 5.2% vasodilation in control and 43.2 +/- 4.9% in endotoxin-exposed segments), so that there was a mean 55% decrease vs controls in coronary and a mean 35% decrease in renal arteries . Endotoxin did not affect superior mesenteric and hepatic arteries . Brief endotoxin exposure of isolated rat arteries may thus inactivate endothelial NO synthase, independent of iNOS . The increase in heterogeneity among endothelium-dependent vasodilation after endotoxin may help to explain early blood flow maldistribution in endotoxin shock.

Virology, 2003 May 25, 310(1), 109 - 17
Effect of selected mutations in the C-terminal region of the vaccinia virus nucleoside triphosphate phosphohydrolase I on binding to the H4L subunit of the viral RNA polymerase and early gene transcription termination in vitro; Piacente SC et al.; Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) is an essential early gene transcription termination factor . The C-terminal end of NPH I binds to the N-terminal end of the H4L subunit (RAP94) of the virion RNA polymerase . This interaction is required for transcription termination and transcript release . To refine our understanding of the specific amino acids in the C-terminal end of NPH I involved in binding to H4L, and to develop a collection of mutations exhibiting various degrees of activity to be employed in in vivo studies, we prepared a set of short deletions, and clustered substitutions of charged amino acids to alanine, or bulky hydrophobic amino acids to alanine mutations . These NPH I mutant proteins were expressed, purified, and tested for ATPase activity, binding to H4L, and transcription termination activity . Most mutations in amino acids 609 to 631 exhibited reduced activity . Deletion of the terminal five amino acids (627-631), or substitution of Y(629) with alanine or glutamic acid, dramatically reduced NPH I mediated transcription termination . Deletion of the terminal F(631), or substitution of F(631) with alanine, reduced binding to H4L and eliminated termination activity . These observations demonstrate that the terminal five amino acids directly participate in binding to RNA polymerase and in early gene transcription termination.

Trends Biotechnol, 2003 Jun, 21(6), 251 - 4
On the complete determination of biological systems; Selinger DW et al.; The nascent field of systems biology ambitiously proposes to integrate information from large-scale biology projects to create computational models that are, in some sense, complete . However, the details of what would constitute a complete systems-level model of an organism are far from clear . To provide a framework for this difficult question it is useful to define a model as a set of rules that maps a set of inputs (e.g . descriptions of the cell's environment) to a set of outputs (e.g . the concentrations of all its RNAs and proteins) . We show how the properties of a model affect the required experimental sampling and estimate the number of experiments needed to "complete" a particular model . Based on these estimates, we suggest that the complete determination of a biological system is a concrete, achievable goal.

FEBS Lett, 2003 Jun 12, 545(1), 31 - 8
Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases; Cecchini G et al.; The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II . The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different . These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.

FEBS Lett, 2003 Jun 12, 545(1), 25 - 30
Protonmotive force generation by a redox loop mechanism; Jormakka M et al.; Respiration involves the oxidation and reduction of substrate for the redox-linked formation of a protonmotive force (PMF) across the inner membrane of mitochondria or the plasma membrane of bacteria . A mechanism for PMF generation was first suggested by Mitchell in his chemiosmotic theory . In the original formulations of the theory, Mitchell envisaged that proton translocation was driven by a 'redox loop' between two catalytically distinct enzyme complexes . Experimental data have shown that this redox loop does not operate in mitochondria, but has been confirmed as an important mechanism in bacteria . The nitrate respiratory pathway in Escherichia coli is a paradigm for a protonmotive redox loop . The structure of one of the enzymes in this two-component system, formate dehydrogenase-N, has revealed the structural basis for the PMF generation by the redox loop mechanism and this forms the basis of this review.

Free Radic Biol Med, 2003 Jun 15, 34(12), 1555 - 62
Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species; Kozlov AV et al.; The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases . In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples . CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo . In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood . By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum . We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum . We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney . Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide . Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO(-) and O(2)(*)(-), as well as other not identified RONS, are detectable by CP-H in tissue . In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues . Increased formation of RONS in response to endotoxin challenge is organ specific.

Bioorg Med Chem, 2003 Jul 3, 11(13), 2783 - 90
Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy; Mahara A et al.; Antisense strategy has high potential for curing diseases and studying gene functions by suppressing the translation step . For the strategy, it is essential to detect acceptor sites of antisense molecules on mRNA under physiological conditions . We propose a new analytical method for the detection of acceptor sites of antisense molecules with high sensitivity . 2'-O-Methyloligoribonucleotide containing 2'-O-(1-pyrenylmethyl)uridine (OMUpy) was chosen as the fluorescence probe . The fluorescence intensity due to the pyrene in single-stranded OMUpy was scarcely observed . When OMUpy was hybridized with the complementary oligoRNA, the fluorescence intensity at 375 nm was remarkably increased . It was found that the increase was derived from the localization of the pyrene by the measurements of time-resolved fluorescence spectroscopy, CD and UV absorption spectra . These results suggest that the change of the fluorescence intensity of OMUpy can be a useful index to monitor hybridization . In this study, we chose Escherichia coli . 16S-rRNA as the model RNA and chose seven regions for probing by OMUpy based on the reported secondary structure of 16S-rRNA . The fluorescence intensity of an equimolar mixture of OMUpy with 16S-rRNA varied depending on the sequence . In particular, the increment in the system of OMUpy-8, which can hybridize with region 887-896 nt of 16S-rRNA, was most significant among the systems . These results indicated that the site targeted by OMUpy-8 was exposed to regulatory molecules, and suggest that the method presented here is useful to design antisense molecules.

Chem Phys Lipids, 2003 Jun, 124(1), 49 - 61
In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups; Sheikh M et al.; A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities . In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity . This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents . In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity . Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.

Biochim Biophys Acta, 2003 Jun 11, 1621(3), 292 - 8
Xerocomus chrysenteron lectin: identification of a new pesticidal protein; Trigueros V et al.; Xerocomus chrysenteron is an edible mushroom with insecticidal properties . In an earlier work, we found that proteins are responsible for this toxicity . Here we describe the purification of a approximately 15 kDa lectin, named XCL, from the mushroom . Its cDNA and gDNA were cloned by PCR strategies and a recombinant form was expressed in Escherichia coli . Sequence alignments and sugar specificity showed that this protein is the third member of a new saline-soluble lectin family present in fungi . This protein, either purified from mushroom or expressed in vitro in E . coli, was found to be toxic to some insects, such as the dipteran Drosophila melanogaster and the hemipteran, Acyrthosiphon pisum . The lectin possesses a high insecticidal activity compared to lectin isolated from leguminosae (Lathyrus ochrus) or from the snowdrop (Galanthus nivalis).

Biochem Pharmacol, 2003 Jun 15, 65(12), 1957 - 65
Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats; Sakaki T et al.; The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3) . The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones . Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues . In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system . In the recombinant E . coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3) . The putative ether was not observed in the recombinant E . coli cells expressing rat CYP24 . These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3) . In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24 . Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor . These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway . Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E . coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E . coli cells appear to exhibit the same metabolism as in human target tissues . Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.

J Mol Biol, 2003 Jun 13, 329(4), 815 - 29
Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein; Baneres JL et al.; We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli . This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2) . Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit . These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha . Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly . This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.

J Mol Biol, 2003 Jun 13, 329(4), 801 - 14
Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1; Baneres JL et al.; We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution . Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies . The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02) . Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4) . We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

J Mol Biol, 2003 Jun 13, 329(4), 779 - 91
Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase; Arai M et al.; The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures . To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR . Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure . Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization . Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates . Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate . This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding . If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.

J Mol Biol, 2003 Jun 13, 329(4), 745 - 61
Structure, stability and dynamics of the central domain of cardiac myosin binding protein C (MyBP-C): implications for multidomain assembly and causes for cardiomyopathy; Idowu SM et al.; The large multidomain muscle protein myosin binding protein C (MyBP-C) has been implicated for some time in cardiac disease while until recently little was known about its structure and function . Here we present a detailed study of the central domain C5 of the cardiac isoform of MyBP-C . This domain is unusual in several aspects . Firstly it contains two sizeable insertions compared to the non-cardiac isoforms . The first insertion comprises the linker between domains cC4 and cC5 that is elongated by ten amino acid residues, the second insertion comprises an elongation of the CD-loop in the middle of the domain by approximately 30 amino acid residues . Secondly two point mutations linked to familial hypertrophic cardiomyopathy (FHC) have been identified in this domain . This work shows that the general fold of cC5 is in agreement with the IgI family of beta-sandwich structures . The long cardiac-specific linker between cC4 and cC5 is not a linker at all but an integral part of the fold of cC5, as evidenced by an unfolded mutant in which this segment was removed . The second insertion is shown to be unstructured, highly dynamic and mostly extended according to NMR relaxation measurements and analytical ultracentrifugation . The loss of several key interactions conserved in the CD-loop of the IgI fold is assumed to be responsible for the low stability of cC5 compared to other IgI domains from titin and MyBP-C itself . The low thermodynamic stability of cC5 is most evident in one of the two FHC-linked mutations, N755K (Asn115 in this construct) which is mainly unfolded with a small proportion of a native-like folded species . In contrast, the second FHC-linked mutation, R654H (Arg14 in this construct) is as well folded and stable as the wild-type . This residue is located in the extended beta-bulge at the N terminus of the protein, pointing towards the surface of the CFGA' beta-sheet . This position is in agreement with recent data pointing to a function of Arg654 in an intermolecular interaction with MyBP-C domain cC8.

