Escherichia coli

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 the E . coli genome dependent on recombination functions-to the full extent . CONCLUSIONS: ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E . coli.

Anal Chem, 2003 Feb 15, 75(4), 709 - 15
Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide; Nath N et al.; We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP) . An ELP is covalently micropatterned on a glass surface against an inert BSA background . The ELP-patterned surface is incubated with the soluble fraction of E . coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface . The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus . The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein . The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution . We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition . The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques . TRAP has application in lab-on-a-chip bioanalytical devices as well as in the fabrication of peptide and protein arrays.

Pol Merkuriusz Lek, 2002 Nov, 13(77), 408 - 9
{Diverticulosis as a background of "abdominal catastrophe" in the course of peritoneal dialysis . Care Report}; Pietrzak B et al.; Peritonitis complicating peritoneal dialysis (PD) may represent a difficult diagnostic and therapeutic problem if it coexists with surgical pathology of intra-abdominal organs defined as "abdominal catastrophe" . The illustration of this problem is the case of 70-year-old patient treated with automated PD, in whom recurrent episodes of peritonitis (Escherichia coli) were typical of "abdominal catastrophe" and were probably caused by microperforations of the colon in the course of diverticulosis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Mar, 35(3), 225 - 9
{An insertion mutant of LeuRS with 116 amino acid residues has full activity}; Huang Y et al.; Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases . It consists of 860 amino acid residues and catalyzes the leucylation of tRNA(Leu) . An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C . Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column . The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues . The kinetic parameters of His(6)-LeuRS-C were determined . The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Mar, 35(3), 219 - 24
Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom; Wei JF et al.; An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogeneity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resource Q ion-exchange chromatography . TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis . The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD . The 24 N-terminal amino acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs . Further studies found that TM-LAO inhibited the growth of E . coli, S . aurues and B . dysenteriae . TM-LAO also showed cytotoxicity and platelet aggregation activity . All the biological activities were eliminated by catalase, a H(2)O(2) scavenger . It was shown that these biological effects were possibly due to the formation of H(2)O(2) produced by TM-LAO.

Exp Physiol, 2003 Mar, 88(2), 201 - 8
Effects of endotoxin exposure on cationic amino acid transporter function in ovine peripheral blood mononuclear cells; Clark MF et al.; Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin . Sheep, when exposed to endotoxin, behave in a manner more similar to humans . Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase . Whether this is true in sheep is not known . We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine . PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin . In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways . In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen . The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB . These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase . Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3137 - 42 Epub 2003 Mar 05.
The 1.6-A crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad; Quigley PM et al.; Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins . EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates . The 1.6-A crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate . The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein . A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding . The alpha-beta sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily . The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.

J Med Microbiol, 2003 Mar, 52(Pt 3), 217 - 22
Identification of immunodominant Helicobacter pylori proteins with reactivity to H . pylori-specific egg-yolk immunoglobulin; Shin JH et al.; The importance of hens eggs as a source of specific antibodies (IgY) is well recognized . The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H . pylori infection . However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY . In the present study, the immunodominant proteins of H . pylori with reactivity to H . pylori-specific IgY (IgY-Hp) were identified . IgY obtained from hens immunized with various fractions of H . pylori proteins was isolated and purified, titres of IgY-Hp against H . pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis . Finally, immunodominant H . pylori proteins were identified by LC/MS analysis . IgY obtained 2 months after immunization with H . pylori whole-cell lysate showed the highest antibody titre . Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease beta-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease alpha-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa) . Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H . pylori.

J Biol Chem, 2003 May 16, 278(20), 18638 - 48 Epub 2003 Mar 05.
The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein; Longhi S et al.; The nucleoprotein of measles virus consists of an N-terminal moiety, N(CORE), resistant to proteolysis and a C-terminal moiety, N(TAIL), hypersensitive to proteolysis and not visible as a distinct domain by electron microscopy . We report the bacterial expression, purification, and characterization of measles virus N(TAIL) . Using nuclear magnetic resonance, circular dichroism, gel filtration, dynamic light scattering, and small angle x-ray scattering, we show that N(TAIL) is not structured in solution . Its sequence and spectroscopic and hydrodynamic properties indicate that N(TAIL) belongs to the premolten globule subfamily within the class of intrinsically disordered proteins . The same epitopes are exposed in N(TAIL) and within the nucleoprotein, which rules out dramatic conformational changes in the isolated N(TAIL) domain compared with the full-length nucleoprotein . Most unstructured proteins undergo some degree of folding upon binding to their partners, a process termed "induced folding." We show that N(TAIL) is able to bind its physiological partner, the phosphoprotein, and that it undergoes such an unstructured-to-structured transition upon binding to the C-terminal moiety of the phosphoprotein . The presence of flexible regions at the surface of the viral nucleocapsid would enable plastic interactions with several partners, whereas the gain of structure arising from induced folding would lead to modulation of these interactions . These results contribute to the study of the emerging field of natively unfolded proteins.

J Biol Chem, 2003 May 16, 278(20), 18401 - 7 Epub 2003 Mar 05.
Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC) . Implications for the catalytic mechanism and anti-malaria drug development; Steinbacher S et al.; 2-C-Methyl-d-erythritol 4-phosphate synthase (IspC) is the first enzyme committed to isoprenoid biosynthesis in the methylerythritol phosphate pathway, which represents an alternative route to the classical mevalonate pathway . As it is present in many pathogens and plants, but not in man, this pathway has attracted considerable interest as a target for novel antibiotics and herbicides . Fosmidomycin represents a specific high-affinity inhibitor of IspC . Very recently, its anti-malaria activity in man has been demonstrated in clinical trials . Here, we present the crystal structure of Escherichia coli IspC in complex with manganese and fosmidomycin at 2.5 A resolution . The (N-formyl-N-hydroxy)amino group provides two oxygen ligands to manganese that is present in a distorted octahedral coordination, whereas the phosphonate group is anchored in a specific pocket by numerous hydrogen bonds . Both sites are connected by a spacer of three methylene groups . The substrate molecule, 1-d-deoxyxylulose 5-phosphate, can be superimposed onto fosmidomycin, explaining the stereochemical course of the reaction.

J Biol Chem, 2003 May 16, 278(20), 18649 - 57 Epub 2003 Mar 05.
The small nuclear RNA-activating protein 190 Myb DNA binding domain stimulates TATA box-binding protein-TATA box recognition; Hinkley CS et al.; Human U6 small nuclear RNA (snRNA) gene transcription by RNA polymerase III requires cooperative promoter binding involving the snRNA-activating protein complex (SNAP(c)) and the TATA-box binding protein (TBP) . To investigate the role of SNAP(c) for TBP function at U6 promoters, TBP recruitment assays were performed using full-length TBP and a mini-SNAP(c) containing SNAP43, SNAP50, and a truncated SNAP190 . Mini-SNAP(c) efficiently recruits TBP to the U6 TATA box, and two SNAP(c) subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain . Truncated SNAP190 containing only the Myb DNA binding domain is sufficient for TBP recruitment to the TATA box . Therefore, the SNAP190 Myb domain functions both to specifically recognize the proximal sequence element present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters . The SNAP190 Myb domain also stimulates complex assembly with TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex . Thus, interactions between the DNA binding domains of SNAP190 and TBP at juxtaposed promoter elements define the assembly of a RNA polymerase III-specific preinitiation complex.

Appl Environ Microbiol, 2003 Mar, 69(3), 1840 - 3
Antigenic hepatitis A virus structures may be produced in Escherichia coli; Sanchez G et al.; The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in Escherichia coli . HAV pentamers (14S) self-assembled into capsids (70S) in vitro . The antibodies induced by these structures recognized and neutralized HAV.

