FEBS Lett, 2003 Mar 13, 538(1-3), 197 - 202 A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9; Kashiwaba M et al.; In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region . The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22% . On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells . PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9. FEBS Lett, 2003 Mar 13, 538(1-3), 35 - 40 Purification of active recombinant trypanosome alternative oxidase; Nihei C et al.; Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes . TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase . These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme-copper terminal oxidase superfamily . The inability to purify stable TAO has severely hampered biochemical studies of the alternative oxidase family . In the present study, we were able to purify recombinant TAO to homogeneity from Escherichia coli membranes using the detergent digitonin . Kinetic analysis of the purified TAO revealed that the specific inhibitor ascofuranone is a competitive inhibitor of ubiquinol oxidase activity. Cytokine, 2002 Dec 21, 20(6), 274 - 82 Cloning, sequencing and expression of porcine CD40 ligand in Escherichia coli and human and porcine cells; Wienhold D et al.; The CD40L ligand (CD40L) plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells . The porcine CD40L encoding gene was isolated from porcine peripheral blood mononuclear cells (PBMC) using RT-PCR . Sequence analysis of the cloned CD40L gene showed an open reading frame of 786 base pairs encoding a 262 amino acid protein with a predicted molecular mass of 29 kD . The deduced amino acid sequence of the porcine CD40L shared 82%, 88% and 93% similarity with the CD40L protein of mouse, human and cattle . The isolated CD40L sequence was expressed as a hexahistidine fusion protein in Escherichia coli and purified by affinity chromatography . The analysis of the CD40L-expression in human 293 and porcine MAX cells by immunofluorescence showed its location on the cell surface . Genet Sel Evol, 2003 Mar-Apr, 35(2), 239 - 47 Substitution of the alpha-lactalbumin transcription unit by a CAT cDNA within a BAC clone silenced the locus in transgenic mice without affecting the physically linked Cyclin T1 gene; Soulier S et al.; We recently reported that a goat bacterial artificial chromosome (BAC) clone conferred site-independent expression in transgenic mice of the two loci present within its insert, the ubiquitously expressed Cyclin T1 and the mammary specific alpha-lactalbumin (alphalac) genes . To assess if this vector could target mammary-restricted expression of cDNA, the CAT ORF was introduced by homologous recombination in Escherichia coli in place of the alphalac transcription unit . The insert of this modified BAC was injected into mice and three transgenic lines were derived . None of these lines expressed the CAT gene suggesting that the use of long genomic inserts is not sufficient to support the expression of intron-less transgenes . The physically linked goat Cyclin T1 locus was found to be active in all three lines . This observation reinforced the hypothesis that the two loci are localised in two separate chromatin domains. Biochem J, 2003 Jul 1, 373(Pt 1), 191 - 200 Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of L-serine biosynthesis; Baek JY et al.; In the present study, we first report two forms of human phosphoserine aminotransferase (PSAT) cDNA (HsPSAT alpha and HsPSAT beta) . HsPSAT alpha has a predicted open reading frame comprising 324 amino acids, encoding a 35.2 kDa protein (PSAT alpha), whereas HsPSAT beta consists of an open reading frame comprising 370 amino acids that encodes a 40 kDa protein (PSAT beta) . PSAT alpha is identical with PSAT beta, except that it lacks 46 amino acids between Val(290) and Ser(337) of PSAT beta, which is encoded by the entire exon 8 (138 bp) . Both PSAT alpha and PSAT beta can functionally rescue the deletion mutation of the Saccharomyces cerevisiae counterpart . Reverse transcriptase-PCR analysis revealed that the expression of PSAT beta mRNA was more dominant when compared with PSAT alpha mRNA in all human cell lines tested . PSAT beta was easily detected in proportion to the level of mRNA; however, PSAT alpha was detected only in K562 and HepG2 cells as a very faint band . The relative enzyme activity of glutathione S-transferase (GST)-PSAT beta expressed in Escherichia coli appeared to be 6.8 times higher than that of GST-PSAT alpha . PSAT mRNA was expressed at high levels (approx . 2.2 kb) in the brain, liver, kidney and pancreas, and very weakly expressed in the thymus, prostate, testis and colon . In U937 cells, the levels of PSAT mRNA and protein appeared to be up-regulated to support proliferation . Accumulation of PSAT mRNA reached a maximum in the S-phase of Jurkat T-cells . These results demonstrate that although two isoforms of human PSAT can be produced by alternative splicing, PSAT beta rather than PSAT alpha is the physiologically functional enzyme required for the phosphorylated pathway, and indicate that the human PSAT gene is regulated depending on tissue specificity as well as cellular proliferation status with a maximum level expression in the S-phase. World J Gastroenterol, 2003 Mar, 9(3), 513 - 5 Expression of RNase H of human hepatitis B virus polymerase in Escherichia coli; Cheng H et al.; AIM: To amplify HBV-RNase H gene fragment and expression of RNase H for further use in the studies of HBV associated liver diseases . METHODS: The encoding gene of HBV-RNase H was separately amplified for the first half and second half (H1 and H2) by PCR from full length HBV gene and cloned into pT7Blue-T vector . Clones were first screened by digestion with XbaI and Hind III enzyme for the correct size, and analyzed further by DNA sequencing . The RNase H1 and H2 fragments isolated from XbaI and Hind III digestion products of pT7 Blue-RNase H plasmid were ligated to the GSTag expressing vectors separately, and expressed in E.coli BL21 . The expressed proteins were checked by PAGE gel and Western blot . RESULTS: Both H1 and H2 nucleotide seqences consisting of known genes and proteins, in correct size, were further confirmed by Western blot to be the GST and RNase H1 or H2 fusion proteins . CONCLUSION: The successful cloning and expression of HBV-RNase H will contribute to further research and application in HBV-associated diseases. Redox Rep, 2003, 8(1), 51 - 6 Thermosensitive phenotype of Escherichia coli mutant lacking NADP+-dependent isocitrate dehydrogenase; Choi IY et al.; Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events . NADP(+)-dependent isocitrate dehydrogenase (ICDH) in Escherichia coli produces NADPH, an essential reducing equivalent for the antioxidant system . The protective role of ICDH against heat shock in E . coli was investigated in wild-type and ICDH-deficient strains . Upon exposure to heat shock, the viability was lower and the protein oxidation was higher in mutant cells as compared to wild-type cells . Induction and inactivation of antioxidant enzymes were observed after their exposure to heat shock both in wild-type and in mutant cells . However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells . These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against heat shock through the removal of reactive oxygen species as well as in the protection of other antioxidant enzymes. Plant J, 2003 Mar, 33(6), 1027 - 35 Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast; Hoyos ME et al.; We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p . The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine . AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11 . The more efficient putative carrier, AtmBAC1, was expressed in E . coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l-amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity) . AtmBAC1 recognized l-histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not . Also different from ORNT1p, AtmBAC1 did not transport citrulline . AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l-forms of arginine, lysine and ornithine . By RT-PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings . Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e . delivery of Arg to mitochondrial arginase . The Km (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons. Eur J Biochem, 2003 Mar, 270(6), 1356 - 62 Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays; Moorhead GB et al.; An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var . botrytis) extracts . The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family . This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+ . The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner . The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase . We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro. Eur J Biochem, 2003 Mar, 270(6), 1277 - 87 Biotinylation in the hyperthermophile Aquifex aeolicus; Clarke DJ et al.; Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific . The exact details of the protein-protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated . Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms . We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP Delta 67) from the extreme hyperthermophile Aquifex aeolicus . In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP Delta 67 at temperatures of up to 70 degrees C . Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus . This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin . Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP Delta 67 complex . Furthermore, this complex was also formed between BPL and a BCCP Delta 67 mutant lacking the lysine residue (BCCP Delta 67 K117L) however, complex formation was considerably reduced using holo-BCCP Delta 67 . These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP Delta 67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition. Eur J Biochem, 2003 Mar, 270(6), 1211 - 21 Membrane targeting of a folded and cofactor-containing protein; Bruser T et al.; Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms . In bacteria, the twin arginine translocation (Tat) system can transport folded proteins . Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli . In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E . coli by a process requiring ATP when the Tat substrate was properly folded . During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state . Membrane targeting did not require a twin arginine motif and known Tat system components . On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane. Eur J Biochem, 2003 Mar, 270(6), 1057 - 64 Plasmoredoxin, a novel redox-active protein unique for malarial parasites; Becker K et al.; Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense . Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics . In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development . Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P . falciparum . As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites . It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree . We amplified the gene from a gametocyte cDNA library and overexpressed it in E . coli . The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin . Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible . Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis . As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites . Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions . It may also represent a specific target for antimalarial drug development and is of phylogenetic interest. Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 183 - 6 Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli; Mukherjee S et al.; The protein-folding catalyst SlyD, a peptidyl prolyl cis-trans isomerase regulated by metal binding, was initially discovered as a major contaminant of non-denaturing immobilized metal-affinity chromatography (IMAC)-based procedures for the purification of heterologously expressed 6xHis-tagged proteins from Escherichia coli . Given its ability to bind weakly to nickel-nitrilotriacetic acid (Ni(2+)-NTA), protocols for the purification of SlyD currently use an initial non-denaturing IMAC step, followed by an ion-exchange chromatographic step and occasionally also other chromatographic steps . Here we demonstrate that using denaturing conditions instead of non-denaturing conditions, and by processing of large quantities of culture through small volumes of Ni(2+)-NTA resin to increase competition for binding, single-step purification of SlyD to homogeneity can be achieved directly from E . coli extracts, as assessed through spectroscopic and electrophoretic methods . The purified, denatured SlyD is shown to be capable of refolding to give rise to a native-like CD spectrum, establishing the utility of the procedure . The procedure also establishes SlyD to be the only E . coli protein capable of contaminating denaturing IMAC-based procedures. Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 109 - 13 Construction of a new tumour necrosis factor fusion-protein expression vector for high-level expression of heterologous genes in Escherichia coli; Han W et al.; We report the construction and application of a new fusion-protein expression plasmid (TNFHis) for Escherichia coli . The plasmid contains both P(R) and P(L) promoters and is optimized to allow a higher level of expression of mature coding sequences . It also contains a six-histidine tag for convenient purification as well as thrombin and hydroxylamine recognition sites for cleaving heterologous protein . The potential use of this expression vector is demonstrated by comparing the expression levels of human tumour necrosis factor (TNF), interferon, interleukin 11, colony-forming factor, osteoprotegrin and interleukin 2 in E . coli . Furthermore, all expressed TNF fusion proteins can be detected by anti-TNF alpha antibody or by specific antibodies and purified by Ni(2+)-nitrilotriacetate beads . The expressed TNF fusion proteins can be cleaved by hydroxylamine. Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 103 - 7 Comparison of cellular stress levels and green-fluorescent-protein expression in several Escherichia coli strains; Seo JH et al.; Constructs comprising stress-gene promoter elements from rpoH (Sigma 32), clpB or dnaK linked to a green-fluorescent-protein (GFP) expression vector were previously used as non-invasive "stress probes" in Escherichia coli . We compared cellular stress responses in four E . coli strains: production hosts JM105 and BL21, and cloning hosts HB101 and TOP10 . When GFP was also used as a model for foreign protein production, we generally observed that the level of expression was inversely proportional to the level of cellular stress . JM105 showed the highest cellular stress level and very low GFP expression, while BL21 exhibited the lowest cellular stress level and the highest GFP expression, in both normal and heat-shock stress environments. Shock, 2003 Mar, 19(3), 245 - 51 Protective effect of 3-deazaadenosine in a rat model of lipopolysaccharide-induced myocardial dysfunction; Braun-Dullaeus RC et al.; Severe sepsis is accompanied by a profound depression of myocardial contractility . Leukocyte adhesion with subsequent local excess nitric oxide and reactive oxygen species production play major roles for this deleterious effect . We hypothesized that 3-deazaadenosine (c3Ado), an adenosine analogue with anti-inflammatory properties, prevents endotoxin-induced myocardial dysfunction . Wistar rats (8 per group) were treated with Escherichia coli lipopoly-saccharide (LPS, 1 mg/kg, i.p., strain 0111:B4) +/- c3Ado (10 mg/kg, i.p.) 8 h before their hearts were harvested for isolated perfusion, histochemical analysis, or electrophoretic mobility shift assay . LPS induced a marked depression of left ventricular contractility . Immunohistochemistry revealed an upregulation of the adhesion molecules VCAM-1, ICAM-1, and P-selectin within the postcapillary venules . c3Ado inhibited VCAM-1 and ICAM-1 upregulation, but not P-selectin, and prevented cardiodepression . Electrophoretic mobility shift assay revealed inactivation of the transcription factor nuclear factor-kappaB and immunohistochemical staining for gp91phox, ED1, and CD11b demonstrated that c3Ado prevented local recruitment of monocytes and polymorph nuclear neutrophils to the myocardium . Accordingly, significantly fewer leukocytes producing nitric oxide or reactive oxygen species accumulated within the myocardium . Intravital microscopy of intestinal venules confirmed that LPS-induced adhesion of leukocytes was prevented by c3Ado . Additionally, c3Ado prevented LPS-induced elevation of serum tumor necrosis factor-alpha levels . Our results imply that c3Ado may prove to have clinical relevance for inflammatory disease processes. Shock, 2003 Mar, 19(3), 223 - 8 Hemodynamic effects of glibenclamide during endotoxemia: contrasting findings in vitro versus in vivo; Preiser JC et al.; The final common pathway involved in the cardiovascular alterations of septic shock is incompletely defined . The opening of KATP channels is associated with vasorelaxation and alterations in cardiac contractility . This event may be triggered during septic shock by increased nitric oxide (NO) production, by a decreased intracellular content of ATP, or by a change in the transmembrane electrical potential . In the present study, we assessed the effects of glibenclamide, an agent that blocks the opening of KATP channels in vitro, on the contractile response of rat aortic rings to norepinephrine, and in vivo in anesthetized dogs, with or without exposure to Escherichia coli endotoxin . In vitro, glibenclamide decreased the contractile response to norepinephrine in the presence of endotoxin, provided that the endothelium was intact . In vivo, administration of 0.15 mg/kg increased systemic vascular resistance (SVR) in the absence of endotoxin only, and increased myocardial performance . A higher dose of 1 mg/kg increased SVR and decreased myocardial performance, both during endotoxic shock and in control conditions . Renal and mesenteric blood flows decreased, but the respective fractional flows were unchanged . Oxygen delivery decreased in both experimental conditions, but oxygen consumption decreased only in control conditions . The in vitro observations suggest that the opening of KATP channels is involved in the regulation of vascular tone during endotoxemia, via an endothelium-dependent mechanism . As different effects of glibenclamide were observed in vivo, the importance of the opening of KATP channels in endotoxic shock may be limited. Zh Mikrobiol Epidemiol Immunobiol, 2003 Jan-Feb, (1), 55 - 9 {Early diagnostics of hemorrhagic fever with renal syndrome on the basis of the use of Pumala virus recombinant nucleocapsid protein}; Veselov SIu et al.; Pumala virus recombinant nucleocapsid protein was used for the early diagnosis of haemorrhagic fever with renal syndrome . Specific IgM in the sera of patients could be determined by the IEA technique as early as on days 2-3 from the onset of the disease . The diagnostic effectiveness of the test-system was 95% and its specificity was 98%. Folia Microbiol (Praha), 2002, 47(6), 641 - 8 Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis; Nguyen LD et al.; Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described . Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E . coli . For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel . A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits . Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed. Nature, 2003 Mar 13, 422(6928), 180 - 5 Epub 2003 Mar 02. Functional analysis of an archaebacterial voltage-dependent K+ channel; Ruta V et al.; All living organisms use ion channels to regulate the transport of ions across cellular membranes . Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage . Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons . Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent . This channel possesses all the functional attributes of classical neuronal K(V) channels . The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels. J Clin Endocrinol Metab, 2003 Mar, 88(3), 1310 - 8 Calcitonin-specific transcription and splicing targets gene-directed enzyme prodrug therapy to medullary thyroid carcinoma cells; Messina M et al.; Recurrent and metastatic medullary thyroid carcinoma (MTC) remains difficult to treat due to its limited responsiveness to chemotherapy, radiotherapy, and imaging . To investigate an alternative therapeutic approach, we examined the feasibility of targeting gene-directed enzyme/prodrug therapy delivered by adenoviral vectors to MTC . We previously described a modified human calcitonin (CT)/CT gene-related peptide promoter that produced increased expression while maintaining specificity for MTC cells . In this study, we introduced an additional level of specificity by using cell-specific splicing and examined whether the selectivity of the gene-directed enzyme/prodrug therapy for MTC was enhanced when both the promoter and splicing features were combined in a single transcription unit . Two replication-defective adenoviruses were constructed that expressed the Escherichia coli purine nucleoside phosphorylase (PNP) gene under the transcriptional control of a modified T2 promoter (Ad.T2-PNP) or the T2 promoter in combination with a CT minigene cassette in which the PNP gene was imbedded within the CT gene exon 4 (Ad.T2-CT/PNP) . The specificity of PNP expression by Ad.T2-PNP, Ad.T2-CT/PNP, and control viruses in the MTC cell line, TT, and in a panel of non-MTC cell lines was evaluated . The highest level of PNP gene expression and the most effective cell killing in the presence of prodrug occurred in TT cells infected with Ad.T2-PNP, followed by Ad.T2-CT/PNP . Infection of most non-MTC cell lines, even with high multiplicities of Ad.T2-PNP, produced only low-level PNP expression that resulted in minimal cell killing in the presence of prodrug . High-level expression of PNP and effective cell killing was observed with both adenoviral gene constructs . The highest level of cell specificity was achieved with the combined use of promoter and splicing regulation in the Ad.T2-CT/PNP virus. EMBO J, 2003 Mar 17, 22(6), 1410 - 8 Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling; Gowers DM et al.; DNA-binding proteins are generally thought to locate their target sites by first associating with the DNA at random and then translocating to the specific site by one-dimensional (1D) diffusion along the DNA . We report here that non-specific DNA conveys proteins to their target sites just as well when held near the target by catenation as when co-linear with the target . Hence, contrary to the prevalent view, proteins move from random to specific sites primarily by three-dimensional (3D) rather than 1D pathways, by multiple dissociation/re-association events within a single DNA molecule . We also uncover a role for DNA supercoiling in target-site location . Proteins find their sites more readily in supercoiled than in relaxed DNA, again indicating 3D rather than 1D routes. EMBO J, 2003 Mar 17, 22(6), 1313 - 24 Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1); Vermeulen L et al.; Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses . NF-kappaB activity has long been thought to be regulated mainly by IkappaB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal . Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF-kappaB is generally accepted . We show that the mitogen-activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen- and stress- activated protein kinase-1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)-mediated stimulation of p65 transactivation capacity . Mutational analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF . Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus-dependent way and phosphorylates p65 at Ser276 . This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF-kappaB-dependent gene expression. EMBO J, 2003 Mar 17, 22(6), 1273 - 81 A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain; Shih SC et al.; Monoubiquitylation is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein . Monoubiquitin signals are likely to be recognized by ubiquitin-binding proteins that transmit the regulatory information conferred by monoubiquitylation . To identify monoubiquitin-binding proteins, we used a mutant ubiquitin that lacks the primary site of polyubiquitin chain formation as bait in a two-hybrid screen . The C-terminus of Vps9, a protein required in the yeast endocytic pathway, interacted specifically with monoubiquitin . The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identified by database searches as similar to parts of the yeast Cue1 and mammalian Tollip proteins . We demonstrate that CUE domains bind directly to monoubiquitin and we have defined crucial interaction surfaces on both binding partners . The Vps9 CUE domain is required to promote monoubiquitylation of Vps9 by the Rsp5 hect domain ubiquitin ligase . Thus, we conclude that the CUE motif is an evolutionarily conserved monoubiquitin-binding domain that mediates intramolecular monoubiquitylation. Vet Immunol Immunopathol, 2003 Mar 20, 92(1-2), 1 - 13 A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L . infantum; Molano I et al.; The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described . The data showed that after i.v . administration of 500,000 parasites of the L . infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens . All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection . One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant . In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection . Four of these dogs became parasite negative by day 634 post-infection . The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis . The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis . The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level . A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs . The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level. Bioorg Med Chem, 2003 Apr 3, 11(7), 1475 - 91 Substituted dibenzo{c,h}cinnolines: topoisomerase I-targeting anticancer agents; Yu Y et al.; Several substituted dibenzo{c,h}cinnolines were synthesized and evaluated for their potential to target topoisomerase I and for their relative cytotoxic activity . Select benzo{i}phenanthridines are capable of stabilizing the cleavable complex formed with topoisomerase I and DNA . This study was initiated to examine whether dibenzo{c,h}cinnolines, which are in essence aza analogues of benzo{i}phenanthridines, possess similar pharmacological properties . 2,3-Dimethoxy-8,9-methylenedioxybenzo{i}phenanthridine is one of the more potent benzo{i}phenanthridine derivatives in regard to topoisomerase I-targeting activity and cytotoxicity . The structure-activity relationship observed with these substituted dibenzo{c,h}cinnolines parallels that observed for benzo{i}phenanthridine derivatives . Compared to similarly substituted benzo{i}phenanthridines, the dibenzo{c,h}cinnoline analogues exhibit more potent topoisomerase I-targeting activity and cytotoxicity . The relative IC(50) values obtained in assessing the cytotoxicity of 2,3-dimethoxy-8,9-methylenedioxydibenzo{c,h}cinnoline and 2,3-dimethoxy-8,9-methylenedioxybenzo{i}phenanthridine in the human lymphoblastma cell line, RPMI8402, are 70 and 400 nM, respectively . In tumor cell lines selected for resistance to camptothecin and known to express mutant topoisomerase I, benzo{i}phenanthridine derivatives were not cross-resistant . In contrast, similarly substituted dibenzo{c,h}cinnolines with significant topoisomerase I-targeting activity did exhibit cross-resistance in these camptothecin-resistant cell lines . The cytotoxicity of these dibenzo{c,h}cinnolines was not diminished in cells overexpressing the efflux transporter, MDR1 . These data indicate that substituted dibenzo{c,h}cinnolines can exhibit potent topoisomerase I-targeting activity and are capable of overcoming the multi-drug resistance associated with this efflux transporter. Bioorg Med Chem, 2003 Apr 3, 11(7), 1433 - 8 Respiratory chain inhibition by fullerene derivatives: hydrogen peroxide production caused by fullerene derivatives and a respiratory chain system; Mashino T et al.; Fullerene is a new type of carbon allotrope . We have shown that the fullerene derivative C(60)-bis(N,N-dimethylpyrrolidinium iodide), a regio isomer mixture, inhibited Escherichia coli growth and dioxygen uptake caused by E . coli and glucose . This result indicates that the mechanism of the bacteriostatic effect is the inhibition of energy metabolism . In this study, we isolated two regio isomers of C(60)-bis(N,N-dimethylpyrrolidinium iodide) and studied their effect on E . coli growth and on respiratory chain activity . In dioxygen uptake caused by the inner-membrane and NADH, the effect of fullerene derivatives was biphasic . At low concentrations of both fullerene derivatives, dioxygen uptake was inhibited, whereas at high concentrations, it was increased . At high concentrations, consumed dioxygen was converted to H(2)O(2) . An electrochemical study revealed that reduced fullerene derivatives react with dioxygen . This activity was closely related to a redox property of the isomers. Bioorg Med Chem, 2003 Apr 3, 11(7), 1311 - 8 4-Hydroxymethyl- and 4-methoxymethylfuro{2,3-h}quinolin-2(1H)-ones: synthesis and biological properties; Chilin A et al.; 4-Hydroxymethyl-1,6,8-trimethylfuro{2,3-h}quinolin-2(1H)-one (HOFQ) was prepared by a new profitable way, which allowed to synthesize also 4-methoxymethyl-1,6,8-trimethylfuro{2,3-h}quinolin-2(1H)-one (MOFQ), and 4-hydroxymethyl-6,8-dimethylfuro{2,3-h}quinolin-2(1H)-one (HOHFQ) . Some biological activities of the three compounds were studied in comparison with 8-MOP . In the dark, they inhibited topoisomerase II, leading to a moderate antiproliferative activity in mammalian cells . The antiproliferative activity was also tested upon UVA irradiation in mammalian cells: all compounds showed higher activity than 8-MOP, without mutagenicity and skin phototoxicity, with the best results for HOFQ . Photobinding to DNA was investigated, demonstrating a different sequence specificity for these furoquinolinones in comparison with furocoumarins . For all these features, HOFQ and the other analogues appeared very promising photochemotherapeutic agents, whose mechanism of action will be further investigated. Bioorg Med Chem, 2003 Apr 3, 11(7), 1207 - 14 Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei; Pasti C et al.; Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver . Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme . A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors . At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target. Fitoterapia, 2003 Feb, 74(1-2), 77 - 83 Effect of meliacine, a plant derived antiviral, on tumor necrosis factor alpha; Petrera E et al.; The effect of meliacine (MAS) and two fractions MAB 1 and MAB 2 obtained from it on the in vitro production of TNF-alpha of murine macrophages induced by bacterial lipopolysaccharide (LPS) (from Escherichia coli) was tested . Simultaneous administration of the above fractions (ranging from 14 to 56 microg/ml) and LPS (10 microg/ml) to a macrophage culture significantly increased the amount of TNF-alpha released at 24 h of induction in a dose-dependent manner . Meliacine alone, at a concentration of 56 microg/ml, is a weak inducer of TNF-alpha production. Comp Biochem Physiol B Biochem Mol Biol, 2003 Mar, 134(3), 407 - 16 Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro; Pedersen GM et al.; This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages . Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column . SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones . Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo . Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation . Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation . Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation . In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation. Trends Biotechnol, 2003 Mar, 21(3), 106 - 8 Escherichia coli gets a new virus but it's nothing to sneeze at; Smith G; For the past two decades virologists have strived to make full-length clones of viral genomes that, on transfection into permissive eukaryotic cells, initiate a productive infection . The large variety of viral RNA and DNA genome structures, as well as different replication strategies, has required investigators to develop new approaches to produce infectious DNA in Escherichia coli . A member of the poxviridae, one of the most complex virus families, has now been made into an infectious clone in E . coli for the first time . Although the isolation was complicated, the infectious clone will greatly simplify future genetic studies of the virus. J Mol Biol, 2003 Mar 21, 327(2), 549 - 60 Importance of substrate and cofactor polarization in the active site of dihydrofolate reductase; Garcia-Viloca M et al.; By using a combined quantum-mechanical and molecular-mechanical potential in molecular dynamics simulations, we have investigated the effects of the enzyme electric field of dihydrofolate reductase on the electronic polarization of its 5-protonated dihydrofolate substrate at various stages of the catalyzed hydride transfer reaction . Energy decomposition of the total electrostatic interaction energy between the ligands and the enzyme shows that the polarization effect is 4% of the total electrostatic interaction energy, and, significantly, it accounts for 9kcal/mol of transition state stabilization relative to the reactant state . Therefore it is essential to take account of substrate polarization for quantitative interpretation of enzymatic function and for calculation of binding free energies of inhibitors to a protein . Atomic polarizations are calculated as the differences in the average atomic charges on the atoms in gas phase and in molecular simulations of the enzyme; this analysis shows that the glutamate tail and the pterin ring are the highly polarized regions of the substrate . Electron density difference plots of the reactant and product complexes at instantaneous configurations in the enzyme active center confirm the inferences made on the basis of partial atomic charges. J Mol Biol, 2003 Mar 21, 327(2), 521 - 36 Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli; Huang YJ et al.; Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein . It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA . RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics . Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods . The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation . RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3 . The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn . In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink . The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field . A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements . Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure . While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein . Both RbfA and NusA are regulated in the same E.coli operon . Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins. J Mol Biol, 2003 Mar 21, 327(2), 383 - 91 Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity; Rimseliene R et al.; Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics . Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far . We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity . MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize . The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure . The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before . This study provides evidence that MABS is a promising technique for generation of REs with new specificities. J Mol Biol, 2003 Mar 21, 327(2), 369 - 81 Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome; Studer SM et al.; Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis . The molecular mechanism for this process is still poorly understood . Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome . In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9 . Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods . Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation . Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome . Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation. Prostaglandins Leukot Essent Fatty Acids, 2003 Apr, 68(4), 273 - 84 Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology; Tsuruta LR et al.; Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system . The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice . Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-{4'-(maleimidomethyl) cyclohexanecarboxamido} butane (Biotin-BMCC) . Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system . Results indicated that the selected anti-11D-TX Fab fragments expressed by E . coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA) . This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody . The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure . According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity. Biochem J, 2003 May 1, 371(Pt 3), 669 - 73 Nucleotide-dependent protein folding in the type II chaperonin from the mesophilic archaeon Methanococcus maripaludis; Kusmierczyk AR et al.; We report the characterization of the first chaperonin (Mm-cpn) from a mesophilic archaeon, Methanococcus maripaludis . The single gene was cloned from genomic DNA and expressed in Escherichia coli to produce a recombinant protein of 543 amino acids . In contrast with other known archaeal chaperonins, Mm-cpn is fully functional in all respects under physiological conditions of 37 degrees C . The complex has Mg(2+)-dependent ATPase activity and can prevent the aggregation of citrate synthase . It promotes a high-yield refolding of guanidinium-chloride-denatured rhodanese in a nucleotide-dependent manner . ATP binding is sufficient to effect folding, but ATP hydrolysis is not essential. Biochemistry, 2003 Mar 18, 42(10), 3096 - 104 HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study; Wojtuszewski K et al.; HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA . In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements . A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex . Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)) . The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend . DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor . From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated . The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU . The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers . Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex . These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism. Biochemistry, 2003 Mar 18, 42(10), 3025 - 31 A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability; Smirnova IN et al.; Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport . The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate . Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced . In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect . (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present . (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment . The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding. Nat Biotechnol, 2003 Apr, 21(4), 435 - 9 Epub 2003 Mar 10. Identification of co-regulated genes through Bayesian clustering of predicted regulatory binding sites; Qin ZS et al.; The identification of co-regulated genes and their transcription-factor binding sites (TFBS) are key steps toward understanding transcription regulation . In addition to effective laboratory assays, various computational approaches for the detection of TFBS in promoter regions of coexpressed genes have been developed . The availability of complete genome sequences combined with the likelihood that transcription factors and their cognate sites are often conserved during evolution has led to the development of phylogenetic footprinting . The modus operandi of this technique is to search for conserved motifs upstream of orthologous genes from closely related species . The method can identify hundreds of TFBS without prior knowledge of co-regulation or coexpression . Because many of these predicted sites are likely to be bound by the same transcription factor, motifs with similar patterns can be put into clusters so as to infer the sets of co-regulated genes, that is, the regulons . This strategy utilizes only genome sequence information and is complementary to and confirmative of gene expression data generated by microarray experiments . However, the limited data available to characterize individual binding patterns, the variation in motif alignment, motif width, and base conservation, and the lack of knowledge of the number and sizes of regulons make this inference problem difficult . We have developed a Gibbs sampling-based Bayesian motif clustering (BMC) algorithm to address these challenges . Tests on simulated data sets show that BMC produces many fewer errors than hierarchical and K-means clustering methods . The application of BMC to hundreds of predicted gamma-proteobacterial motifs correctly identified many experimentally reported regulons, inferred the existence of previously unreported members of these regulons, and suggested novel regulons. Crit Care Med, 2003 Mar, 31(3), 683 - 8 Leukotriene-mediated coronary vasoconstriction and loss of myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused rat hearts; Sibelius U et al.; OBJECTIVE: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis . We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow . DESIGN: Prospective, experimental study . SETTING: Research laboratory at a university hospital . SUBJECTS: Isolated hearts from male Wistar rats . INTERVENTIONS: Isolated hearts were perfused with purified hemolysin for 60 min . MEASUREMENTS AND MAIN RESULTS: Low concentrations of the toxin in the perfusate (0.1-0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase) . Moreover, 0.2 hemolytic units/mL hemolysin evoked ventricular fibrillation within 10 mins of toxin application . These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate . The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase . In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective . In addition, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886 . CONCLUSIONS: Low doses of hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature . Bacterial exotoxins such as hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock. Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3143 - 8 Epub 2003 Mar 07. Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution; Williams GJ et al.; Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates . Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase . After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k(cat)/K(m) toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity . (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon-carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate . This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics. Nucleic Acids Res . 2003 Mar 15;31(6):e31. Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog; Nagatsugi F et al.; The specific recognition of homopurine-homo pyrimidine regions in duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation . Alkylation of nucleobases with functionalized TFOs would have the potential for site-directed mutagenesis . Recently, we demonstrated that a TFO bearing 2-amino-6-vinylpurine derivative, 1, achieves triplex-mediated reaction with high selectivity toward the cytosine of the G-C target site . In this report, we have investigated the use of this reagent to target mutations to a specific site in a shuttle vector plasmid, which replicates in mammalian cells . TFOs bearing 1 produced adducts at the complementary position of 1 and thereby introduced mutations at that site during replication/repair of the plasmid in mammalian cells . Reagents that produce covalent cytosine modifications are relatively rare . These TFOs enable the preparation of templates carrying targeted cytosine adducts for in vitro and in vivo studies . The ability to target mutations may prove useful as a tool for studying DNA repair, and as a technique for gene therapy and genetic engineering. Nucleic Acids Res, 2003 Mar 15, 31(6), 1790 - 5 Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells; Nakamura J et al.; Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells . Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H2O2) . Interestingly, a bimodal H2O2 dose-response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells . Furthermore, it has been demonstrated that H2O2 causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose-response curve . Here we show that H2O2 produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H2O2 were much more efficient at inducing ADLs than higher concentrations . This dose-response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H2O2 . Interestingly, serial treatment of submillimolar levels of H2O2 induced a massive accumulation of ADLs . The toxicity arising from H2O2 determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs . The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H2O2 . These results suggest that ADLs induced by submillimolar levels of H2O2 may be due to a Fenton-type reaction between H2O2 and intracellular iron ions in mammalian cells. Nucleic Acids Res, 2003 Mar 15, 31(6), 1683 - 92 Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties; Gojkovic Z et al.; Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri . The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme . In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency . The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-beta-alanine . At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring . Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes . Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap . Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones. Nucleic Acids Res, 2003 Mar 15, 31(6), 1633 - 9 Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix; Bunting KA et al.; Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences . We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5'-C-OH-3' 5'-p-T-p-A-p-G-p-G-3'/3'-G-p-G-p-T-p(Me5)C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix . The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites. Nucleic Acids Res, 2003 Mar 15, 31(6), 1585 - 96 The enzymatic basis of processivity in lambda exonuclease; Subramanian K et al.; Lambda exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA . The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination . The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity . lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism . We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA . The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes . By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated . We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation. Microbiol Mol Biol Rev, 2003 Mar, 67(1), 66 - 85, table of contents Two families of mechanosensitive channel proteins; Pivetti CD et al.; Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya . Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families . A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes . In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain . Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins . We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure . The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin. J Immunol, 2003 Mar 15, 170(6), 3273 - 8 Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis; Maus UA et al.; Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses . We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic . Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2(+) alveolar macrophage population, and CCR2-deficient mice transfused with CCR2(+) mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin . Our data show that alveolar monocyte recruitment is strictly dependent on CCR2 . LPS-induced neutrophil migration to the lungs is CCR2 independent . However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified . We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation. J Biol Chem, 2003 May 23, 278(21), 19102 - 10 Epub 2003 Mar 07. Biochemical properties of CikA, an unusual phytochrome-like histidine protein kinase that resets the circadian clock in Synechococcus elongatus PCC 7942; Mutsuda M et al.; We recently described the cikA (circadian input kinase A) gene, whose product supplies environmental information to the circadian oscillator in the cyanobacterium Synechococcus elongatus PCC 7942 . CikA possesses three distinct domains: a GAF, a histidine protein kinase (HPK), and a receiver domain similar to those of the response regulator family . To determine how CikA functions in providing circadian input, we constructed modified alleles to tag and truncate the protein, allowing analysis of each domain individually . CikA covalently bound bilin chromophores in vitro, even though it lacks the expected ligand residues, and the GAF domain influenced but did not entirely account for this function . Full-length CikA and truncated variants that carry the HPK domain showed autophosphorylation activity . Deletion of the GAF domain or the N-terminal region adjacent to GAF dramatically reduced autophosphorylation, whereas elimination of the receiver domain increased activity 10-fold . Assays to test phosphorelay from the HPK to the cryptic receiver domain, which lacks the conserved aspartyl residue that serves as a phosphoryl acceptor in response regulators, were negative . We propose that the cryptic receiver is a regulatory domain that interacts with an unknown protein partner to modulate the autokinase activity of CikA but does not work as bona fide receiver domain in a phosphorelay. FASEB J, 2003 May, 17(8), 884 - 6 Epub 2003 Mar 05. Exercise and IL-6 infusion inhibit endotoxin-induced TNF-alpha production in humans; Starkie R et al.; During "nondamaging" exercise, skeletal muscle markedly releases interleukin (IL)-6, and it has been suggested that one biological role of this phenomenon is to inhibit the production of tumor necrosis factor (TNF)- alpha, which is known to cause pathogenesis such as insulin resistance and atherosclerosis . To test this hypothesis, we performed three experiments in which eight healthy males either rested (CON), rode a bicycle for 3 h (EX), or were infused with recombinant human IL-6 (rhIL-6) for 3 h while they rested . After 2.5 h, the volunteers received a bolus of Escherichia coli lipopolysaccharide endotoxin (0.06 ng/kg) i.v . to induce low-grade inflammation . In CON, plasma TNF-alpha increased significantly in response to endotoxin . In contrast, during EX, which resulted in elevated IL-6, and rhIL-6, the endotoxin-induced increase in TNF-alpha was totally attenuated . In conclusion, physical exercise and rhIL-6 infusion at physiological concentrations inhibit endotoxin-induced TNF-alpha production in humans . Hence, these data provide the first experimental evidence that physical activity mediates antiinflammatory activity and suggest that the mechanism include IL-6, which is produced by and released from exercising muscles. Antioxid Redox Signal, 2003 Feb, 5(1), 15 - 22 Characterization of the redox properties of poplar glutaredoxin; Rouhier N et al.; The presence of glutaredoxins in plants is now well recognized, but their functions and natural substrates remain largely unknown . Recently, a poplar glutaredoxin has been biochemically characterized and several mutants have been engineered in order to explore its reactivity . This work focuses on some physiological functions of the enzyme . According to our findings, the poplar glutaredoxin can serve as an electron donor to the bacterial 3'-phosphoadenylylsulfate reductase as it supports both the catalysis by the enzyme in vitro and complements a methionine auxotroph strain of Escherichia coli . In addition, poplar glutaredoxin is able to reduce the Escherichia coli ribonucleotide reductase 1a (in vitro reduction of cytidine diphosphate) . Although this glutaredoxin is described as an electron donor to a phloem-located peroxiredoxin, whose function is to detoxify hydroperoxides, we found that it does not directly reduce hydrogen peroxide or other alkyl hydroperoxides as described for yeast and rice glutaredoxins . However, the poplar glutaredoxin may be involved in the response to oxidative stress as its overexpression in Escherichia coli resulted in a higher resistance toward hydrogen peroxide, menadione, and tert-butyl hydroperoxide. Protein Pept Lett, 2003 Feb, 10(1), 61 - 72 A structure-function analysis of glial cell-line-derived neurotrophic factor receptor alpha1; Wang LM et al.; The GFRalpha1 cDNA was amplified by RT-PCR from fetal rat hippocampus . The soluble recombinant GFRalpha1 and its mutants were obtained from an Escherichia coli expression system . The biological activity of soluble GFRalpha1 and its mutants were evaluated in PC12 cells . The results suggest that the central domain of GFRalpha1 is a crucial determinant for ligand binding . This established a solid basis for further study to find the key amino acid mediating the binding of GDNF and GFRalpha1. Protein Pept Lett, 2003 Feb, 10(1), 19 - 26 Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides; Khumthong R et al.; The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein . Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions . Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained . The data presented here show that NS2B-NS3(pro) expressed in recombinant E . coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site . A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites. Technol Cancer Res Treat, 2002 Oct, 1(5), 319 - 28 Factors controlling electropermeabilisation of cell membranes; Rols MP et al.; Electric field pulses are a new approach for drug and gene delivery for cancer therapy . They induce a localized structural alteration of cell membranes . The associated physical mechanisms are well explained and can be safely controlled . A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell . Electric field pulses with an overcritical intensity evoke a local membrane alteration . A free exchange of hydrophilic low molecular weight molecules takes place across the membrane . A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained . The fraction of the cell surface which is competent for exchange is a function of the field intensity . The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses . The permeabilised state is long lived . Its lifetime is under the control of the cumulated pulse duration . Cell viability can be preserved . Gene transfer is obtained but its mechanism is not a free diffusion . Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect . After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression . Molecular structural and metabolic changes in cells remain mostly poorly understood . Nevertheless, while most studies were established on cells in culture (in vitro), recent experiments show that similar effects are obtained on tissue (in vivo) . Transfer remains controlled by the physical parameters of the electrical treatment. Mol Biol (Mosk), 2003 Jan-Feb, 37(1), 121 - 7 {The effect of modification of nucleotide-37 on the interaction of aminoacyl-tRNA with the A-site of the 70S ribosome}; Soboleva NG et al.; To estimate the effect of modified nucleotide-37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAK+YPhe and Phe-tRNAK-YPhe) with the A site of complex {70S.poly(U).deacylated tRNA(Phe) in the P site} was assayed at 0-20 degrees C . As comparisons with native Phe-tRNAK+YPhe showed, removal of the Y base decreased the association constant of Phe-tRNAK-YPhe and the complex by an order of magnitude at any temperature, and increased the enthalpy of their interaction by 23 kJ/mol . When the Y base was present in the anticodon loop of deacylated tRNA(Phe) bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAK-YPhe but not for Phe-tRNAK+YPhe . Thus, the modified nucleotide 3' of the Phe-tRNA(Phe) anticodon stabilized the codon-anticodon interaction both in the A and in the P sites of the 70S ribosome. Planta, 2003 Mar, 216(5), 752 - 61 Epub 2002 Nov 26. Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme; Jones AL et al.; A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves . The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein . The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer . Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin . A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations . This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site . A full-length cDNA for the pea KAS III was isolated . This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification . Demonstrated activity in preparations from E . coli confirmed that the cDNA encoded a KAS III enzyme . Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent . The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp . (62%), Synechocystis spp . (65%) and various bacteria (42-65%) . The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353) . In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs . Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism. Planta, 2003 Mar, 216(5), 745 - 51 Epub 2002 Oct 25. Functional identification of AtTPS03 as (E)-beta-ocimene synthase: a monoterpene synthase catalyzing jasmonate- and wound-induced volatile formation in Arabidopsis thaliana; Faldt J et al.; (E)-beta-Ocimene is one of the most commonly found monoterpenes of the volatile blends that are emitted from leaves in response to damage by herbivores or mechanical wounding . (E)-beta-Ocimene is also a component of many floral scents . Airborne (E)-beta-ocimene emitted from plants can serve as a chemical cue for the attraction of parasitoids or predators of plant herbivores and also as an attractant for pollinating insects . Furthermore, exposure of plants to (E)-beta-ocimene can activate defense gene expression . In this paper, we describe cDNA cloning and functional characterization of a gene encoding a highly specialized (E)-beta-ocimene synthase, AtTPS03, from Arabidopsis thaliana (L.) Heynh . AtTPS03 was identified as a member of the large AtTPS gene family of putative terpene synthases . A cDNA for AtTPS03 was expressed in Escherichia coli and the enzyme function identified in vitro . The A . thaliana (E)-beta-ocimene synthase produces almost exclusively (E)-beta-ocimene (94%) with minor amounts of the related acyclic monoterpenes (Z)-beta-ocimene (4%) and myrcene (2%) . Transcripts for AtTPS03 were up-regulated in leaves of Arabidopsis in response to mechanical wounding and