J Mol Biol, 2003 Jun 13, 329(4), 645 - 54
Response delays and the structure of transcription networks; Rosenfeld N et al.; Sensory transcription networks generally control rapid and reversible gene expression responses to external stimuli . Developmental transcription networks carry out slow and irreversible temporal programs of gene expression during development . It is important to understand the design principles that underlie the structure of sensory and developmental transcription networks . Cascades, which are chains of regulatory reactions, are a basic structural element of transcription networks . When comparing databases of sensory and developmental transcription networks, a striking difference is found in the distribution of cascade lengths . Here, we suggest that delay times in the responses of the network present a design constraint that influences the network architecture . We experimentally studied the response times in simple cascades constructed of well-characterized repressors in Escherichia coli . Accurate kinetics at high temporal resolution was measured using green fluorescent protein (GFP) reporters . We find that transcription cascades can show long delays of about one cell-cycle time per cascade step . Mathematical analysis suggests that such a delay is characteristic of cascades that are designed to minimize the response times for both turning-on and turning-off gene expression . The need to achieve rapid reversible responses in sensory transcription networks may help explain the finding that long cascades are very rare in databases of E.coli and Saccharomyces cerevisiae sensory transcription networks . In contrast, long cascades are common in developmental transcription networks from sea urchin and from Drosophila melanogaster . Response delay constraints are likely to be less important for developmental networks, since they control irreversible processes on the timescale of cell-cycles . This study highlights a fundamental difference between the architecture of sensory and developmental transcription networks.

Cell, 2003 May 30, 113(5), 597 - 607
Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli; Atkinson MR et al.; Analysis of the system design principles of signaling systems requires model systems where all components and regulatory interactions are known . Components of the Lac and Ntr systems were used to construct genetic circuits that display toggle switch or oscillatory behavior . Both devices contain an "activator module" consisting of a modified glnA promoter with lac operators, driving the expression of the activator, NRI . Since NRI activates the glnA promoter, this creates an autoactivated circuit repressible by LacI . The oscillator contains a "repressor module" consisting of the NRI-activated glnK promoter driving LacI expression . This circuitry produced synchronous damped oscillations in turbidostat cultures, with periods much longer than the cell cycle . For the toggle switch, LacI was provided constitutively; the level of active repressor was controlled by using a lacY mutant and varying the concentration of IPTG . This circuitry provided nearly discontinuous expression of activator.

Cell, 2003 May 30, 113(5), 556 - 7
Periplasmic chaperones--preservers of subunit folding energy for organelle assembly; Behrens S; The periplasmic PapD-like chaperones have long been known to be necessary for the assembly of bacterial surface organelles . New structural work now suggests that they control assembly by arresting subunit folding . This step may be required to preserve energy for fiber formation.

BMC Struct Biol . 2003 Jun 04;3(1):5.
Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment; Hughes SJ et al.; BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate . The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP . Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme . The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study . RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters . An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process . Two lysines are found to bind per dimer, and trigger a large conformational change . Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations.Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site . Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy . Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice . This strongly suggests the involvement of an anti-cooperative mechanism . Pathways for relaying information between the two active sites are proposed . CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role . We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism.

Mol Microbiol, 2003 Jun, 48(5), 1267 - 74
Ribosome bypassing elicited by tRNA depletion; Lindsley D et al.; Ribosome bypassing refers to the ability of the ribosome::peptidyl-tRNA complex to slide down the message without translation to a site several or dozens of nucleotides downstream and resume protein chain elongation there . The product is an isoform of a protein with a 'coding' gap corresponding to the region of the message which was bypassed . Previous work showed that ribosome bypassing was strongly stimulated at 'hungry' codons calling for a tRNA whose aminoacylation was limited . We have now used the 'minigene' phenomenon to ascertain whether depletion of the pool of specific isoacceptors has a similar effect . High level expression of plasmid-borne minigenes results in the sequestration as peptidyl-tRNA of tRNA cognate to the last triplet of the minigene, thereby limiting protein synthesis for lack of the tRNA in question . We find that induction of a minigene ending in AUA stimulates bypassing at an AUA codon, but not in a control sequence with AGA at the test position; induction of a minigene ending in AGA stimulates bypassing at the latter but not the former . Induction of the AUA minigene also stimulates both leftward and rightward frameshifting at 'shifty' sequences containing an AUA codon . The normal, background frequency of bypassing at an AUA codon is markedly reduced by increasing the cellular level of the tRNA which reads the codon . Thus, the frequency of bypassing can be increased or decreased by lowering or raising the concentration of a relevant tRNA isoacceptor . These observations suggest that the occurrence of ribosome bypassing reflects the length of the pause at a given codon.

Mol Microbiol, 2003 Jun, 48(5), 1253 - 65
The DEAD-box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli; Charollais J et al.; Ribosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs . Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non-ribosomal factors facilitate assembly in vivo . Here, we show that SrmB, a putative DEAD-box RNA helicase, is involved in ribosome assembly . The deletion of the srmB gene causes a slow-growth phenotype at low temperature . Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S . Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled . In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro . Sucrose gradient fractionation also shows that, in wild-type cells, SrmB associates with a pre50S particle . From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13 . This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.

Mol Microbiol, 2003 Jun, 48(5), 1241 - 52
A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector; Jonuscheit M et al.; Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea . Although a number of in vitro systems have been established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile . In this work, we have developed a stable and selectable shuttle vector based on the virus SSV1 of Sulfolobus shibatae . We have introduced pUC18 for propagation in Escherichia coli and the genes pyrEF coding for orotidine-5'-monophosphate pyrophosphorylase and orotidine-5'-monophosphate decarboxylase of Sulfolobus solfataricus as selectable marker to complement pyrimidine auxotrophic mutants . Furthermore, the beta-galactosidase gene (lacS) was introduced into this vector as a reporter under the control of the strong and heat-inducible promoter of the Sulfolobus chaperonin (thermosome) . After transformation of a S . solfataricus pyrEF/lacS double mutant, the vector was found to reside as a single-copy vector, stably integrated into the host chromosome via the site-specific recombination system of SSV1 . Specific beta-galactosidase activities in transformants were found to be fourfold higher than in wild-type S . solfataricus cells, and increased to more than 10-fold after heat shock . Greatly increased levels of lacS mRNA were detected in Northern analyses, demonstrating that this reporter gene system is suitable for the study of regulated promoters in Sulfolobus and that the vector can also be used for the high-level expression of genes from hyperthermophilic archaea.

Mol Microbiol, 2003 Jun, 48(5), 1225 - 39
Promoter analysis of Helicobacter pylori genes with enhanced expression at low pH; McGowan CC et al.; To identify Helicobacter pylori genes with expression that is enhanced under low pH conditions, we used subtractive hybridization methodology . We identified 28 acid-induced genes, of which 18 have known or putative functions . Six pairs of genes were co-transcribed . Primer extension analysis identified single or multiple transcriptional start points (tsp) for 14 of the 22 loci . Sequence analysis of the -10 regions upstream of the tsps revealed consensus motifs for multiple RNA polymerase sigma factors present in H . pylori (sigma80, sigma54 and sigma28) . No sequences resembling the -35 Escherichia coli consensus sequence (TTGACA) were present upstream of any of the genes . Both increased gene transcription and decreased mRNA decay contribute to the observed increase in H . pylori transcript abundance at acid pH . These studies document the complex response of H . pylori to environmental pH changes, and provide insight into mechanisms used for intragastric survival.

Mol Microbiol, 2003 Jun, 48(5), 1171 - 82
The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway; Bernhardt TG et al.; The N-acetylmuramoyl-l-alanine amidases of Escherichia coli (AmiA, B and C) are periplasmic enzymes that remove murein cross-links by cleaving the peptide moiety from N-acetylmuramic acid . Ami- cells form chains, indicating that the amidases help to split the septal murein . Interestingly, cells defective in the twin-arginine protein transport (Tat) pathway show a similar division defect . We find that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat- division phenotype . Taking advantage of the ability of Tat to export prefolded (fluorescent) green fluorescent protein (GFP) to the periplasm, we sublocalized AmiA and AmiC in live cells using functional fusions to GFP . Interestingly, the periplasmic localization of the fusions differed markedly . AmiA-GFP appeared to be dispersed throughout the periplasm in all cells . AmiC-GFP similarly appeared throughout the periplasm in small cells, but was concentrated almost exclusively at the septal ring in constricting cells . Recruitment of AmiC to the ring was mediated by an N-terminal non-amidase targeting domain and required the septal ring component FtsN . AmiC therefore replaces FtsN as the latest known recruit to the septal ring and is the first entirely periplasmic component to be localized.