Appl Environ Microbiol, 2003 Mar, 69(3), 1759 - 74
DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate; Polen T et al.; In its natural environment, Escherichia coli is exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations . DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH . The adaptive responses to acetate and propionate were similar and involved genes in three categories . First, the RNA levels for chemotaxis and flagellum genes increased . Accordingly, the expression of chromosomal fliC'-'lacZ and flhDC'-'lacZ fusions and swimming motility increased after adaptation to acetate or propionate . Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate . Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response . Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes . The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response of E . coli to either compound.

Appl Environ Microbiol, 2003 Mar, 69(3), 1527 - 31
Fnr is involved in oxygen control of Herbaspirillum seropedicae N-truncated NifA protein activity in Escherichia coli; Monteiro RA et al.; Herbaspirillum seropedicae is an endophytic diazotroph belonging to the beta-subclass of the class Proteobacteria, which colonizes many members of the Gramineae . The activity of the NifA protein, a transcriptional activator of nif genes in H . seropedicae, is controlled by ammonium ions through its N-terminal domain and by oxygen through mechanisms that are not well understood . Here we report that the NifA protein of H . seropedicae is inactive and more susceptible to degradation in an fnr Escherichia coli background . Both effects correlate with oxygen exposure and iron deprivation . Our results suggest that the oxygen sensitivity and iron requirement for H . seropedicae NifA activity involve the Fnr protein.

J Inorg Biochem, 2003 Feb 1, 94(1-2), 155 - 60
The effects of tungstophosphate and tungstosilicate on various stress promoters transformed in Escherichia coli; Tajima Y; Although tungsten is an important material in some industrial and chemical processes, the biological and biochemical effects, including the toxicity, of tungsten compounds are not known well . In this study, a reporter gene assay using special strains of Escherichia coli was performed to investigate the mode of action of two polyoxotungstates, i.e . undecatungstophosphate (PW(11)) and undecatungstosilicate (SiW(11)) . When the bacterial cells were cultured with PW(11), osmY (a stress promoter gene sensitive to osmotic signals) was induced to some extent, while other stress promoters were expressed only slightly . SiW(11) gave similar results, but clpB (an analogue of human heat shock protein) was more strongly induced . It is possible that PW(11) and SiW(11) can produce an osmotic signal at lower concentrations without increasing ionic strength . Since the constituents of PW(11)/SiW(11) (i.e . HPO(4)(2-), SiO(3)(2-), WO(4)(2-)) showed almost no effect, a chemical feature unique to PW(11)/SiW(11) and originating from neither of their constituents, i.e . a polyanionic characteristic, may play an important role in their biological effects.

J Inorg Biochem, 2003 Feb 1, 94(1-2), 94 - 9
Thermodynamic investigation of M-DNA: a novel metal ion-DNA complex; Wettig SD et al.; The thermodynamics of formation of a novel divalent metal ion-DNA complex known as M-DNA have been investigated using an ethidium bromide (EB) fluorescence assay, and with isothermal titration calorimetry . The process of M-DNA formation was observed from the EB assay to be strongly temperature-dependent . The binding of Zn(2+) to calf thymus (42% GC content) and Escherichia coli (50% GC content) DNA at pH 8.5 exhibited an endothermic cooperative binding process at Zn(2+) concentrations of approximately 0.1 mM, indicating an entropy driven process . This binding process is consistent with a site-specific binding interaction, similar in nature to Z-DNA formation; however, the interaction occurs at much lower metal ion concentrations . The enthalpy of M-DNA formation for calf thymus DNA was determined to be 10.5+/-0.7 and 9+/-2 kJ/mbp at DNA concentrations of 100 and 50 microg ml(-1), respectively . An enthalpy of 13+/-3 kJ/mbp was obtained for M-DNA formation for 50 microg ml(-1) E . coli DNA . No evidence of M-DNA formation was observed in either DNA at pH 7.5 with Zn(2+) or at either pH 7.5 or 8.5 with Mg(2+).

Phytochemistry, 2003 Feb, 62(3), 313 - 23
Aromatic and pyrone polyketides synthesized by a stilbene synthase from Rheum tataricum; Samappito S et al.; A cDNA encoding a stilbene synthase, RtSTS, was isolated from the rhizomes of Tatar rhubarb, Rheum tataricum L . (Polygonaceae), a medicinal plant containing stilbenes and other polyketides . Recombinant RtSTS was expressed in E . coli and assayed with acetyl-coenzyme A (CoA), n-butyryl-CoA, isovaleryl-CoA, n-hexanoyl-CoA, cinnamoyl-CoA and p-coumaroyl-CoA as primers of polyketide synthesis . RtSTS synthesized resveratrol and a trace amount of naringenin chalcone from p-coumaroyl-CoA, supporting the enzyme's identification as a resveratrol-type stilbene synthase (EC 2.3.1.95) . Bis-noryangonin and p-coumaroyl triacetic acid lactone (CTAL)-type pyrones were observed in minor amounts in the reaction with p-coumaroyl-CoA and as major products with cinnamoyl CoA . As well, such pyrones, and not aromatic polyketides, were identified as the only products in assays with aliphatic and benzoyl CoA esters . Acetonyl-4-hydroxy-2-pyrone, a pyrone synthesized from acetyl-CoA, was identified as a new product of a stilbene synthase . Using Northern blot analysis, RtSTS transcript was found to be highly expressed in R . tataricum rhizomes, with low transcript levels also present in young leaves . This expression pattern correlated with the occurrence of resveratrol, which was detected in higher amounts in R . tataricum rhizomes compared with leaves and petioles using HPLC . Few stilbene synthases have been found in plants, and the identification of RtSTS provides additional sequence and catalytic information with which to study the evolution of plant polyketide synthases.

Phytochemistry, 2003 Feb, 62(3), 287 - 92
Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase; Lukacin R et al.; Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e . dihydrokaempferol, to the corresponding flavonols (kaempferol) . Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3beta-hydroxylase and flavonol synthase activities to the enzyme . In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate . The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (-)-trans-dihydrokaempferol . The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.

Curr Biol, 2003 Mar 4, 13(5), R183 - 5
Mechanosensitive channels: stress relief; Biggin PC et al.; Bacterial responses to decreasing osmolality involve mechanosensitive channels . The crystal structure has been determined of the small conductance mechanosensitive channel (MscS) from Escherichia coli, providing new insights into mechanical and voltage sensing by this and other channel proteins.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 211 - 3
Construction of a vector plasmid for use in Gluconobacter oxydans; Tonouchi N et al.; A host vector system in Gluconobacter oxydans was constructed . An Acetobacter-Escherichia coli shuttle vector was introduced with the efficiency of 10(4) transformants/microg of DNA . Next, aiming for a self-cloning vector, we found a cryptic plasmid (which we named pAG5) of 5648 bp in G . oxydans strain IFO 3171, and sequenced the nucleotides . The plasmid seemed to have only one open reading flame (ORF) for a possible replication protein . Shuttle vectors of Gluconobacter-E . coli were constructed with the plasmid pAG5 and an E . coli vector, pUC18.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 77 - 82
Identification and characterization of Scp15, a protein from Streptomyces coelicolor A3(2) inducing neurites in PC12 cells; Nakashima S et al.; We previously showed that a fungal protein, p15, induces neurite outgrowth and differentiation of rat pheochromocytoma PC12 cells . We report here the identification and characterization of a protein similar to p15, found in Streptomyces coelicolor A3(2) . This hypothetical protein, tentatively named Scp15, has significant similarity with p15, including conserved positions of four cysteine residues involved in the formation of essential disulfide bonds in p15 . Hexahistidine-tagged recombinant Scp15 proteins were produced in Escherichia coli, purified, and analyzed for their neurite-inducing activity . Although they were less active than p15, they dose-dependently induced neurites and the expression of neurofilament M . Neurite outgrowth by Scp15 was inhibited by nicardipine, suggesting that Scp15 induces neurites via activation of a calcium signaling pathway.