treatment with jasmonic acid, concurrent with induced emission of (E)-beta-ocimene . AtTPS03 provides an important gene for probing plant-insect and possibly plant-plant interactions mediated by terpenoid volatiles. Planta, 2003 Mar, 216(5), 736 - 44 Epub 2002 Oct 22. Two isoforms of Rubisco activase in cotton, the products of separate genes not alternative splicing; Salvucci ME et al.; In several plant species, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA . Two forms of activase corresponding to the longer, redox-regulated alpha and the shorter, beta forms were detected immunologically in cotton (Gossypium hirsutum L.) leaves, but their N-termini differed in 4 of 14 residues . The cDNAs for the alpha and beta forms of cotton activase diverged throughout the translated and 3'-untranslated regions, including variations that accounted for the differences in N-terminal amino acid sequence . Analysis of genomic DNA confirmed that separate genes encoded the alpha and beta forms of cotton activase . Separate activase genes were also detected in diploid species of cotton containing the different progenitor genomes of the cultivated allotetraploid, indicating that the occurrence of separate alpha- and beta-form genes in cotton predates the merger of the diploid genomes . The deduced amino acid sequences of the two forms of cotton activase exhibited 84% identity and both forms were active after expression in Escherichia coli . The recombinant alpha and beta forms exhibited similar affinities for ATP and only minor differences in thermotolerance, but their ATPase specific activities differed . The results show for the first time a plant species with two forms of activase that are structurally and functionally equivalent to the alternatively spliced alpha and beta forms in other plants, but that are encoded by separate genes . That cotton still expresses both forms of activase, even without alternative splicing, suggests that each form has a required function in photosynthesis. Amino Acids, 2003, 24(1-2), 19 - 41 Proteomic analysis of the cell envelope fraction of Escherichia coli; Fountoulakis M et al.; We applied proteomics technologies to analyze a membrane preparation of Escherichia coli, wild type strain and of transformants expressing human cytochrome P450s . The proteins were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry . The membrane proteins were solubilized with both mild detergents such as CHAPS and strong detergents, such as sodium and lithium dodecyl sulfate, sodium cholate and sodium deoxycholate . In the E . colimembrane fraction, 394 different gene products were identified . Approximately 28% of them were predicted to be integral membrane proteins, of which 100 proteins have been predicted to carry one transmembrane region, ten proteins to carry two, and two proteins to include three transmembrane domains . The remaining are probably membrane-associated and cytosolic proteins . Cytochrome P450s did not enter the immobilized pH gradient strips but were efficiently analyzed in a two-dimensional, two-detergent system . Use of strong solubilizing agents resulted in the detection of about 20 membrane proteins, which were not detected following extraction with mild detergents and chaotropes . The present database is one of the largest for membrane proteins. Am J Nephrol, 2003 May-Jun, 23(3), 140 - 51 Epub 2003 Mar 06. Uropathogenic Escherichia coli toxins induce caspase-independent apoptosis in renal proximal tubular cells via ERK signaling; Chen M et al.; BACKGROUND: Pyelonephritis is a risk factor for renal tubular epithelial cell damage . Recent studies have shown that Escherichia coli and/or its toxins may stimulate apoptotic cell death in renal tubular cells, but the underlying molecular mechanisms remain to be elucidated . METHODS: Confluent LLC-PK(1) cells were exposed to E . coli toxins from overnight cultures of the uropathogenic O6K13H1 (O6) and the nonpathogenic W3110 . The cell death was studied with morphological and biological assay . RESULTS: E . coli soluble toxins from uropathogenic O6:K13:H1(O6) strain were found to induce apoptosis in a dose- and time-dependent manner in LLC-PK1 cells . The expression of FasR and the phosphorylation of ERK1/2 were significantly upregulated by O6 soluble toxins in a time-dependent manner . Cell death was completely inhibited by two specific ERK1/2 inhibitors, but not by a broad caspase inhibitor, zVAD-fmk, implicating a caspase-independent pathway via ERK . Moreover, we found that lysophosphatidic acid could trigger a survival signal through G-proteins and PI3K . CONCLUSION: We demonstrate that apoptosis induced by uropathogenic E . coli toxins is dependent on ERK1/2 . Caspases, although being activated, are not necessary for cell death, and they act after the ERK signaling at which point cells become committed to cell death or can be rescued . Microbiology, 2003 Feb, 149(Pt 2), 431 - 44 Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp strain 12S; Yoshida T et al.; Methylobacillus sp . strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose . Twenty-four ORFs flanking a Tn5 insertion site in an EPS-deficient mutant were identified, and 21 genes (epsCBAKLDEFGHIJMNOPQRSTU) were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence . Gene disruption analyses revealed that epsABCEFGIJNOP and epsR are required for methanolan synthesis, whereas epsKD and epsH are not essential . EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing Escherichia coli, suggesting that methanolan was synthesized via a Wzy-like biosynthesis system . This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system . EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier . In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety . On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface . E . coli strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan . EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides . EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators . In particular, EpsA seems to positively regulate the transcription of methanolan synthesis genes, since the constitutive expression of epsA in strain 12S increased the EPS production . Interestingly, EpsD showed homology with peptidyl prolyl cis-trans isomerases that catalyse the folding of proteins following translocation across the cytoplasmic membrane. Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3209 - 14 Epub 2003 Mar 06. Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data; Hogbom M et al.; The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis . X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein . Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122(*)) . We further report on subsequent high-field EPR experiments on the radical-containing crystals . A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame . The EPR data are discussed in comparison with a 1.42-A x-ray structure of the met (oxidized) form of the protein, also presented in this paper . Comparison of the orientation of the radical Y122(*) obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state . Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed . In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography. J Biol Chem, 2003 May 16, 278(20), 17615 - 24 Epub 2003 Mar 06. Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity; Mogk A et al.; ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system . ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region . Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity . Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer . Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner . Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity . The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo . In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity. J Biol Chem, 2003 May 16, 278(20), 18557 - 62 Epub 2003 Mar 06. Differential and simultaneous adenosine di- and triphosphate binding by MutS; Bjornson KP et al.; The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood . As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS . Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent . Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein . Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range . These findings suggest that the protein exists primarily as the ATP.MutS.ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo. J Biol Chem, 2003 May 16, 278(20), 18440 - 7 Epub 2003 Mar 06. Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2; Horike N et al.; Salt-inducible kinase (SIK), first cloned from the adrenal glands of rats fed a high salt diet, is a serine/threonine protein kinase belonging to an AMP-activated protein kinase family . Induced in Y1 cells at an early stage of ACTH stimulation, it regulated the initial steps of steroidogenesis . Here we report the identification of its isoform SIK2 . When a green fluorescent protein-fused SIK2 was expressed in 3T3-L1 preadipocytes, it was mostly present in the cytoplasm . When coexpressed in cAMP-responsive element-reporter assay systems, SIK2 could repress the cAMP-responsive element-dependent transcription, although the degree of repression seemed weaker than that by SIK1 . SIK2 was specifically expressed in adipose tissues . When 3T3-L1 cells were treated with the adipose differentiation mixture, SIK2 mRNA was induced within 1 h, the time of induction almost coinciding with that of c/EBPbeta mRNA . Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser(794) of human IRS-1 . Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser(789), the mouse equivalent of human Ser(794) . Moreover, the activity and content of SIK2 were elevated in white adipose tissues of db/db diabetic mice . These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser(794) of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals. J Biol Chem, 2003 May 9, 278(19), 17053 - 9 Epub 2003 Mar 06. Structure of the GTPase-binding domain of Sec5 and elucidation of its Ral binding site; Mott HR et al.; The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock . The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation . Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst . This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles . We have determined the structure of the Ral-binding domain of Sec5 and shown that it adopts a fold that has not been observed in a G protein effector before . This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains . We have mapped the Ral binding site on this domain and found that it overlaps with protein-protein interaction sites on other IPT domains but that it is completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor . This mapping, along with available site-directed mutagenesis data, allows us to predict how Ral and Sec5 may interact. J Clin Microbiol, 2003 Mar, 41(3), 1225 - 34 Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates; Carvalho VM et al.; Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries . While diarrhea is also a major problem among primates kept in captivity, the role of E . coli is unclear . This study was designed to characterize diarrheagenic E . coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.) . Seventeen of the 56 primates had signs of diarrhea and/or enteritis . E . coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E . coli and for patterns of adherence to HeLa cells . In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions . PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis . Colonies of eae(+) E . coli were serotyped and examined by PCR for genes encoding EPEC virulence markers . The eae(+) E . coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC . The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation . The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans. J Clin Microbiol, 2003 Mar, 41(3), 1147 - 51 High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis; Huang X et al.; The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t) . Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity . Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B . equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates . The specific antibodies in B . equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection) . Horse sera from Jilin Province, China, were examined by the two tests . The seroprevalence of B . equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t . The correspondence was 98.4% (62 of 63 sera) between the two tests . The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield. Microb Pathog, 2003 Feb, 34(2), 81 - 90 Immunogenicity of a 16.7 kDa Mycobacterium paratuberculosis antigen; Mullerad J et al.; Mycobacterium paratuberculosis (MPT), the agent of paratuberculosis is a slow growing mycobacteria that causes important economic losses mainly due to lower weight gains and drastic decrease in milk production . Existing paratuberculosis vaccines are not completely protective and induce antibodies/delayed type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals . New potent acellular vaccines that allow discrimination between infected and vaccinated animals are needed to improve the control of this disease . We have identified, expressed and purified a hypothetical thiol peroxidase of MPT (MPT-TP) in mice . We also characterized the immunogenicity of this antigen in mice . The recombinant MPT-TP (rMPT-TP) antigen induced a high production of IFNgamma, IL-6, and NO and a low production of IL-10 by spleen cells of immunized mice . Addition of Ribi adjuvant to rMPT-TP resulted in lower IFNgamma secretion and higher NO production in spleen cells . A similar level of proliferation of spleen cells exposed to rMPT-TP was found in immunized groups (rMPT-TP and rMPT-TP emulsified in Ribi) . DTH responses in mice footpads were observed only in mice immunized with rMPT-TP emulsified in Ribi . Addition of Ribi adjuvant clearly induced a significantly higher anti-rMPT-TP antibody production of all classes tested and decreased the IgG1/IgG2a ratio . MPT-TP demonstrated antigenic characteristics that make this antigen a potential component in the development of a future subunit vaccine against paratuberculosis. Tuberculosis (Edinb), 2002, 82(6), 283 - 91 Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains; Al-Zarouni M et al.; SETTING: Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression . OBJECTIVES: To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains . DESIGN: Plasmids were constructed using mycobacterial promoters (hsp60, 19-kDa antigen, 85A antigen--from the Mycobacterium tuberculosis complex--and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes . RESULTS: The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product . Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters . BCG Moreau appeared to be more susceptible to deletions than other BCG strains . CONCLUSIONS: The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product . Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation . With intact plasmid there was no marked difference in expression between BCG strains. Arch Biochem Biophys, 2003 Mar 15, 411(2), 277 - 88 Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699; Xu J et al.; The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase . Frameshift inactivation of rif orf14 generated a mutant of A . mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV) . Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W . 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S . The purified enzyme does not require a divalent cation for catalytic activity . While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory . The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1) . The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction . They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product. Arch Biochem Biophys, 2003 Mar 15, 411(2), 267 - 76 cDNA isolation, functional expression, and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis; Phillips MA et al.; The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens . Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine . The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions . Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes . Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases . The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms . The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed. Structure (Camb), 2003 Mar, 11(3), 253 - 63 Nucleotide-induced conformational changes in an isolated Escherichia coli DNA polymerase III clamp loader subunit; Podobnik M et al.; Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes . The structure of the E . coli clamp loader in a nucleotide-free state has been determined previously . We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E . coli clamp loader, in nucleotide-free and bound forms . The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked . The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated . This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process. Chin Med J (Engl), 2002 Dec, 115(12), 1785 - 9 High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell; Chen W et al.; OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E . coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell . METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed . The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments . The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments . The expression level of desired protein was assayed by SDS-PAGE and laser density scanning . Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR) . RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed . Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein . In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein . As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P < 0.01) . Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P > 0.05) and restricted to some extent . CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein . Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E . coli strains under identical incubation condition. Mol Microbiol, 2003 Mar, 47(6), 1653 - 67 Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein; Landraud L et al.; Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD-cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain . We examined if this association could reflect a co-regulation of the production of these toxins . Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine-Dalgarno sequence present in the cnf1 coding sequence {fold-back inhibition (cnf1 fbi)} . The cnf1 fbi was not regulated by elements present in the igs . An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain . This RNA could be part of a HlyCABD mRNA . Transcription of the haemolysin operon requires RfaH antitermination activity . Inactivation of rfaH in J96 resulted in a 100-fold reduction of the CNF1 content of bacteria . The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter . This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH . This constitutes a novel system of bacterial virulence factors co-regulation. Genes Cells, 2003 Mar, 8(3), 251 - 61 ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication; Tanaka T et al.; BACKGROUNDS: PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication . However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood . RESULTS: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs . All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein . They also rescued the low viability and UV-sensitivity of priA null cells . However, they did not rescue iSDR or cSDR-alternative modes of chromosomal DNA replication of t