Pediatr Allergy Immunol, 2003 Jun, 14(3), 222 - 8
Early acquisition of serum and saliva antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins by infants living in an endemic area; Carbonare CB et al.; Enteropathogenic Escherichia coli (EPEC) is the most common etiological agent of acute diarrhea among infants living in poor social conditions in Brazil and other developing countries . This infection is rare in breast-fed infants, as well as in children older than 2 years . Over the past few years, our group has attempted to identify antibodies to EPEC virulence proteins in human milk and to establish the in vitro protective role of these antibodies . In the present study, we report the identification of antibodies to EPEC virulence proteins in sera and saliva from children of different ages, living in slums in the city of Sao Paulo, Brazil . Using EPEC and bacterial constructs (pET) for immunoblotting (IB) analysis, antibodies reacting to the main adhesins (intimin, bundle-forming pilli) and cell-signaling proteins (EPEC secreted proteins - Esp A, Esp B) were detected in sera from adults and children older than 1 year . Almost all children older than 1 year presented recognition patterns similar to those of adults in IB assays for serum IgG and secretory IgA antibodies, using EPEC outer membrane and other antigenic preparations . As previously observed for human milk, all samples from adults and older children recognized the 94 kDa molecular weight adhesin intimin strongly . In most children, previous EPEC symptomatic diarrhea could not be confirmed; however, almost all of them have presented one or more diarrhea episodes during their lifetime . These results suggest that reduction of EPEC infection frequency after 2 years of age may be associated with the development of anti-EPEC antibody repertoires.

Eur J Biochem, 2003 Jun, 270(12), 2689 - 95
Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead?
Villarino A, Rager MN, Grimont PA, Bouvet OM.
Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability . As a consequence, the interpretation of the state of 'nonculturable' cells is often ambiguous . Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells . Cells inactivated by the UV1 dose lost 'culturability' but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis . The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose . Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden-Meyerhof and pentose-phosphate pathways . However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities . Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively.

Eur J Biochem, 2003 Jun, 270(12), 2644 - 51
Dynamic model of Escherichia coli tryptophan operon shows an optimal structural design; Bhartiya S et al.; A mathematical model has been developed to study the effect of external tryptophan on the trp operon . The model accounts for the effect of feedback repression by tryptophan through the Hill equation . We demonstrate that the trp operon maintains an intracellular steady-state concentration in a fivefold range irrespective of extracellular conditions . Dynamic behavior of the trp operon corresponding to varying levels of extracellular tryptophan illustrates the adaptive nature of regulation . Depending on the external tryptophan level in the medium, the transient response ranges from a rapid and underdamped to a sluggish and highly overdamped response . To test model fidelity, simulation results are compared with experimental data available in the literature . We further demonstrate the significance of the biological structure of the operon on the overall performance . Our analysis suggests that the tryptophan operon has evolved to a truly optimal design.

Eur J Biochem, 2003 Jun, 270(12), 2633 - 43
Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome; Maeda K et al.; Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS . HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo . During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo . On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR . The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g . rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2) . Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified using a three-step procedure . The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley alpha-amylase/subtilisin inhibitor as substrates . HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR) . The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a Km of 13 micro m compared with 44 micro m for HvTrxh2 . Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles.

Eur J Biochem, 2003 Jun, 270(12), 2612 - 21
Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding . Relaxation kinetic studies with coenzyme analogues; Gutierrez A et al.; The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy . Previous studies {Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K . Biochemistry (2002) 41, 4626-4637} have shown that the observed rate, 1/tau, of interflavin electron transfer (FADsq - FMNsq-->FADox - FMNhq) in CPR reduced at the two-electron level with NADPH is 55 +/- 2 s-1, whereas with dithionite-reduced enzyme the observed rate is 11 +/- 0.5 s-1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer . In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/tau = 18 +/- 0.7 s-1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2'-phosphate is important for enhancing internal electron transfer . To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2',5'-ADP and 2'-AMP . These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/tau = 11 s-1 to 35 +/- 0.2 s-1 and 32 +/- 0.6 s-1, respectively . Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump . Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2',5'-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments . Addition of 2',5'-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to perturbation of the optical spectra of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors . The binding of 2',5'-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentration (i.e . enhanced at low coenzyme concentration) observed in stopped-flow studies . The data are discussed in the context of previous kinetic studies and of the crystallographic structure of rat CPR.

Eur J Biochem, 2003 Jun, 270(12), 2593 - 604
AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death; Belenghi B et al.; In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens . AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly) . Recombinant A . thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified . AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases . The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex . AtCYS1 is constitutively expressed in roots and in developing siliques of A . thaliana . In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO) . The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A . thaliana suspension cultured cells and in transgenic tobacco plants . The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.

Eur J Biochem, 2003 Jun, 270(12), 2583 - 92
Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein; Hanada K et al.; Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation . As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction . However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood . We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein . Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein . To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed . The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants . Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide . Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.

Eur J Biochem, 2003 Jun, 270(12), 2576 - 82
Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor; Carlsson K et al.; Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF) . TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa . The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI . We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI . We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region . Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e . the residues 104 and 197, participates in the interaction with FXa in the quaternary complex . Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

Eur J Oral Sci, 2003 Jun, 111(3), 228 - 34
Effect of lipopolysaccharides on vascular endothelial growth factor expression in mouse pulp cells and macrophages; Botero TM et al.; Vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor, might regulate the neovascularization observed in the pulp of teeth with deep caries . The purpose of this in vitro study was to evaluate the effect of bacterial lipopolysaccharides (LPS) on VEGF expression in dental pulp cells . Mouse odontoblast-like cells (MDPC-23) or undifferentiated pulp cells (OD-21) were exposed to 0-20 microg ml-1Escherichia coli LPS or 0-80 microg ml-1Prevotella intermedia LPS . As controls, mouse macrophages or gingival fibroblasts were exposed to LPS, since these cells are known to secrete VEGF . The VEGF expression was evaluated by reverse transcriptase polymerase chain reaction or enzyme-linked immunosorbent assay . The baseline expression levels of VEGF protein were higher in MDPC-23 and OD-21 than in fibroblasts or macrophages . Vascular endothelial growth factor protein expression was upregulated in MDPC-23 and macrophages exposed to E . coli LPS, but not in OD-21 cells or fibroblasts . Higher concentrations of P . intermedia LPS were required to induce VEGF expression in MDPC-23 cells . Treatment with LPS did not affect VEGF expression at the mRNA level in any of the cells evaluated . These results demonstrate that bacterial LPS upregulates VEGF expression in odontoblast-like cells and macrophages, and suggest that the regulation of VEGF expression occurs primarily at a post-transcriptional level.

Biochem J, 2003 Sep 15, 374(Pt 3), 785 - 92
Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases; Karantzeni I et al.; Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy . The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain . Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively . Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously . Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis . However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase . In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5 . Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately . The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain . Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment . Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq . Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins . Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step . Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

Shock, 2003 Jun, 19(6), 538 - 46
Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle; Lang CH et al.; The presence of increased levels of proinflammatory cytokines in the blood is associated with decreased muscle protein synthesis and the erosion of lean body mass in many catabolic conditions . However, little is known regarding the role of endogenous cytokine synthesis in muscle per se . The purpose of the present study was to characterize the cytokine expression profile of skeletal muscle in response to an in vivo injection of endotoxin (lipopolysaccharide, LPS) . Intraperitoneal injection of a nonlethal dose of LPS (1,000 microg/kg Escherichia coli) into male rats increased the mRNA content of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in gastrocnemius muscle as early as 1 h; IL-6 mRNA was not increased until 2 h post-LPS . Expression of TNF-alpha and IL-1beta peaked at 2 h (10- and 80-fold, respectively), whereas the increased IL-6 mRNA content (150-fold) peaked later at 4 h . The abundance of all measured cytokine mRNAs in skeletal muscle declined thereafter . The LPS-induced increase in muscle mRNA content for TNF-alpha, IL-6, and IL-1beta was dose-dependent with elevations being seen with as little as 10 microg/kg of LPS (2.5-, 8-, and 9-fold, respectively) . In general, pretreatment of rats with dexamethasone attenuated but did not completely prevent the LPS-induced increase in muscle cytokine mRNA . LPS increased muscle TNF-alpha protein content approximately 2-fold and this increase was prevented by pretreatment with dexamethasone . LPS-induced increases in muscle IL-1beta and IL-6 protein were not detected . LPS also produced a 2-fold increase in the mRNA content of the high-mobility-group protein-1, a late-phase cytokine, in muscle at 12-24 h . Finally, although skeletal muscle was found to contain both the toll-like receptor (TLR)-2 and TLR4, LPS did not alter the mRNA content of TLR4 and produced a small (50%) but significant increase in TLR2 mRNA . These changes in TLRs were less dramatic than those observed for liver, spleen or cardiac muscle . Collectively these data indicate that skeletal muscle possesses many of the components of the innate immune system, including increases in both early- and late-phase cytokines and the presence of toll-like receptors.