Yonsei Med J, 2003 Feb, 44(1), 58 - 64
Anti-HER2/neu peptide producing vector system for biologic therapy - is it possible to mass-produce the peptide?
Park BW, Kim KS, Heo MK, Lee KS, Kim EK, Kim KS.
A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties . A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential . Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers . We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al . Nat . Biotechnol, 2000, 18:194-198), as a reference peptide . We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17 . Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells . The proliferation inhibition shown by V2 was slightly better than that shown by V1 . Recombinant peptides V2 and V3 were produced on a large scale in an E . coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30% . Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E . coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells . Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

Genes Chromosomes Cancer, 2003 Apr, 36(4), 361 - 74
DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones; Fiegler H et al.; We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays . A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E . coli DNA, a common contaminant of DNA preparations from large insert clones . We chose the three most selective primers for use in printing DNA microarrays . DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E . coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW . The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH . We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes . Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet .

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2316 - 21
CloQ, a prenyltransferase involved in clorobiocin biosynthesis; Pojer F et al.; Ring A (3-dimethylallyl-4-hydroxybenzoic acid) is a structural moiety of the aminocoumarin antibiotics novobiocin and clorobiocin . In the present study, the prenyltransferase involved in the biosynthesis of this moiety was identified from the clorobiocin producer (Streptomyces roseochromogenes), overexpressed, and purified . It is a soluble, monomeric 35-kDa protein, encoded by the structural gene cloQ . 4-Hydroxyphenylpyruvate and dimethylallyl diphosphate were identified as the substrates of this enzyme, with K(m) values determined as 25 and 35 microM, respectively . A gene inactivation experiment confirmed that cloQ is essential for ring A biosynthesis . Database searches did not reveal any similarity of CloQ to known prenyltransferases, and the enzyme did not contain the typical prenyl diphosphate binding site (N/D)DXXD . In contrast to most of the known prenyltransferases, the enzymatic activity was not dependent on the presence of magnesium, and in contrast to the membrane-bound polyprenyltransferases involved in ubiquinone biosynthesis, CloQ did not accept 4-hydroxybenzoic acid as substrate . CloQ and the similar NovQ from the novobiocin producer seem to belong to a new class of prenyltransferases.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2255 - 60
A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein; Hong SH et al.; We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT . The specific labeling takes advantage of two different modification reactions . One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purification system, the other on the observation that one human MT isoform (1b) contains an additional cysteine at position 32 . It is located in the linker region of the molecule, allowing the introduction of a probe between the two domains . An S32C mutation was introduced into hMT-2 . Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the additional cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metalthiolate clusters . MT containing only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT containing only zinc . Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal beta-domain of the protein.

J Bacteriol, 2003 Mar, 185(6), 2046 - 50
Identification of an unknown promoter, OUTIIp, within the IS10R element; Martinez-Garcia E et al.; A novel promoter in IS10R (OUTIIp) has been found in one of its ends in an inverted position relative to promoter pOUT . OUTIIp shows characteristics similar to those of rpoS-dependent promoters such as a gearbox expression pattern . It is under catabolite repression and positively regulated by ppGpp or conditioned media . This opens new challenges in IS10R transposition.

J Bacteriol, 2003 Mar, 185(6), 2042 - 5
Effects of ISSo2 insertions in structural and regulatory genes of the trimethylamine oxide reductase of Shewanella oneidensis; Bordi C et al.; We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration . The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS . Although TorA is not the sole enzyme reducing TMAO in S . oneidensis, growth analysis showed that it is the main respiratory TMAO reductase . Use of a plasmid-borne torE'-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.

J Bacteriol, 2003 Mar, 185(6), 2017 - 21
Definition of the Escherichia coli MC4100 genome by use of a DNA array; Peters JE et al.; We have used an Escherichia coli K-12 whole-genome array based on the DNA sequence of strain MG1655 as a tool to identify deletions in another E . coli K-12 strain, MC4100, by probing the array with labeled chromosomal DNA . Despite the continued widespread use of MC4100 as an experimental system, the specific genetic relationship of this strain to the sequenced K-12 derivative MG1655 has not been resolved . MC4100 was found to contain four deletions, ranging from 1 to 97 kb in size . The exact nature of three of the deletions was previously unresolved, and the fourth deletion was altogether unknown.

J Bacteriol, 2003 Mar, 185(6), 1958 - 66
Molecular analysis of the multiple GroEL proteins of Chlamydiae; Karunakaran KP et al.; Genome sequencing revealed that all six chlamydiae genomes contain three groEL-like genes (groEL1, groEL2, and groEL3) . Phylogenetic analysis of groEL1, groEL2, and groEL3 indicates that these genes are likely to have been present in chlamydiae since the beginning of the lineage . Comparison of deduced amino acid sequences of the three groEL genes with those of other organisms showed high homology only for groEL1, although comparison of critical amino acid residues that are required for polypeptide binding of the Escherichia coli chaperonin GroEL revealed substantial conservation in all three chlamydial GroELs . This was further supported by three-dimensional structural predictions . All three genes are expressed constitutively throughout the developmental cycle of Chlamydia trachomatis, although groEL1 is expressed at much higher levels than are groEL2 and groEL3 . Transcription of groEL1, but not groEL2 and groEL3, was elevated when HeLa cells infected with C . trachomatis were subjected to heat shock . Western blot analysis with polyclonal antibodies raised against recombinant GroEL1, GroEL2, and GroEL3 demonstrated the presence of the three proteins in C . trachomatis elementary bodies, with GroEL1 being present in the largest amount . Only C . trachomatis groEL1 and groES together complemented a temperature-sensitive E . coli groEL mutant . Complementation did not occur with groEL2 or groEL3 alone or together with groES . The role for each of the three GroELs in the chlamydial developmental cycle and in disease pathogenesis requires further study.

J Bacteriol, 2003 Mar, 185(6), 1942 - 50
High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction; Park S et al.; Escherichia coli is generally resistant to H(2)O(2), with >75% of cells surviving a 3-min challenge with 2.5 mM H(2)O(2) . However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H(2)O(2), with <1% surviving the challenge . Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay . Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H(2)O(2) into a hydroxyl radical, the direct effector of DNA damage . The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold . In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed . This action permits free iron to redox cycle rapidly in the presence of H(2)O(2), thereby augmenting the rate at which hydroxyl radicals are formed . During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage . Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants . More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state.

J Bacteriol, 2003 Mar, 185(6), 1886 - 94
Leucine-responsive regulatory protein-mediated repression of clp (encoding CS31A) expression by L-leucine and L-alanine in Escherichia coli; Crost C et al.; CS31A produced by septicemic and diarrheic Escherichia coli belongs to the Pap-regulatory family of adhesive factors, which are under methylation-dependent transcriptional regulation . Common features of operons encoding members of this family include two conserved GATC sites in the upstream regulatory region, and transcriptional regulators homologue to the PapB and PapI proteins . Methylation protection of GATC sites was previously shown to be dependent on the leucine-responsive regulatory protein (Lrp) . Lrp and ClpB, the PapB equivalent, repressed clp basal transcription . A PapI homologue (AfaF) was required together with Lrp to establish the phase variation control, which gave rise to phase-ON cells that expressed CS31A and phase-OFF cells that did not express CS31A . In phase-OFF cells, the GATC(dist) site was methylated and the GATC(prox) site was protected from methylation, whereas in phase-ON cells, the inverse situation was found . Unlike Pap fimbriae, CS31A synthesis was dramatically reduced in media containing L-alanine or L-leucine . L-Alanine prevented the OFF-to-ON switch, locking clp expression in the OFF phase, whereas L-leucine repressed transcription without obvious effect on the switch frequency of phase variation . In phase-variable cells, leucine and alanine promoted methylation of GATC(dist) and methylation protection of GATC(prox), increasing the methylation pattern characteristic of repressed cells . Furthermore, alanine prevented the AfaF-dependent methylation protection of GATC(dist) and thus the appearance of phase-ON cells . In addition, analysis of clp expression in a Lrp-negative background indicated that alanine and leucine also repressed clp transcription by a methylation-independent mechanism.

J Bacteriol, 2003 Mar, 185(6), 1870 - 85
Interactions between the outer membrane ferric citrate transporter FecA and TonB: studies of the FecA TonB box; Ogierman M et al.; Both induction of transcription of the ferric citrate transport genes and transport of ferric citrate by the Escherichia coli outer membrane receptor FecA require energy derived from the proton motive force (PMF) of the inner membrane . The energy is transduced to FecA by the inner membrane complex, TonB, ExbB, and ExbD . Region 160 of TonB and the conserved TonB box of other TonB-dependent receptors are implicated as sites of interaction . In the present study, the postulated TonB box (D(80)A(81)L(82)T(83)V(84)) of FecA was deleted in frame, with a subsequent loss of both FecA functions . DALTV of FecA could be functionally replaced with the core TonB boxes of FhuA (DTITV) and FepA (DTIVV) . Each residue of the TonB box of FecA was sequentially replaced with cysteine residues, and only the D80C replacement showed a loss (reduction) of both FecA functions . A physical interaction between TonB and FecA was demonstrated using both in vivo site-specific disulfide bond cross-linking and nonspecific formaldehyde (FA) cross-linking . Pairwise combinations of FecA (DALTV)/Cys substitutions were cross-linked via disulfide bond formation with TonBQ160C, TonBQ162C, and TonBY163C . Unexpectedly, this cross-linking was not enhanced by substrate (ferric citrate) . In contrast, the TonB-FecA interaction was enhanced by ferric citrate in the FA-cross-linking assay . Energy derived from the PMF was not required for the TonB-FecA interaction in either the disulfide- or FA-cross-linking assay . TonB/CysExbB/ExbD(D25N) was still able to cross-link with the FecA (DALTV)/Cys derivatives in a tonB tolQ background, even though ExbD25N renders the TonB/ExbBD complex nonfunctional (V . Braun, S . Gaisser, C . Herrmann, K . Kampfenkel, H . Killmann, and I . Traub, J . Bacteriol . 178:2836-2845, 1996) . TonB cross-linked to FecA via FA was not inhibited by either carbonylcyanide-m-chlorophenylhydrazone or 1 mM 2,4-dinitrophenol, which dissipate the electrochemical potential of the cytoplasmic membrane and disrupt both FecA functions . The studies shown here demonstrate the significance of the TonB box for FecA functions and are consistent with the view that it is the structure and not the sequence of the TonB box that is important for activity . Demonstrated here for the first time is the physical interaction of TonB and FecA, which is enhanced by ferric citrate.

J Bacteriol, 2003 Mar, 185(6), 1817 - 24
Identification of a novel membrane-associated gene product that suppresses toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein; Kim PD et al.; The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli . The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form . The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E . coli . Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added . Recently, the Dp was independently shown to help prevent overinitiation in E . coli and was termed Hda (S . Kato and T . Katayama, EMBO J . 20:4253-4262, 2001).

J Bacteriol, 2003 Mar, 185(6), 1783 - 95
The activator of GntII genes for gluconate metabolism, GntH, exerts negative control of GntR-regulated GntI genes in Escherichia coli; Tsunedomi R et al.; Gluconate is one of the preferred carbon sources of Escherichia coli, and two sets of gnt genes (encoding the GntI and GntII systems) are involved in its transport and metabolism . GntR represses the GntI genes gntKU and gntT, whereas GntH was previously suggested to be an activator for the GntII genes gntV and idnDO-gntWH . The helix-turn-helix residues of the two regulators GntR and GntH exhibit extensive homologies . The similarity between the two regulators prompted analysis of the cross-regulation of the GntI genes by GntH . Repression of gntKU and gntT by GntH, as well as GntR, was indeed observed using transcriptional fusions and RNA analysis . High GntH expression, from cloned gntH or induced through 5-ketogluconate, was required to observe repression of GntI genes . Two GntR-binding elements were identified in the promoter-operator region of gntKU and were also shown to be the target sites of GntH by mutational analysis . However, the GntI genes were not induced by gluconate in the presence of enhanced amounts of GntH, whereas repression by GntR was relieved by gluconate . The repression of GntI genes by GntH is thus unusual in that it is not relieved by the availability of substrate . These results led us to propose that GntH activates GntII and represses the GntI genes in the presence of metabolites derived from gluconate, allowing the organism to switch from the GntI to the GntII system . This cross-regulation may explain the progressive changes in gnt gene expression along with phases of cell growth in the presence of gluconate.

J Biol Chem, 2003 May 23, 278(21), 19378 - 86 Epub 2003 Mar 04.
Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor; Imamura H et al.; Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic alpha-1,4-glucan (cycloamylose) from linear amylose . We determined the crystal structure of TLGT with and without an inhibitor, acarbose . TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (beta/alpha)7 barrel fold, and a C-terminal domain (domain II), which has a twisted beta-sandwich fold . In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT . The acarbose-bound structure also clarified that Glu123 and Asp214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding . It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft . Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi alpha-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38 . Furthermore, the catalytic residue of the two enzymes is located in the same position . These observations suggest that families 57 and 38 evolved from a common ancestor.

J Biol Chem, 2003 May 9, 278(19), 17093 - 102 Epub 2003 Mar 04.
Requirement of dimerization for RNA editing activity of adenosine deaminases acting on RNA; Cho DS et al.; Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA . Three vertebrate ADAR gene family members, ADAR1, ADAR2, and ADAR3, have been identified . The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1 . Homodimerization is essential for the enzyme activity of those cytidine deaminases . In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography . Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form . No heterodimer complex formation among different ADAR gene family members was detected . Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex . Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization . Homodimer formation may be necessary for ADAR to act as active deaminases . Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing.

J Biol Chem, 2003 May 9, 278(19), 17075 - 83 Epub 2003 Mar 04.
Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei . In vitro RNA interference and in vivo inhibition studies; Montalvetti A et al.; We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of Trypanosoma brucei . The protein (TbFPPS) is an attractive target for drug development because the growth of T . brucei has been shown to be inhibited by analogs of its substrates, the nitrogen containing bisphosphonates currently in use in bone resorption therapy . The protein predicted from the nucleotide sequence of the gene has 367 amino acids and a molecular mass of 42 kDa . Several sequence motifs found in other FPPSs are present in TbFPPS, including an 11-mer peptide insertion present also in the Trypanosoma cruzi FPPS . Heterologous expression of TbFPPS in Escherichia coli produced a functional enzyme that was inhibited by several nitrogen-containing bisphosphonates, such as pamidronate and risedronate . Risedronate was active in vivo against T . brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective . The essential nature of TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene . Expression of TbFPPS double-stranded RNA in procyclic trypomastigotes caused specific degradation of mRNA . After 4 days of RNAi, the parasite growth rate declined and the cells subsequently died . Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in this case was leaky and mRNA levels and parasites recovered with time . Molecular modeling and structure-activity investigations of enzyme and in vitro growth inhibition data resulted in similar pharmacophores, further validating TbFPPS as the target for bisphosphonates . These results establish that FPPS is essential for parasite viability and validate this enzyme as a target for drug development.

Genetics, 2003 Feb, 163(2), 485 - 94
RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli; Ivancic-Bace I et al.; The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli . One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA . There are, however, previously published contradictory data on the recombination proficiency of this mutant . In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient . A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme . We tested this hypothesis by using three in vivo assays . We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants . We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme . According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.