Shock, 2003 Jun, 19(6), 503 - 7
Adenosine treatment attenuates cytokine interleukin-6 responses to endotoxin challenge in healthy volunteers; Soop A et al.; Anti-inflammatory effects of adenosine were evaluated in two randomized, double-blind, placebo-controlled studies . In one study healthy male volunteers received no endotoxin (adenosine study, n = 10), in the other intravenous endotoxin (4 ng/kg, endotoxin study n = 11) was given . All subjects were treated with adenosine infusion (40 microg/kg/min) and placebo (saline) infusion in a crossover design . Heart rate, body temperature, blood pressure and plasma cytokines (tumor necrosis factor {TNF}-alpha, interleukin {IL}-6, IL-10, and soluble TNF receptors I and II), nitric oxide oxidation products, nitrite and nitrate, as well as superoxide anions were determined . There were no significant changes of any measured parameter after adenosine treatment alone . Endotoxin elicited clinical signs of an inflammatory reaction, prominent release of all cytokines and O2- synthesis by neutrophils (N-formyl-methionin-leucyl-phenylalanin-stimulated cells measured by cytochrome C reduction) . The plasma IL-6 response to endotoxin was attenuated by adenosine, as IL-6 increased from 0.9 (0.8-1.6) to 1345 (743-1906) pg/mL (median; 25-75th percentiles) with adenosine infusion, and from 0.8 (0.5-1) to 1,959 (1,344-2,505) pg/mL with placebo (P = 0.0065) . There was no significant influence of adenosine infusion on the other variables examined . In conclusion, systemic adenosine infusion counteracts the release of IL-6 in healthy volunteers, indicating an anti-inflammatory effect of adenosine which should be further explored.

Lipids, 2003 Mar, 38(3), 191 - 9
Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli; Wu M et al.; The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles . The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides . The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases . However, this protein had a low homology with lipases of P . camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level . The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation . A distinct band with a M.W . of 33 kDa was detected on SDS-PAGE . Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase . Its catalytic properties were not changed when compared with its natural counterpart.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 919 - 22
Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure; Hashimoto M et al.; We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli . However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3- . This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 667 - 76
Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit; Kaomek M et al.; Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening . Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids . Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate . The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase . The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity . It also inhibited growth of 13 of the 14 fungal strains tested.

Mol Reprod Dev, 2003 Jul, 65(3), 309 - 17
Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding; Sivapurapu N et al.; To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed . In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347) . The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli . Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa . The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions . The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA . In addition, the immune sera also reacted with E . coli expressed recombinant bmZP1, bmZP2, and bmZP3 . In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human . The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA) . These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding .

Proteins, 2003 Jun 1, 51(4), 562 - 8
Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens; Kimber MS et al.; Protein crystallization is a major bottleneck in protein X-ray crystallography, the workhorse of most structural proteomics projects . Because the principles that govern protein crystallization are too poorly understood to allow them to be used in a strongly predictive sense, the most common crystallization strategy entails screening a wide variety of solution conditions to identify the small subset that will support crystal nucleation and growth . We tested the hypothesis that more efficient crystallization strategies could be formulated by extracting useful patterns and correlations from the large data sets of crystallization trials created in structural proteomics projects . A database of crystallization conditions was constructed for 755 different proteins purified and crystallized under uniform conditions . Forty-five percent of the proteins formed crystals . Data mining identified the conditions that crystallize the most proteins, revealed that many conditions are highly correlated in their behavior, and showed that the crystallization success rate is markedly dependent on the organism from which proteins derive . Of the proteins that crystallized in a 48-condition experiment, 60% could be crystallized in as few as 6 conditions and 94% in 24 conditions . Consideration of the full range of information coming from crystal screening trials allows one to design screens that are maximally productive while consuming minimal resources, and also suggests further useful conditions for extending existing screens .

J Biol Chem, 2003 Jul 25, 278(30), 27966 - 70 Epub 2003 Jun 03.
Crystal structure of the ectodomain of human FcalphaRI; Ding Y et al.; Human FcalphaRI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum . Although FcalphaRI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes . The genes of most FcRs are located on chromosome 1 at 1q21-23, whereas FcalphaRI is on chromosome 19, at 19q13.4, a region called the leukocyte receptor complex, because it is clustered with several leukocyte receptor families including killer cell inhibitory receptors (KIRs) and leukocyte Ig-like receptors (LIRs) . The amino acid sequence of FcalphaRI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs . In this work, we analyzed the crystal structure of the ectodomain of FcalphaRI and examined structure similarities between FcalphaRI and KIR2DL1, KIR2DL2 and LIR-1 . Our data show that FcalphaRI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and FcalphaRI is evolutionally closer to LIR than KIR.

J Biol Chem, 2003 Aug 8, 278(32), 30193 - 8 Epub 2003 Jun 03.
Novel antioxidant role of alcohol dehydrogenase E from Escherichia coli; Echave P et al.; Alcohol dehydrogenase E (AdhE) is an Fe-enzyme that, under anaerobic conditions, is involved in dissimilation of glucose . The enzyme is also present under aerobic conditions, its amount is about one-third and its activity is only one-tenth of the values observed under anaerobic conditions . Nevertheless, its function in the presence of oxygen remained ignored . The data presented in this paper led us to propose that the enzyme has a protective role against oxidative stress . Our results indicated that cells deleted in adhE gene could not grow aerobically in minimal media, were extremely sensitive to oxidative stress and showed division defects . In addition, compared with wild type, mutant cells displayed increased levels of internal peroxides (even higher than those found in a Delta katG strain) and increased protein carbonyl content . This pleiotropic phenotype disappeared when the adhE gene was reintroduced into the defective strain . The purified enzyme was highly reactive with hydrogen peroxide (with a Ki of 5 microM), causing inactivation due to a metal-catalyzed oxidation reaction . It is possible to prevent this reactivity to hydrogen peroxide by zinc, which can replace the iron atom at the catalytic site of AdhE . This can also be achieved by addition of ZnSO4 to cell cultures . In such conditions, addition of hydrogen peroxide resulted in reduced cell viability compared with that obtained without the Zn treatment . We therefore propose that AdhE acts as a H2O2 scavenger in Escherichia coli cells grown under aerobic conditions.

Zhonghua Wai Ke Za Zhi, 2003 Feb, 41(2), 90 - 2
{Construction and screening of suppression subtractive hybridization library of renal cell carcinoma}; Zhang Y et al.; OBJECTIVE: To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC) . METHODS: Poly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively . SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E . coli TOP10F' . All positive clones picked out were digested and some of which were sequenced . RESULTS: The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb . Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes . CONCLUSION: The quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.

J Am Chem Soc, 2003 Jun 11, 125(23), 6900 - 6
Incorporation of trifluoroisoleucine into proteins in vivo; Wang P et al.; Two fluorinated derivatives of isoleucine: d,l-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and d,l-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid (5TFI, 3) were prepared . 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine . Incorporation of 5TFI was confirmed by tryptic peptide analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product . Amino acid analysis showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI . Measurement of the rate of activation of 5TFI by the E . coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity constant (k(cat)/K(m)) 134-fold lower than that for isoleucine . 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions . The concentration of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC(50) = 2.70 ng/mL) . The maximal responses are equivalent for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures . 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.

Planta, 2003 Jun, 217(2), 290 - 8 Epub 2003 Feb 15.
Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco; Hara M et al.; Citrus ( Citrus unshiu Marcov.) dehydrin in response to chilling stress was overexpressed in tobacco ( Nicotiana tabacum L.), and the cold stress tolerance of transgenics at low temperature was analyzed . The freezing at -4 degrees C for 3 h of 24 independent lines indicated that a phenotype expressing citrus dehydrin showed less electrolyte leakage than the control . Dehydrin protein content was correlated with freezing tolerance in transgenics . Dehydrin-expressing tobacco exhibited earlier germination and better seedling growth than the control at 15 degrees C . Cell fractionation experiments suggested that the protein was predominantly expressed in mitochondria and the soluble fraction . Malondialdehyde production enhanced by chilling stress was lower in tobacco plants expressing citrus dehydrin than in control phenotypes . Dehydrin protein, purified from Escherichia coli expressing citrus dehydrin cDNA, prevented peroxidation of soybean ( Glycine max L.) liposomes in vitro . The inhibitory activity of dehydrin against liposome oxidation was stronger than that of albumin, glutathione, proline, glycine betaine, and sucrose . These results suggest that dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress.

J Biol Chem, 2003 Aug 15, 278(33), 31412 - 8 Epub 2003 Jun 01.
Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase; Lee DC et al.; The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger . Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity . The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell . We have determined the crystal structure of E . coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity . The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution . In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases . Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter . Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.

Biosens Bioelectron, 2003 Aug 1, 18(8), 1015 - 21
A bioluminescent sensor for high throughput toxicity classification; Kim BC et al.; A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon . The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage . In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples . As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased . This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range . In particular, each test requires only 4 h to give clear bioluminescent response signature . Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response . The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.

FEBS Lett, 2003 Jun 5, 544(1-3), 268 - 73
Monovalent cation dependence and preference of GHKL ATPases and kinases; Hu X et al.; The GHKL phosphotransferase superfamily, characterized by four sequence motifs that form the ATP-binding site, consists of the ATPase domains of type II DNA topoisomerases, Hsp90, and MutL, and bacterial and mitochondrial protein kinases . In addition to a magnesium ion, which is essential for catalysis, a potassium ion bound adjacent to the triphosphate moiety of ATP in a rat mitochondrial protein kinase, BCK (branched-chain alpha-ketoacid dehydrogenase kinase), has been shown to be indispensable for nucleotide binding and hydrolysis . Using X-ray crystallographic, biochemical, and genetic analyses, we find that the monovalent cation-binding site is conserved in MutL, but both Na(+) and K(+) support the MutL ATPase activity . When Ala100 of MutL is substituted by proline, mimicking the K(+)-binding environment in BCK, the mutant MutL protein becomes exclusively dependent on Na(+) for the ATPase activity . The coordination of this Na(+) ion is identical to that of the K(+) ion in BCK and involves four carbonyl oxygen atoms emanating from the hinges of the ATP lid and a non-bridging oxygen of the bound nucleotide . A similar monovalent cation-binding site is found in DNA gyrase with additional coordination by a serine side chain . The conserved and protein-specific monovalent cation-binding site is unique to the GHKL superfamily and probably essential for both ATPase and kinase activity . Dependence on different monovalent cations for catalysis may be exploited for future drug design specifically targeting each individual member of the GHKL superfamily.