Genetics, 2003 Feb, 163(2), 475 - 83
Molecular evolution of the Escherichia coli chromosome . VI . Two regions of high effective recombination; Milkman R et al.; Two 6- to 8-min regions, centered respectively near 45 min (O-antigen region) and 99 min (restriction-modification region) on the Escherichia coli chromosome, display unusually high variability among 11 otherwise very similar strains . This variation, revealed by restriction fragment length polymorphism (RFLP) and nucleotide sequence comparisons, appears to be due to a great local increase in the retention frequency of recombinant replacements . We infer a two-step mechanism . The first step is the acquisition of a small stretch of DNA from a phylogenetically distant source . The second is the successful retransmission of the imported DNA, together with flanking native DNA, to other strains of E . coli . Each cell containing the newly transferred DNA has a very high selective advantage until it reaches a high frequency and (in the O-antigen case) is recognized by the new host's immune system . A high selective advantage increases the probability of retention greatly; the effective recombination rate is the product of the basic recombination rate and the probability of retention . Nearby nucleotide sequences clockwise from the O-antigen (rfb) region are correlated with specific O antigens, confirming local hitchhiking . Comparable selection involving imported restriction endonuclease genes is proposed for the region near 99 min.

Genome Res, 2003 Mar, 13(3), 476 - 84
A highly efficient recombineering-based method for generating conditional knockout mutations; Liu P et al.; Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases . This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies . Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations . This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA . Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology . We also make use of several new E . coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter . We also describe two new Neo selection cassettes that work well in both E . coli and mouse ES cells . Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk . This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

Genome Res, 2003 Mar, 13(3), 422 - 7
The phylogenetic extent of metabolic enzymes and pathways; Peregrin-Alvarez JM et al.; The evolution of metabolic enzymes and pathways has been a subject of intense study for more than half a century . Yet, so far, previous studies have focused on a small number of enzyme families or biochemical pathways . Here, we examine the phylogenetic distribution of the full-known metabolic complement of Escherichia coli, using sequence comparison against taxa-specific databases . Half of the metabolic enzymes have homologs in all domains of life, representing families involved in some of the most fundamental cellular processes . We thus show for the first time and in a comprehensive way that metabolism is conserved at the enzyme level . In addition, our analysis suggests that despite the sequence conservation and the extensive phylogenetic distribution of metabolic enzymes, their groupings into biochemical pathways are much more variable than previously thought.

Am J Physiol Endocrinol Metab, 2003 Jul, 285(1), E106 - 15 Epub 2003 Mar 04.
Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation; Moon B et al.; Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes . However, its role in skeletal muscle metabolism is unknown . In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene . Transfected clones expressed intracellular resistin, which was released in the medium . Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake . The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane . Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity . Insulin-stimulated phosphorylation of Akt/protein kinase B-alpha, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin . Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling . We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.

Cell Signal, 2003 Apr, 15(4), 347 - 54
CaMBOT: profiling and characterizing calmodulin-binding proteins; O'Day DH; Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin-binding proteins; CaMBPs) that carry out its critical signalling functions . In spite of the central importance of CaM in Ca(2+)-mediated signal transduction pathways in all eukaryotes, many CaMBPs remain to be identified and characterized . SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin-binding Overlay Technique, CaMBOT) is a valuable method for following behavioural, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study . CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms . Recently, the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators . To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins . This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome-proteins regulated by calmodulin.

BMC Microbiol . 2003 Jan 31;3(1):2 {Epub ahead of print}
Molecular cloning and characterization of Escherichia coli K12 ygjG gene; Samsonova NN et al.; BACKGROUND: Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source . Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into gamma-aminobutyrate . But genes coding for these enzymes have not been identified so far . RESULTS: The 1.8-kbp DNA fragment containing E . coli K12 ygjG gene with aer-ygjG intergenic region was examined . It was found that the fragment contains sigma54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa . The cytidine (C) residue localized 10 bp downstream of the sigma54 promoter sequence was identified as the first mRNA base . The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start . The YgjG was expressed as a his6-tag fused protein and purified to homogeneity . The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29) . The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively . The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0 . CONCLUSION: Expression of E . coli K12 ygjG coding region revealed sigma54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity . The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.

Biochem J, 2003 Jun 1, 372(Pt 2), 503 - 15
The structure and regulation of the human and mouse matrix metalloproteinase-21 gene and protein; Marchenko GN et al.; Matrix metalloproteinases (MMPs) play key roles in tissue remodelling under normal development and, especially, in diseases ranging from malignancies to stroke . We cloned and thoroughly characterized the novel human and mouse MMP gene encoding MMP-21 . MMP-21 is the last uncharacterized MMP coded by the human genome . Human and mouse MMP-21 is the orthologue of Xenopus laevis X-MMP . The latent proenzyme of MMP-21 (569 amino acid residues) consists of the prodomain, the catalytic domain and the haemopexin-like domain, and is potentially capable of being activated in its secretory pathway to the extracellular milieu by furin-like proprotein convertases . Human MMP-21 is the probable target gene of the Wnt pathway . In addition, the expression of MMP-21 is controlled uniquely by Pax and Notch transcription factors known to be critical for organogenesis . MMP-21 is expressed transiently in mouse embryogenesis and increased in embryonic neuronal tissues . Our observations clearly indicate that there is an important specific function for MMP-21 in embryogenesis, especially in neuronal cells.

J Am Chem Soc, 2003 Mar 12, 125(10), 2902 - 12
Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics; Clore GM et al.; A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure . The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation . The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations . The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers . For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A . For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor . The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality . This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.

Laryngoscope, 2003 Mar, 113(3), 393 - 400
Potential biomarkers for head and neck squamous cell carcinoma; Brown JJ et al.; OBJECTIVE: The purpose of the study was twofold: 1) to search for potential biomarkers that were overexpressed in cell lines that could represent both a clinical premalignant (immortalized) and a malignant state, and 2) to attempt to correlate metallothionein gene expression with clinical outcome in laryngeal carcinoma . STUDY DESIGN: A series of in vitro experiments were used to unearth differentially expressed genes among normal, immortalized and tumorigenic cell lines . Secondarily, a retrospective analysis was undertaken . METHODS: Differential display analysis was conducted to identify differentially expressed genes between human papillomavirus-infected immortalized HOK16B and benzo{ }pyrene-derived tumorigenic cell line, HOK16B-BaP-T . The cell-specific expressions were examined by Northern blot analysis and compared with other known immortalized and cancer cell lines . Immunohistochemical staining was also conducted to localize metallothionein (MT I/II) protein expression among the different cell lines studied . A retrospective analysis of laryngeal specimens from archival tissues of 29 cancer patients who underwent primary surgical resection was also undertaken after immunohistochemical staining . RESULTS: Twenty-one differentially expressed complementary cDNA clones, both novel and known, were identified using the differential display analysis . Northern blot analysis confirmed that clone 6 hybridized to a 1.6-kb RNA in HOK16B-Bap-T cell line . Clone 4 showed decreased expression in immortalized and cancer cell compared with NHOK . MT I/II transcript was observed in HOK16B, which was further elevated in HOK16B-Bap-T . Retrospective analysis showed that high immunoreactivity to MT I/II in surgically resected laryngeal cancer specimen correlated with increased frequency of recurrence within 2 years of surgery . CONCLUSION: These findings suggest that clone 4 may potentially function as a tumor suppressor gene, which may be significant in tumor progression and invasion . Clone 6 may participate in viral-mediated oncogenic transformation of normal cells . Clone 6 may also have potential as a tumor maker differentiating normal from malignant tissue, as in the determination of surgical resection margins . MT I/II gene product may serve as a prognostic biomarker for laryngeal squamous cell carcinoma . The differentially expressed genes and gene products may serve as sensitive biomarkers for improved early detection, diagnosis, and prognosis of head and neck squamous cell carcinoma.

J Biol Chem, 2003 May 16, 278(20), 18330 - 5 Epub 2003 Mar 03.
Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains; Muller C et al.; The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied . The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains . It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step . The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways . In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers . As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein . These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers . The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process . Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.

J Struct Biol, 2003 Feb, 141(2), 103 - 14
A comparative three-dimensional model of the carboxy-terminal domain of the lambda repressor and its use to build intact repressor tetramer models bound to adjacent operator sites; Chattopadhyaya R et al.; A model for residues 93-236 of the lambda repressor (1gfx) was predicted, based on the UmuD(') crystal structure, as part of four intact repressor molecules bound to two adjacent operator sites . The structure of region 136-230 in 1gfx was found to be nearly identical to the independently determined crystal structure of the 132-236 fragment, 1f39, released later by the PDB . Later, two more tetrameric models of the lambda repressor tetramer bound to two adjacent operator sites were constructed by us; in one of these, 1j5g, the N-domain and C-domain coordinates and hence monomer-monomer and dimer-dimer interactions are almost the same as in 1gfx, but the structure of the linker region is partly based on the linker region of the LexA dimer in 1jhe; in the other, 1lwq, the crystalline tetramer for region 140-236 has been coopted from the crystal structure deposited in 1kca, the operator DNA and N-domain coordinates of which are same as those in 1gfx and 1j5g, but the linker region is partly based on the LexA dimer structures 1jhe and 1jhh . Monomer-monomer interactions at the same operator site are stabilized by exposed hydrophobic side chains in beta-strands while cooperative interactions are mostly confined to beta(6) and some adjacent residues in both 1gfx and 1j5g . Mutational data, existence of a twofold axis relating two C-domains within a dimer, and minimization of DNA distortion between adjacent operator sites allow us to roughly position the C-domain with respect to the N-domain for both 1gfx and 1j5g . The study correlates these models with functional, biochemical, biophysical, and immunological data on the repressor in the literature . The oligomerization mode observed in the crystal structure of 132-236 may not exist in the intact repressor bound to the operator since it is shown to contradict several published biochemical data on the intact repressor.

Burns, 2003 Mar, 29(2), 133 - 8
Escherichia coli endotoxin enhances acute renal failure in rats after thermal injury; Yamaguchi H et al.; This study was designed to evaluate the burned rat model to determine whether there are any differences in endotoxin-sensitive kidney functions between an infant rat (10-day-old pup) and an adult rat (10-week-old rat) . Renal failure was observed in the infant burned rat and histological changes showed the adhesion of inflammatory cells in the glomerular capillaries and vacuolar changes in the renal proximal tubular cell . A horseradish peroxidase (HRP) tracer experiment suggested that the intestinal barrier damage of the infant burned rat was more severe than that of the adult burned rat . Therefore, more bacterial translocation of the intestinal flora, rich in endotoxin, might be expected in the infant versus the adult rats . Renal failure was not observed in the adult burned rat, so we investigated to determine the effects of endotoxin on the kidney function of the adult burned rat with low lethal lipopolysaccharide (LPS) or carrageenan (CAR) . CAR is known to increase sensitivity to the lethal effects of endotoxin in rodents . Our present data demonstrated that renal failure was observed in the LPS- or CAR-treated adult burned rat and LPS- and CAR-treated adult rat (non-burned) . These results show the possibility that endotoxin enhances renal failure in a burned rat model and provide additional support for the hypothesis that postburn renal failure is mediated, in part, by endotoxin associated with bacterial translocation.

Vaccine, 2003 Mar 28, 21(13-14), 1445 - 54
Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives; Petrovska L et al.; Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood . This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells . Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells . LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells . LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants . LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran . Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments . This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules.

Vaccine, 2003 Mar 28, 21(13-14), 1409 - 14
Development of a foot-and-mouth disease NSP ELISA and its comparison with differential diagnostic methods; Kweon CH et al.; The gene encoding the nonstructural protein (NSP) of O/SKR/2000 foot-and-mouth disease virus (FMDV) was constructed to express under the polyhedron promoter of baculovirus . The expression of NSP was confirmed by indirect immunofluorescence assay (IFA) and Western blotting . The expressed NSP was applied as a diagnostic antigen for indirect-trapping ELISA (I-ELISA) . An I-ELISA using monoclonal antibody (Mab) against 3A as trapping antibody was developed to differentiate infected from vaccinated cattle.The diagnostic efficiency of Mab linked I-ELISA was compared and evaluated with baculovirus expressed 3ABC I-ELISA from USDA and Mab (3A) linked E . coli expressed 3ABC I-ELISA from IZSLE through retrospective sero-surveillance . Compared with the two different I-ELISA methods, Mab (3A) linked I-ELISA using baculovirus expressed NSP showed the same level of sensitivity and specificity, indicating that this method is suitable for a differential diagnostic method in cattle.

Biochim Biophys Acta, 2003 Mar 6, 1557(1-3), 109 - 17
Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin alpha; Markova OV et al.; Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones . On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis {FEBS Lett . 467 (2000) 150} . It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other . In this paper, such an interaction is shown . (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay . (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins . The masses increase when the cytochrome c/tPT ratio increases . High concentration of KCl prevents the complex formation . (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited . (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase . Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant . A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.

Biochim Biophys Acta, 2003 Mar 6, 1557(1-3), 67 - 76
The menD and menE homologs code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate synthase and O-succinylbenzoic acid-CoA synthase in the phylloquinone biosynthetic pathway of Synechocystis sp . PCC 6803; Wade Johnson T et al.; The genome of the cyanobacterium Synechocystis sp . PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid-CoA ligase in the menaquinone biosynthetic pathway . In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I . The menD(-) and menE(-) strains were generated, and both were found to lack phylloquinone . Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl-CoA . Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700(+) {F(A)/F(B)}(-) backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A(1) site of PS I . At a light intensity of 40 microE m(-2) s(-1), the menD(-) and menE(-) mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type . Both of which are sensitive to high light intensities . Low-temperature fluorescence studies show that in the menD(-) and menE(-) mutants, the ratio of PS I to PS II is reduced relative to the wild type . Whole-chain electron transfer rates in the menD(-) and menE(-) mutant cells are correspondingly higher on a chlorophyll basis . The slower growth rate and high-light sensitivity of the menD(-) and menE(-) mutants are therefore attributed to a lower content of PS I per cell.

Mol Biochem Parasitol, 2003 Feb, 126(2), 251 - 62
Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi; Caceres AJ et al.; The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli . Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human . It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins . The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV . The natural enzyme was purified to homogeneity from T . cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form . Its kinetic properties were compared with those of the purified recombinant protein . Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase . However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively) . None of these enzymes presented activity with fructose . As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P) . In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM) .

Mol Biochem Parasitol, 2003 Feb, 126(2), 231 - 8
Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs; Loyevsky M et al.; Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species . We have previously demonstrated that erythrocyte P . falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status . In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs . To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E . coli, which carries extra copies of tRNA genes usually found in organisms such as P . falciparum whose genome is (A+T)-rich . The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin . We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria . In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE . The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding . Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels . Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa .

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 620 - 4
Production of recombinant xenotransplantation antigen in Escherichia coli; Bettler E et al.; The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug . As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection . An E . coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA . This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 469 - 75
Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion; Wang E et al.; Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins . They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV . Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus . However, in other members, only HR2 gives strong inhibition whereas HR1 does not . Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately . Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known . In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed . It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 442 - 7
Predicted molecular structure of the mammalian cell entry protein Mce1A of Mycobacterium tuberculosis; Das AK et al.; The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells . The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading . Mce1A, as the model predicts, is an alpha/beta protein consisting of two major (alpha and beta) domains, connected by a long alpha helix . The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn . The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region . A mouse monoclonal antibody, TB1-5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells . We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD . Upstream flanking residues, KRR also contributed to binding . Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake . The epitope was located at the surface of the Mce1A model at the distal beta strand-loop region in the beta domain . The localization of this epitope in the model confirms its potential role in promoting uptake of M . tuberculosis in host cells.