FEBS Lett, 2003 Jun 5, 544(1-3), 206 - 9
Defined subcomplexes of the A1 ATPase from the archaeon Methanosarcina mazei Gö1: biochemical properties and redox regulation; Lemker T et al.; The potential A(1) ATPase genes ahaA, ahaB, ahaC, ahaD, ahaE, ahaF, and ahaG from the anaerobic archaeon Methanosarcina mazei Go1 were overexpressed in Escherichia coli DK8 (pTL2) . An A(1) complex was purified to apparent homogeneity and shown by Western blot and N-terminal sequence analyses to contain subunits A, B, C, D, and F but to be devoid of subunits E and G . Further removal of subunit C was without effect on ATPase activity . The enzyme was most active at pH 5.2 and required bisulfite and acetate for maximal activity . Kinetic studies confirmed three new inhibitors for A(1) ATPases (diethylstilbestrol and its derivatives hexestrol and dienestrol) and identified redox modulation as a new type of regulation of archaeal A(1) ATPases.

FEBS Lett, 2003 Jun 5, 544(1-3), 199 - 205
Substitutions in the Escherichia coli RNA polymerase sigma70 factor that affect recognition of extended -10 elements at promoters; Sanderson A et al.; Previous work has shown that the base sequence of the DNA segment immediately upstream of the -10 hexamer at bacterial promoters (the extended -10 element) can make a significant contribution to promoter strength . Guided by recently published structural information, we used alanine scanning and suppression mutagenesis of Region 2.4 and Region 3.0 of the Escherichia coli RNA polymerase sigma(70) subunit to identify amino acid sidechains that play a role in recognition of this element . Our study shows that changes in these regions of the sigma(70) subunit can affect the recognition of different extended -10 element sequences.

FEBS Lett, 2003 Jun 5, 544(1-3), 33 - 7
Critical regions for the sweetness of brazzein; Jin Z et al.; Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues . Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness . To assess their sweetness, psychophysical experiments were carried out with 14 human subjects . First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein . Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.

J Theor Biol, 2003 Jul 7, 223(1), 45 - 53
Centers of complex networks; Wuchty S et al.; The central vertices in complex networks are of particular interest because they might play the role of organizational hubs . Here, we consider three different geometric centrality measures, excentricity, status, and centroid value, that were originally used in the context of resource placement problems . We show that these quantities lead to useful descriptions of the centers of biological networks which often, but not always, correlate with a purely local notion of centrality such as the vertex degree . We introduce the notion of local centers as local optima of a centrality value "landscape" on a network and discuss briefly their role.

Toxicon, 2003 Jun, 41(7), 793 - 801
Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp; Liu WH et al.; A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp . using degenerate oligonucleotide primers and 3',5'-RACE . The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway . The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62 . The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C.The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli . The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification . The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence . The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix.Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.) . The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5% . The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

Anal Biochem, 2003 Jul 1, 318(1), 71 - 9
Screening methods to determine biophysical properties of proteins in structural genomics; Woestenenk EA et al.; We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy . The method constitutes an extension of previously described protocols for gene expression and protein solubility screening {M . Hammarstrom et al., (2002), Protein Science 11, 313} . Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day . The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner . Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations . The purification method can be automated, scaled up or down, and extended to a traditional purification scheme . We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.

Anal Biochem, 2003 Jul 1, 318(1), 30 - 8
Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry; Binet MR et al.; Metals bound to proteins play key roles in structure stabilization, catalysis, and metal transport in cells, but metals may also be toxic . As a consequence, cells have developed mechanisms to control metal concentrations through binding to proteins . We have used a hyphenated strategy linking gel electrophoresis with laser ablation-inductively coupled plasma-mass spectrometry in order to detect, map, and quantify metal-binding proteins synthesized in Escherichia coli under zinc- and cadmium-stress conditions . We report the development of a powerful analytical method suitable for detection and characterization of metalloproteins in complex, unfractionated bacterial cell extracts . The approach was validated by using an E . coli strain overexpressing the cyanobacterial metallothionein protein SmtA . We observed induction of SmtA synthesis by zinc and binding of both zinc and cadmium cations by this protein . A profile of zinc- and cadmium-binding proteins was obtained from E . coli cytoplasmic fractions . Analysis of induction patterns and metal contents demonstrated the presence of proteins with high metal content which, on further study, should lead to the identification of novel metal-binding proteins.

Comp Biochem Physiol B Biochem Mol Biol, 2003 May, 135(1), 109 - 16
cDNA cloning of growth hormone from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli; Liao MJ et al.; A cDNA encoding Ailuropoda melanoleuca growth hormone (AmGH) was isolated from pituitary total RNA using RT-PCR and expressed in Escherichia coli . This is the first report of a GH nucleotide and amino acid (aa) sequence from giant panda . The open reading frame of AmGH (651 bp) encodes a precursor of 216 aa comprising a 26 aa signal peptide and a 190 aa mature protein with four cysteine residues similar to the typical primary structure of mammalian GH precursor . AmGH shares a high degree of identity (54-98.9%) with that of mammals, birds and amphibians, but a very low identity with bony fish GH (only 20-30%) . The mature AmGH exhibits striking similarity to that of putative ancestral GH with a difference of only two residues, indicating a very slow basal rate of molecular evolution . The DNA fragment encoding mature AmGH was then subcloned into the pGEX-4T-1 expression vector and highly expressed in E . coli host BL21 with IPTG induction . The expressed proteins fused to GST were found to be sequestered into inclusion bodies and therefore the NaOH method was employed to solubilize the inclusion bodies; the proteins were further purified by Glutathione Sepharose 4B affinity chromatography . The production and purification of GST-AmGH reported here provide a basis for further studies on the biological activity of AmGH.

Arch Biochem Biophys, 2003 Jun 15, 414(2), 244 - 54
Identification of epitopes on cytochrome P450 3A4/5 recognized by monoclonal antibodies; Parimoo B et al.; In this study we describe the mapping of epitopes on CYP3A4/5 recognized by a panel of monoclonal antibodies (MAbs) . CYP3A4 and CYP3A5 cDNAs were cloned in GST expression vectors and the fusion proteins were subjected to Western blot . Eight MAbs reacted with the full-length GST-3A4 fusion protein as well as baculovirus cDNA-expressed CYP3A4, while six of these reacted with baculovirus cDNA-expressed CYP3A5 . Five (MAb 347, 351, 352, 354, and 357) out of 8 MAbs were inhibitory in a metabolic assay using quinine as substrate . MAbs 352, 354, and 357 brought about a moderate inhibition of quinine metabolism (60-70%) while MAb 347 inhibited quinine 3- hydroxylation in human liver microsomes (n=6) by more than 70% . MAb 347 was a potent inhibitor of baculovirus-expressed CYP3A5-catalyzed metabolism of quinine (95%) at </=0.20 mg IgG/nmol P450 but only moderately inhibited CYP3A4 at much higher ratios of MAb to P450 . This MAb was mapped to a region of 283 to 504 amino acids on CYP3A4 protein and to an identical region on CYP3A5 protein . The region that was identified on the CYP3A5 construct was further validated based on the ability of the construct harboring the epitope to reverse the inhibition of hydroxylation of quinine by MAb 347 . Our experiments clearly demonstrate that a spatial antigenic determinant is responsible for the inhibitory potency of MAb 347.

Arch Biochem Biophys, 2003 Jun 15, 414(2), 232 - 43
The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain; Xu L et al.; 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway . This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation . Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase . However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal . In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR . These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.

Arch Biochem Biophys, 2003 Jun 15, 414(2), 170 - 9
Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms; Kai Y et al.; Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and Pi using Mg2+ or Mn2+ as a cofactor . PEPC plays a key role in photosynthesis by C4 and Crassulacean acid metabolism plants, in addition to its many anaplerotic functions . Recently, three-dimensional structures of PEPC from Escherichia coli and the C4 plant maize (Zea mays) were elucidated by X-ray crystallographic analysis . These structures reveal an overall square arrangement of the four identical subunits, making up a "dimer-of-dimers" and an eight-stranded beta barrel structure . At the C-terminal region of the beta barrel, the Mn2+ and a PEP analog interact with catalytically essential residues, confirmed by site-directed mutagenesis studies . At about 20A from the beta barrel, an allosteric inhibitor (aspartate) was found to be tightly bound to down-regulate the activity of the E . coli enzyme . In the case of maize C4-PEPC, the putative binding site for an allosteric activator (glucose 6-phosphate) was also revealed . Detailed comparison of the various structures of E . coli PEPC in its inactive state with maize PEPC in its active state shows that the relative orientations of the two subunits in the basal "dimer" are different, implicating an allosteric transition . Dynamic movements were observed for several loops due to the binding of either an allosteric inhibitor, a metal cofactor, a PEP analog, or a sulfate anion, indicating the functional significance of these mobile loops in catalysis and regulation . Information derived from these three-dimensional structures, combined with related biochemical studies, has established models for the reaction mechanism and allosteric regulation of this important C-fixing enzyme.

J Theor Biol, 2003 Jun 21, 222(4), 407 - 23
Dynamic responses of protein homeostatic regulatory mechanisms to perturbations from steady state; Yang Q et al.; Nineteen hypothetical protein homeostatic regulatory mechanisms were constructed and analysed in terms of the rate at which they recovered from a perturbation in the steady-state concentration of any component . Systems were constructed to symbolize transcription/translation processes of the average protein from Escherichia coli (1000 copies of protein P along with 1 gene G per cell) . In some model systems, G catalysed the synthesis of P directly, while in others G catalysed the synthesis of mRNA (called M), and M catalysed the synthesis of P in a subsequent step . Recovery rates for each regulatory mechanism were obtained by generating the corresponding system of differential equations, linearizing the system about the steady state, and determining eigenvalues of the associated coefficient matrix . The optimal rate of recovery for a given mechanism, R(D), was determined by combining random and gradient search approaches to find rate constants for which the system recovered fastest . Regulatory elements that improved dynamic regulation were identified . These consisted of negative feedback relationships that involved P binding to either G (to shut off the synthesis of P) or M (to stimulate its degradation) . Regulation improved as increasing numbers of P's bound to either G or M; however, the binding to M was more effective . In other mechanisms PP dimers bound G . Dimer-binding mechanisms were roughly twice as effective in terms of regulation as those that bound P monomers . The effect of linking two regulatory "modules" was also investigated . Linking had no effect on R(D), but optimal rate constants for the linked system were similar to those of the unlinked modules, suggesting that it may be feasible to construct regulatory networks by linking individual modules of this type.

Vet Microbiol, 2003 Jul 1, 94(2), 159 - 65
Prevalence and identity of cdt-related sequences in necrotoxigenic Escherichia coli; Mainil JG et al.; The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture . Escherichia coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes . The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in necrotoxigenic E . coli (NTEC) . A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E . coli . cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive . The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates) . After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1 . The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E . coli.

Bioorg Med Chem Lett, 2003 Jun 16, 13(12), 1955 - 8
Synthesis, stereochemical assignment and biological activity of a novel series of C-4" modified aza-macrolides; Bronk BS et al.; Modification of the cladinose C-4" position via manipulation of the corresponding keto derivatives afforded two stereochemically pure series of compounds . The synthesis and structure determination of these compounds is described within . The in vitro and in vivo biological activity of this novel series of C-4" modified macrolides is also described.

Cell Microbiol, 2003 Jun, 5(6), 359 - 72
Adhesion of enteropathogenic Escherichia coli to host cells; Nougayrede JP et al.; Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect . Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence . Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir . Tir is produced by the bacteria and delivered to the host cell via a type III secretion system . In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors . Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.

Chaos, 2001 Mar, 11(1), 261 - 268
Dynamic behavior in mathematical models of the tryptophan operon; Santillan M et al.; This paper surveys the general theory of operon regulation as first formulated by Goodwin and Griffith, and then goes on to consider in detail models of regulation of tryptophan production by Bliss, Sinha, and Santillan and Mackey, and the interrelationships between them . We further give a linear stability analysis of the Santillan and Mackey model for wild type E . coli as well as three different mutant strains that have been previously studied in the literature . This stability analysis indicates that the tryptophan production systems should be stable, which is in accord with our numerical results . (c) 2001 American Institute of Physics.

Biochemistry, 2003 Jun 10, 42(22), 6921 - 8
Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity; Li J et al.; The involvement of Tax oncoprotein in the INK4-CDK4/6-Rb pathway has been regarded as a key factor for immortalization and transformation of human T-cell leukemia virus 1 (HTLV-1) infected cells . In both p16 -/- and +/+ cells, expression of Tax has been correlated with an increase in CDK4 activity, which subsequently increases the phosphorylation of Rb and drives the infected cells into cell cycle progression . In relation to these effects, Tax has been shown to interact with two components of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s) . While Tax competes with CDK4 for p16 binding, thus suppressing p16 inhibition of CDK4, Tax also binds to cyclin D(s) with concomitant increases in both CDK4 activity and the phosphorylation of cyclin D(s) . Here we show that both Tax and residues 1-40 of the N-terminus of Tax, Tax40N, bind to and activate CDK4 in vitro . In the presence of INK4 proteins, binding of Tax and Tax40N to CDK4 counteracts against the inhibition of p16 and p18 and acts as the major path to regulate Tax-mediated activation of CDK4 . We also report that Tax40N retains the transactivation ability . These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.

Biochemistry, 2003 Jun 10, 42(22), 6863 - 70
Mechanism of posttranslational regulation of phenol sulfotransferase: expression of two enzyme forms through redox modification and nucleotide binding; Su TM et al.; Sulfotransferase catalyzes sulfuryl group transfer between a nucleotide and a variety of nucleophiles that may be sugar, protein, xenobiotics, and other small molecules . Nucleotides may serve as cosubstrate, cofactor, inhibitor, or regulator in an enzyme catalyzed sulfuryl group transfer reaction . We are trying to understand how nucleotide regulates the activity of phenol sulfotransferase (PST) through the expression of two enzyme forms . The homogeneous rat recombinant PST was obtained from Escherichia coli, and the nucleotide copurified was examined . The nucleotide was completely removed from inactive PST in high salt and oxidative condition . Total enzyme activity was recovered following incubation in reductive environment . Many nucleotides are known to tightly bind to PST but only one nucleotide, 3'-phosphoadenosine 5'-phosphate (PAP), was identified to combine with PST by ion-pair RP-HPLC, UV-visible spectra, (31)P NMR, and ESI-MS and MS-MS spectrometry . In addition to the presence or absence of PAP, oxidation following reduction of PST was required to completely interconvert the two forms of PST . According to the experimental results, a mechanism for the formation of the two enzyme forms was proposed.

Biochemistry, 2003 Jun 10, 42(22), 6804 - 13
Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase; Zhang H et al.; NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor . Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein . More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized . In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM . The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions . The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1 . Residual FAD accounted for less than 5% of the total flavins . Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein . The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV) . The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5) . Reduction of the cytochromes and oxidation of the reductase occurred simultaneously . The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1) . Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.

Biochemistry, 2003 Jun 10, 42(22), 6735 - 46
A new strategy for the site-specific modification of proteins in vivo; Zhang Z et al.; We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon . Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E . coli . We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells . The labeling reactions are selective and in general proceed with yields of >75% . In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes . The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Biochemistry, 2003 Jun 10, 42(22), 6656 - 63
D18G transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?
Hammarstrom P, Sekijima Y, White JT, Wiseman RL, Lim A, Costello CE, Altland K, Garzuly F, Budka H, Kelly JW.
Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS) . In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis . D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date . Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR . Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT . It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade . Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation; however, benign systemic deposition is also observed . Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR . The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease . CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate . Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T(4)) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T(4) CSF concentration . Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.

Dig Liver Dis, 2003 Mar, 35(3), 193 - 6
Lower intestinal bleeding due to aorto-enteric fistula; Maiolo C et al.; The case is described of a man who complained of intermittent fever and fatigue . After three digestive endoscopies and computed tomography, a 99m technetium-HM-PAO-labelled white cell scan was usefully employed to establish diagnosis . Anaerobic aortic Graft infection and anaemia due to lower intermittent occult intestinal bleeding were found . The intestinal bleeding was caused by secondary aorto-jejunal fistula . This condition is rare, but should be suspected whenever a patient with aortic prosthesis presents with occult digestive bleeding and unexplained fever.

J Immunoassay Immunochem, 2003, 24(2), 219 - 32
Development and application of a pig IL-8 ELISA detection system; Splichal I et al.; Interleukin 8 (IL-8) is a chemotactic and activating chemokine, especially for neutrophils, which plays an important role in inflammatory process . A pig IL-8 specific enzyme-linked immunosorbent assay (ELISA) was developed to measure IL-8 concentrations in cell culture supernatants and biological fluids . A streptavidin-biotin amplified sandwich method uses mouse capture mAb IZ8.03 and detection biotinylated mouse mAb IZ8.04 against recombinant pig IL-8 . The assay specifically and reproducibly recognizes both recombinant and natural pig IL-8 . A working range of the assay is 16-1000 pg/mL and takes a mere 3.5 h of incubation time . This pig IL-8 ELISA is a suitable alternative way of measurement of IL-8 concentrations to time consuming and laborious IL-8 bioassays.