Biol Psychiatry, 2003 Mar 1, 53(5), 361 - 75
Studies characterizing 60 kda autoantibodies in subjects with schizophrenia; Wang XF et al.; BACKGROUND: Studies suggest that schizophrenic patients have an increased prevalence of serum antibodies to neuroblastoma cell proteins migrating at 60 kilodaltons (kDa) . We present work identifying and characterizing 60 kDa antigen-antibody interactions . METHODS: Sera from schizophrenic subjects and normal volunteers were screened by Western blotting . Proteins migrating at 60 kDa were characterized by two-dimensional gel electrophoresis and indirect immunofluorescent staining of human epithelial cell (HEp-2) slides . Human brain and bladder cell complementary deoxyribonucleic acid libraries were screened with immunoaffinity-purified antibodies . Complementary deoxyribonucleic acid clones were sequenced and compared with published databases . Proteins were generated by in vitro transcription/translation and expression in an Escherichia coli BL21 system . Immunoprecipitation and immunohistochemistry studies were performed . RESULTS: Fifteen percent (17/117) of schizophrenic subjects and 3% (2/62) of normal volunteers had autoantibodies that reacted with 60 kDa proteins {chi(2)(1) = 4.4, p =.037} . Five percent of subjects had autoantibodies directed against 60 kDa heat shock protein (HSP60) {chi(2)(1) = 3.3, p =.100) . Two-dimensional gel electrophoresis identified 13 different proteins migrating at 60 kDa; 5 were splice variants of HSP60, and 2 corresponded with a protein associated with MYC (PAM) . CONCLUSIONS: There is an increased prevalence of autoantibodies that bind to proteins migrating at 60 kDa in subjects with schizophrenia . Potential target antigens include HSP60 and PAM.

Diagn Microbiol Infect Dis, 2003 Feb, 45(2), 97 - 105
Development and evaluation of genotypic assays for the detection and characterization of enterotoxigenic Escherichia coli; Steinsland H et al.; We developed and evaluated a method to genotypically identify enterotoxigenic Escherichia coli (ETEC) and to characterize these organisms with respect to 18 of 21 known colonization factors (CFs) . The method, which is based on polynucleotide DNA-DNA colony hybridization, includes a pooled toxin probe assay to identify ETEC, and individual probe assays to detect the enterotoxins STp, STh, and LT, and the CFs CFA/I, CS1-CS8, CS12-CS15, CS17-CS19, CS21, and CS22 . We evaluated the pooled toxin probe assay during a cohort study of childhood diarrhea, and the individual probe assays against 33 reference strains and 92 clinical ETEC isolates . There was close to a complete agreement between the pooled toxin probe assay and the individual toxin probe assays, and between the individual CF probe assays and the corresponding phenotypic assays.

Bioorg Med Chem, 2003 Mar 20, 11(6), 869 - 73
Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase; Fishlock D et al.; Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized . These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS) . Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity . Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS . These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS) . Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS . These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.

J Mol Biol, 2003 Mar 14, 327(1), 239 - 49
FRET-based in vivo screening for protein folding and increased protein stability; Philipps B et al.; Fluorescence resonance energy transfer (FRET) was used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the cytoplasm of Escherichia coli . The system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue-fluorescent protein (BFP) to the N terminus of protein X . Efficient FRET from BFP to GFP in the ternary fusion protein is observed in vivo only when protein X is folded and brings BFP and GFP into close proximity, while FRET is lost when BFP and GFP are far apart due to unfolding or intracellular degradation of protein X . The screening system was validated by identification of antibody V(L) intradomains with increased thermodynamic stabilities from expression libraries after random mutagenesis, bacterial cell sorting, and colony screening.

J Mol Biol, 2003 Mar 14, 327(1), 183 - 91
Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy; Inobe T et al.; We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique . With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time . The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence . Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C . Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process . The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.

J Mol Biol, 2003 Mar 14, 327(1), 7 - 18
Spontaneous hotspot mutations resistant to mismatch correction in Escherichia coli: transcription-dependent mutagenesis involving template-switching mechanisms; Yoshiyama K et al.; The generation and stabilization of spontaneous mutations are affected by many factors, including the accuracy of DNA replication, the generation of spontaneous DNA lesions, and the capacity of mutation-avoidance systems . However, little is known about the causes of spontaneous mutations in cells with fully active mutation-avoidance systems . Using the rpsL forward mutation assay, we previously found that the directionality of replication fork movement significantly affects spontaneous mutagenesis in Escherichia coli . In particular, sequence substitutions and a hotspot type of single-base frameshift, both of which are caused by quasipalindrome-directed mutagenesis, appeared to depend on the directionality of the replication fork . These mutations are also resistant to post-replicative mismatch correction . Here, we show that the level of transcription of the rpsL gene strongly affects spontaneous mutagenesis at two mutational hotspot sites in the target sequence, one for a T-->G base substitution and the other for a+1 single-base frameshift . Mutation frequencies at the hotspot sites were below a detectable level when the transcription of the target sequence was tightly suppressed, but were dramatically increased when the target sequence was highly transcribed . Both of the hotspot mutations were also dependent on the directionality of the replication fork and were caused by quasipalindrome-directed mutagenesis . The frequencies of the hotspot mutations were unchanged in a mismatch-repair deficient strain, indicating that the hotspot mutations are resistant to the mismatch correction . Based on these findings, we propose a novel mutagenic process for these hotspot mutations that depends on transcription and involves template-switching mechanisms induced by spontaneous DNA lesions.

J Comput Biol, 2002, 9(6), 761 - 73
Occurrence probability of structured motifs in random sequences; Robin S et al.; The problem of extracting from a set of nucleic acid sequences motifs which may have biological function is more and more important . In this paper, we are interested in particular motifs that may be implicated in the transcription process . These motifs, called structured motifs, are composed of two ordered parts separated by a variable distance and allowing for substitutions . In order to assess their statistical significance, we propose approximations of the probability of occurrences of such a structured motif in a given sequence . An application of our method to evaluate candidate promoters in E . coli and B . subtilis is presented . Simulations show the goodness of the approximations.

Am J Transplant, 2003 Mar, 3(3), 324 - 7
Thrombotic micro-angiopathy with sirolimus-based immunosuppression: potentiation of calcineurin-inhibitor-induced endothelial damage?
Robson M, Cote I, Abbs I, Koffman G, Goldsmith D.
Thrombotic microangiopathy is a rare but important finding in the context of organ transplantation . Acute renal insufficiency in the setting of hemolysis and thrombocytopenia, a triad that constitutes 'hemolytic uremic syndrome', can be associated with, or triggered by, conditions such as verocytotoxin-producing Escherichia coli, viral infections, malignant hypertension, scleroderma, allograft rejection, lupus erythematosus, pregnancy, and medications including mitomycin C, calcineurin inhibitors, and oral contraceptives . After renal transplantation, it can occur, as either a de novo episode, or recurrent disease . Calcineurin inhibitors have long been associated with post-transplantation thrombotic microangiopathy . Sirolimus has been used as a primary immunosuppressant in patients transplanted with a history of earlier hemolytic-uremic syndrome, and also as rescue therapy in patients with calcineurin-inhibitor-associated thrombotic microangiopathy . We describe four cases where there was significant thrombotic microangiopathy in the context of contemporaneous or contiguous calcineurin inhibitor and sirolimus usage . As the intrarenal cyclosporin concentration is thought to be significantly elevated when cyclosporin and sirolimus are used together, this may explain these findings, and mandates caution in their co-administration.

Biochem J, 2003 May 15, 372(Pt 1), 253 - 61
Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus; Kanai A et al.; We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method . One gene product, which we named FAU-1 (P . furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence . The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced . The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids . The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli . Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand . Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer . These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

Biochemistry, 2003 Mar 11, 42(9), 2708 - 19
Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB; Ugulava NB et al.; Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin . To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin . The enzyme from Escherichia coli is purified as a dimer that contains one {2Fe-2S}2+ cluster per monomer and can be reconstituted in vitro to contain an additional {4Fe-4S}2+ cluster per monomer . Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface . To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase . We find that both the {2Fe-2S}2+ cluster and the {4Fe-4S}2+ cluster must be present for tight substrate binding . Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet . The stoichiometry of binding is ca . 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer . AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at approximately 400 nm that we attribute to changes in the coordination environment of the {4Fe-4S}2+ cluster . Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex . This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB . Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5'-deoxyadenosyl radicals in the absence of the correct substrate.