Eur J Immunol, 2003 Jun, 33(6), 1586 - 92
The generalized endotoxic principle; Seydel U et al.; Bacterial lipopolysaccharides (endotoxins, LPS) belong to the most potent immunostimulators in mammals . The endotoxic principle of LPS is located in its lipid A moiety, which for Escherichia coli-type LPS consists of a hexaacylated diphosphoryl diglucosamine backbone . This lipid A adopts a cubic inverted aggregate structure from which a conical shape of the molecule can be deduced, whereas the tetraacyl lipid A precursor IVa adopts a cylindrical shape and is endotoxically inactive, but antagonizes active LPS . We hypothesize that non-lipid A amphiphiles with similar physicochemical properties of amphiphilicity, charge, and shape, might mimic the respective lipid A . To test this hypothesis, phospholipid-like amphiphiles with six acyl chains attached to a bisphosphorylated serine-like backbone of varying length replacing the diglucosamine backbone were synthesized . The compound with a short backbone fulfills all criteria of an endotoxic agonist, and that with long backbone fulfills those of an antagonist . This holds true for the human as well as for the murine system . Interestingly, these compounds are inactive in the Limulus amebocyte lysate test which is specific for LPS diglucosamine backbone . These results define a general endotoxic principle and, furthermore, provide new insights into an understanding of early steps of endotoxin action.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 916 - 8 Epub 2003 Apr 25.
Crystallization and preliminary crystallographic study of a pheromone-binding protein from the cockroach Leucophaea maderae; Lartigue A et al.; Pheromone-binding proteins (PBPs) are small helical proteins (13-18 kDa) present in various sensory organs of moths and other insect species . An antennal protein from the cockroach Leucophaea maderae (LmaPBP) has been found to share all the hallmarks of the PBP family and is expressed specifically in the female adult antennae, the gender that perceives the sex pheromone . Here, the crystallization of LmaPBP expressed as a recombinant protein in Escherichia coli periplasm is reported . Crystals of LmaPBP were obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot . The protein crystallizes in two different crystal forms . Form 1 belongs to space group P1, with unit-cell parameters a = 43.2, b = 45.1, c = 45.7 A, alpha = 118.6, beta = 93.0, gamma = 106.9 degrees . With two molecules in the asymmetric unit, V(M) is 2.7 A(3) Da(-1) and the solvent content is 47% . A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble) . Form 2 was obtained in the presence of the pheromone (3-hydroxy-butan-2-one) and belongs to space group P2(1), with unit-cell parameters a = 38.2, b = 62.2, c = 45.1 A, beta = 93.0 degrees . With two molecules in the asymmetric unit, V(M) is 2.0 A(3) Da(-1) and the solvent content is 39% . A complete data set has been collected at 1.7 A resolution on beamline BM14 (ESRF, Grenoble) . SeMet expression has been performed with a view to solving the structure by MAD data collection using the Se absorption edge.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 910 - 2 Epub 2003 Apr 25.
A single mutation of restriction endonuclease EcoRII led to a new crystal form that diffracts to 2.1 A resolution; Zhou XE et al.; R88A, a mutant of the type IIE restriction endonuclease EcoRII, has been crystallized in space group P2(1), with unit-cell parameters a = 58.7, b = 92.4, c = 88.3 A, beta = 108.1 degrees . There are two monomers in the asymmetric unit and the solvent content is estimated to be 50% by volume . The crystals diffract to 2.1 A resolution, which is much higher than that of the wild type, which diffracted to 2.8 A resolution . The mutant crystals have been used in the identification of an excellent heavy-atom derivative.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 897 - 902 Epub 2003 Apr 25.
An orthorhombic form of Escherichia coli aminopeptidase P at 2.4 A resolution; Graham SC et al.; Aminopeptidase P (AMPP) from Escherichia coli cleaves the N-terminal residue from an oligopeptide if the second residue is proline . The active site contains a dinuclear metal centre . Following earlier structural analyses of crystals in space groups P6(4)22 and I4(1)22, the structure of AMPP has been solved and refined in the orthorhombic space group C222(1) at 2.4 A resolution . There are six subunits in the asymmetric unit . These are arranged in two types of tetramer . One tetramer comprises four crystallographically independent subunits, while the other comprises two pairs of subunits related by a crystallographic twofold axis . The final model of 20 994 protein atoms, 1618 water molecules and 12 metal atoms refined to residuals R = 0.195 and R(free) = 0.215 . The molecular structure confirms most of the previously reported features, including the subunit-subunit interfaces in the tetramer and persistent disorder at some residues . The metal-ligand bond lengths at the active site suggest that one of the two Mn atoms is five-coordinate rather than six-coordinate.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 876 - 80 Epub 2003 Apr 25.
Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution; Longenecker K et al.; Mutants of the small G protein RhoA that are deficient in GTPase activity and thereby exhibit constitutive molecular signaling activity are commonly used to discover its cellular functions . In particular, two such mutants, Gly14-->Val (G14V) and Gln63-->Leu (Q63L), are often used interchangeably for such studies . However, while their in vitro rates of GTP hydrolysis are very similar, differences are observed in their other functional properties . The structure of G14V-RhoA is known; in order to assess whether structural variations are responsible for functional differences, the crystal structure of a Q63L-RhoA bound to the GTP-analog 5'-guanylylimidodiphosphate (GMPPNP) was determined at 1.5 A resolution . Overall, the structure is very similar to that of G14V-RhoA, but the significantly higher resolution data permit an improved basis for structural analysis and comparison . The data support the notion that differences observed between the mutants in vivo are likely to arise from altered affinities for RhoGDI and not from direct structural differences.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 815 - 22 Epub 2003 Apr 25.
Structure of DNA helicase RepA in complex with sulfate at 1.95 A resolution implicates structural changes to an "open" form; Xu H et al.; The structure of a new crystal form (space group C2), grown at pH 8.0 and diffracting to 1.95 A resolution, of the replicative homo-hexameric DNA helicase RepA encoded by plasmid RSF1010 is reported . In contrast to previous crystals grown at pH 6.0 in space group P2(1) (Niedenzu et al., 2001), only one half (a trimer) of the RepA hexamer occupies the asymmetric unit of the space-group C2 crystals . The new crystal packing explains the pH-dependent hexamer-hexamer association mechanism of RepA . The C-terminus (264)VLERQRKSKGVPRGEA(279), which could not be modelled in the previous structure, is clearly defined in the present electron density except for the last four amino acids . Sulfate anions occupy the six ATPase active sites of RepA at positions where the product phosphates are supposed to bind . Binding of sulfate anions induces conformational changes both at the ATPase active sites and throughout the whole molecular structure . In agreement with electron microscopy, the above studies implicate structural changes to an "open" form that may occur upon binding and hydrolysis of nucleotide 5'-triphosphates and could be essential for DNA duplex-unwinding activity.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1109 - 10 Epub 2003 May 23.
Purification, crystallization and preliminary X-ray studies of GMP reductase 2 from human; Ji CN et al.; GMP reductase 2 from human has been expressed in Escherichia coli, purified and crystallized . The crystals belong to space group P3(2)21, with unit-cell parameters a = b = 110.6, c = 209.8 A, alpha = beta = 90, gamma = 120 degrees . Diffraction data were collected to 3.0 A with a completeness of 100% (100% for the last shell), an R(merge) value of 0.089 (0.189) and an I/sigma(I) value of 7.3 (3.2).

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1106 - 8 Epub 2003 May 23.
Purification, nanocrystallization and preliminary X-ray analysis of a C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans; Ding H et al.; The C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans was expressed in Escherichia coli and purified to homogeneity . Optimized from the initial nanoscreen, crystals grew to dimensions of 0.25 x 0.15 x 0.15 mm at 277 K using 28.0%(v/v) PEG 400 as the precipitant by the hanging-drop vapor-diffusion technique . A data set of 94.9% completeness was collected to a resolution of 1.98 A at 100 K using a synchrotron X-ray source (SER-CAT) . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 31.7, b = 50.6, c = 107.1 A, and contained one molecule per asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1103 - 5 Epub 2003 May 23.
Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1; Kaiser L et al.; The domestic cat (Felis domesticus) is an important cause of allergic disease worldwide . The major cat allergen 1 (Fel d 1) has been expressed in Escherichia coli, purified and refolded in a soluble form . Crystals of Fel d 1 were obtained in 13% 2-methyl-2,4-pentanediol, 0.1 M sodium acetate pH 4.8 . The Fel d 1 crystals belong to space group P2(1), with unit-cell parameters a = 43.3, b = 51.5, c = 67.7 A, and diffract to 1.9 A resolution.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1100 - 2 Epub 2003 May 23.
Crystallization of butyrate kinase 2 from Thermotoga maritima mediated by vapor diffusion of acetic acid; Diao J et al.; The sitting-drop method of crystallization uses the evaporation of water to increase the concentration of the protein and precipitant in the drop . The presence of other volatile components, such as acetic acid, can have a marked impact on crystallization . A member of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of proteins, isobutyrate kinase (Buk2) from Thermotoga maritima, was expressed in Escherichia coli with six histidine residues added to the C-terminus . The purified protein was crystallized in a sitting drop with a well solution consisting of 1.7-3.0 M sodium formate, with the pH of the well solution alone adjusted to 4.5 with acetic acid . Diffraction data collected at 100 K show that the crystals diffract to 3.1 A and belong to space group I422, with unit-cell parameters a = b = 198.12, c = 58.93 A . Both the crystal form and the results of dynamic light-scattering studies suggest that Buk2 is an octomer, the first to be identified in the ASKHA superfamily.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1084 - 6 Epub 2003 May 23.
Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina; Premkumar L et al.; An extracellular alpha-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations . To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method . The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 A, beta = 94.2 degrees . Data from a single crystal were collected to 2.4 A resolution under cryogenic conditions (120 K) using an R-AXIS IV(++) detector mounted on a Rigaku RU-H3R rotating-anode generator . The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 2.65 A(3) Da(-1) and a solvent content of 52.7%.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1079 - 80 Epub 2003 May 23.
Crystallization and preliminary X-ray characterization of a novel calcium-binding protein AtCBL2 from Arabidopsis thaliana; Nagae M et al.; A new family of calcineurin B-like calcium-binding proteins has recently been identified in Arabidopsis thaliana . AtCBL2, a member of this family, has been crystallized in the presence of calcium ions using polyethylene glycol as a precipitant at 293 K . The crystals belong to space group C222(1), with unit-cell parameters a = 83.9, b = 118.1, c = 49.1 A . The asymmetric unit contains one molecule, with a V(M) of 2.36 A(3) Da(-1) and a solvent content of 48% . Native diffraction data to 2.1 A resolution have been collected using synchrotron radiation at SPring-8.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1070 - 2 Epub 2003 May 23.
Crystallization and preliminary X-ray diffraction studies of the eukaryotic iron superoxide dismutase (FeSOD) from Vigna unguiculata; Munoz IG et al.; Eukaryotic iron superoxide dismutases (FeSODs) are homodimeric proteins that constitute a fundamental protection against free radicals, which can damage essential cellular mechanisms . The protein was cloned and overexpressed in Escherichia coli with an N-terminal His tag . Crystallization experiments of the protein resulted, after several refined screenings, in crystals suitable for X-ray diffraction analysis . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 82.54, b = 48.41, c = 64.28 A, alpha = gamma = 90, beta = 119.66 degrees, and contain one molecule per asymmetric unit . At cryogenic temperatures, the crystals diffracted to a resolution limit of 1.80 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF).

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1058 - 60 Epub 2003 May 23.
Expression, purification, crystallization and preliminary X-ray characterization of the class B acid phosphatase (AphA) from Escherichia coli; Forleo C et al.; The class B non-specific acid phosphatase AphA from Escherichia coli has been expressed in E . coli and purified following a new protocol . ESI mass spectroscopy shows that the purified enzyme solution contains two polypeptides with molecular weights differing by 185 Da corresponding to two different cleavage sites of the signal peptide from the AphA E . coli precursor . Despite the solution heterogeneity, X-ray quality crystals have been obtained . However, the crystals have a tendency to give polymorphs and to lose long-range order with time while maintaining an intact crystal habit . Crystals have been grown in space groups I222 and C2 with three different unit cells and different asymmetric unit contents . Diffraction data to 1.6 A resolution have been collected with synchrotron radiation at ESRF and DESY.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1049 - 51 Epub 2003 May 23.
Crystallization and preliminary X-ray diffraction analysis of Langat virus envelope protein domain III; White MA et al.; The putative receptor-binding domain (domain III) of the flavivirus Langat envelope glycoprotein has been crystallized using the hanging-drop vapor-diffusion method at 277 K . Two distinct crystal morphologies were observed to grow under the same conditions . The crystal forms both belong to a trigonal space group, P3(1)21 or P3(2)21, with unit-cell parameters a = 80.93, c = 132.1 A and a = 104.8, c = 219.5 A for forms I and II, respectively . Complete data sets to 2.9 and 3.35 A, respectively, have been collected at 100 K with Cu Kalpha X-rays from a rotating-anode generator.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1046 - 8 Epub 2003 May 23.
Crystallization and preliminary X-ray analysis of Der f 2, a potent allergen derived from the house dust mite (Dermatophagoides farinae); Roeber D et al.; Although a number of allergens have been identified and isolated, the underlying molecular basis for the potent immune response is poorly understood . House dust mites (Dermatophagoides sp.) are ubiquitous contributors to atopy in developed countries . The rhinitis, dermatitis and asthma associated with allergic reactions to these arthropods are frequently caused by relatively small (125-129 amino acids) mite proteins of unknown biological function . Der f 2, a major allergen from the mite D . farinae, has been recombinantly expressed, characterized and crystallized . The crystals belong to the tetragonal space group I4(1)22, with unit-cell parameters a = b = 95.2, c = 103.3 A . An essentially complete (97.2%) data set has been collected to 2.4 A at a synchrotron source . Attempts to solve the crystal structure of Der f 2 by molecular replacement using the NMR coordinates for either Der f 2 or Der p 2 (the homologous protein from D . pteronyssinus) failed, but preliminary searches using the crystalline Der p 2 atomic coordinates appear to be promising.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1020 - 7 Epub 2003 May 23.
Phasing on anomalous signal of sulfurs: what is the limit?
Ramagopal UA, Dauter M, Dauter Z.
Recent years have witnessed significant advancements in X-ray data-acquisition techniques and phasing algorithms, which have made possible the successful use of a very small anomalous diffraction signal for the solution of crystal structures of macromolecules . Two crystal structures, a 44 kDa glucose isomerase containing nine sulfurs and a 33 kDa xylanase containing five sulfurs, have been solved from single-wavelength anomalous data using widely available methods and programs . These two enzymes contain less sulfur than most proteins in the bacterial or eukaryotic proteomes, providing a Bijvoet ratio of about 0.6% . For glucose isomerase the automatically interpretable electron-density maps could be obtained at high as well as low resolution . The S-SAD approach relies on the anomalous signal of sulfur naturally occurring in proteins and alleviates all need for sample derivatization . It may therefore be applicable to all protein crystals able to provide accurate diffraction data.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1004 - 11 Epub 2003 May 23.
Structure of an RNA dodecamer containing a fragment from SRP domain IV of Escherichia coli; Deng J et al.; The crystal structure of an RNA dodecamer, r(GCGUCAGGUC(Br)CG)/r(CGGAAGCAG(Br)CGC), containing a fragment from the signal recognition particle (SRP) RNA (domain IV) of Escherichia coli, has been determined at 1.7 A resolution with 21 666 independent reflections and an R(work) and R(free) of 20.1 and 22.5%, respectively . The structure exhibits a novel crystal packing pattern for RNA oligomer duplexes: one end of the duplex adopts the stacking interaction, while the other end adopts the abutting interaction in the minor groove . The symmetric loop of the SRP, r(CAGG)/r(AGCA), in the center of the dodecamer forms two different conformations of the A.C mismatch, a sheared G.G and a symmetrical G.A mismatch . These four mismatches present a unique surface for the abutting interaction . The involvement of the two A.C mismatches in the abutting interaction implies that these mismatches are the important sites for interaction with proteins . The conformation of the symmetric loop is greatly stabilized by hydrated metal ions, which display flexibility in adjusting their geometry and coordination in interaction with nucleic acids . Comparison with other crystal structures of fragments of 4.5S RNA indicates that the conformation of the symmetric loop is independent of the asymmetrical loop in domain IV.

Rheumatology (Oxford), 2003 Oct, 42(10), 1155 - 63 Epub 2003 May 30.
Protein interaction for an interferon-inducible systemic lupus associated gene, IFIT1; Ye S et al.; OBJECTIVE: To identify disease-related genes and immune-regulatory pathways in the pathogenesis of systemic lupus erythematosus (SLE) by using gene expression profiling and protein-protein interaction analysis . METHODS: Peripheral white blood cell gene expression profiles of 10 SLE patients were determined by oligonucleotide microarray analysis . Clustering of the gene expression profile was compared with the clinical immune phenotype . SLE-induced genes that were over- or under-expressed were determined and independently validated using a real-time polymerase chain reaction (PCR) method . To study their potential function and the possible pathways involved, a candidate gene was cloned and a GST (glutathione S-transferase) fusion protein was expressed in Escherichia coli . The fusion protein was further purified using the glutathione Sepharose 4B system, and was treated as bait to capture prey from SLE peripheral white blood cell lysate . MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry was then performed to determine the prey protein . RESULTS: Similarity was found between the gene expression profile and the immune phenotype clusters of the SLE patients . More than 20 disease-associated genes were identified, some of which have not been related to SLE previously . Of these genes, a cluster of interferon-induced genes were highly correlated . IFIT1 (interferon-induced with tetratricopeptide repeats 1) was one of these genes, and overexpression of its mRNA was confirmed independently by real-time PCR in a larger population (40 SLE patients and 29 normal controls) . An IFIT1 protein- protein interaction study showed that IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor . CONCLUSION: The gene expression profile seems to be the molecular basis of the diverse immune phenotype of SLE . On the basis of the SLE-related genes found in this study, we suggest that the interferon-related immune pathway is important in the pathogenesis of SLE . IFIT1 is the first gene described as a candidate gene for SLE, and may function by activating Rho proteins through interaction with Rho/Rac guanine nucleotide exchange factor . IFIT1 and the interferon-related pathway may provide potential targets for novel interventions in the treatment of SLE.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7277 - 82 Epub 2003 May 30.
Primary hyperoxaluria type 1 in the Canary Islands: a conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase; Santana A et al.; Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44) . Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 --> Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation . Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect . The consequences of these amino acid changes were investigated . Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 --> Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts . Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers . The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation . This aggregation was temperature-sensitive . AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity . Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates . In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

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