J Dairy Sci, 2003 Jan, 86(1), 146 - 51
Opsonic activity of serum and whey from cows immunized with the ferric citrate receptor; Wise AJ et al.; The effects of immunizing dairy cows with the ferric citrate receptor, FecA, on the opsonic activity of serum and whey were measured in a phagocytosis assay . Fifteen cows were assigned to five blocks of three cows based on date of expected parturition . Cows within a block were randomly assigned to one of three treatments: 1) FecA immunization, 2) immunization with a commercially available Escherichia coli J5 bacterin, and 3) unimmunized controls . Cows were challenged at approximately 21 DIM by intramammary infusion of E . coli 727 into one mammary quarter . Escherichia coli 727 were opsonized for the phagocytosis assay with either 10% heat-inactivated serum or 50% heat-inactivated whey collected from each cow at calving, immediately before challenge and 7 d after challenge . Cows immunized with FecA or the E . coli J5 bacterin had increased IgG titers against FecA and E . coli 727 compared with unimmunized control cows . However, sera and whey collected from cows immunized with FecA did not enhance opsonization of E . coli 727 compared with sera and whey from control cows . Immunization with the E . coli J5 bacterin increased opsonization of sera greater than immunization with FecA . Immunoglobulin M antibody titer against E . coli 727 in whey and phagocytic indexes were positively correlated . The phagocytic index of whey immediately before challenge and 7 d after challenge were negatively associated with peak bacterial counts in mammary quarters challenged with E . coli 727 . Results of the current trial suggest that the immune response resulting from immunization with FecA did not enhance opsonization and in vitro phagocytosis of E . coli 727.

J Dairy Sci, 2003 Jan, 86(1), 133 - 7
Effect of immunoglobulin G from cows immunized with ferric citrate receptor (FecA) on iron uptake by Escherichia coli; Takemura K et al.; The effects of immunoglobulin (Ig) G from cows immunized with the ferric citrate receptor (FecA) on iron uptake by Escherichia coli were investigated . Receptor FecA was purified from E . coli UT5600/pSV66 . Cows were immunized with 400 microg purified FecA three times at 21 d intervals during late lactation and the nonlactating period . Immunoglobulin G was purified by protein G affinity chromatography from colostral whey from cows immunized with FecA and from unimmunized control cows . The purified IgG from FecA immunized cows had higher IgG titers against FecA compared with control IgG . Fifteen E . coli isolated from intramammary infections and E . coli UT5600/pSV66 were grown in an iron-depleted medium containing 1 mM citrate to induce FecA . The bacterial cells were mixed with 0, 2, and 4 mg/ml purified IgG, and 55Fe was added to the assay . After 5, 10, and 15 min incubations at 37 degrees C, samples were passed through 0.45-pm pore size filters . Filters were washed with saline three times, and the radioactivity of 55Fe taken up by the bacterial cells on the filters was measured by a liquid scintillation counter . The measurements were expressed as numbers of 55Fe atoms per colony-forming unit and transformed to log10 . The assay was repeated three times for each isolate in a partially balanced incomplete block design . The presence of IgG decreased 55Fe uptake by E . coli mastitis isolates and E . coli UT5600/pSV66 . Anti-FecA IgG reduced 55Fe uptake by E . coli greater than IgG from unimmunized cows.

J Oral Sci, 2002 Dec, 44(3-4), 155 - 9
The effect of parachlorophenol and camphorated parachlorophenol on nitric oxide production by a murine macrophage cell line, RAW264.7; Barid I et al.; The aim of this study was to determine the effect of parachlorophenol (PCP) and camphorated parachlorophenol (CMCP) on nitric oxide (NO) production by a murine macrophage cell line, RAW264.7 . The cells were incubated on plastic disks with either PCP or CMCP . Plastic adherent and nonadherent cells were subsequently stimulated with recombinant mouse IFN-gamma or bacterial lipopolysaccharide (LPS) . Nitric oxide (NO) levels detected from the culture supernatants were determined by the Griess reaction . The results showed that PCP and CMCP diluted at 10(-1) but not at 10(-3) suppressed NO production by both plastic adherent and nonadherent cells, suggesting that both phenolic compounds may suppress NO production by murine macrophages in a dose-dependent manner.

Nat Rev Cancer, 2003 Mar, 3(3), 169 - 78
RecQ helicases: caretakers of the genome; Hickson ID; RecQ helicases are highly conserved from bacteria to man . Germline mutations in three of the five known family members in humans give rise to debilitating disorders that are characterized by, amongst other things, a predisposition to the development of cancer . One of these disorders--Bloom's syndrome--is uniquely associated with a predisposition to cancers of all types . So how do RecQ helicases protect against cancer? They seem to maintain genomic stability by functioning at the interface between DNA replication and DNA repair.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3197 - 202 Epub 2003 Feb 28.
Watching proteins fold one molecule at a time; Rhoades E et al.; Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape . We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique . Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques . The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels . Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity . The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution . This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape . The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete . These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.

Pediatr Res, 2003 Apr, 53(4), 679 - 83 Epub 2003 Jan 29.
Intraamniotic endotoxin increases lung antioxidant enzyme activity in preterm lambs; Sosenko IR et al.; Proinflammatory stimulation resulting from intraamniotic endotoxin improves lung function, increases surfactant protein mRNA expression and protein content, increases alveolar and lung saturated phosphatidylcholine pools, and accelerates lung morphometric maturation in fetal sheep . The mechanism for induction of lung maturation does not involve an increase in fetal cortisol . The effect of endotoxin on the maturation of a different lung system, the antioxidant enzyme (AOE) system, has not been examined . Therefore, we hypothesized that intraamniotic endotoxin would produce acceleration of AOE activity in fetal sheep at similar doses and schedule of administration to those producing lung functional and surfactant maturation . In a dose-response study, intraamniotic injections of 1, 4, 20, or 100 mg of Escherichia coli 055:beta5 endotoxin were administered 7 d before preterm delivery of sheep at 125 d gestation . In a study examining time interval of administration before delivery, 20 mg of endotoxin was injected at either 1-, 2-, 4-, 7-, or 15-d intervals before preterm delivery at 125 d . Doses of 1-100 mg of endotoxin produced significant increases in glutathione peroxidase activity; doses of 4-100 mg significantly increased catalase activity, whereas doses of 20-100 mg resulted in significant increases in total superoxide dismutase activity . Glutathione peroxidase activity was elevated within 2 d, whereas superoxide dismutase was increased by 4 d and catalase activity increased by 7 d after endotoxin . No AOE increases were sustained for 15 d . Endotoxin increased fetal lung AOE activity at similar dosing amounts and intervals to those producing maturation of lung function and surfactant . Thus, mechanisms involving proinflammatory stimulation, unrelated to glucocorticoid hormones, can induce maturation of the AOE system of the fetal lung.

Mol Cancer Res, 2003 Feb, 1(4), 290 - 9
8-hydroxydeoxyguanosine causes death of human leukemia cells deficient in 8-oxoguanine glycosylase 1 activity by inducing apoptosis; Hyun JW et al.; Our previous study showed that KG-1, a human acute leukemia cell line, has mutational loss of 8-oxoguanine (8-hydroxyguanine; oh(8)Gua) glycosylase 1 (OGG1) activity and that its viability is severely affected by 8-hydroxydeoxyguanosine (8-oxodeoxyguanosine; oh(8)dG) . In the present study, the nature of the killing action of oh(8)dG on KG-1 was investigated . Signs observed in oh(8)dG-treated KG-1 cells indicated that death was due to apoptosis, as demonstrated by: increased sub-G(1) hypodiploid (apoptotic) cells, DNA fragmentation, and apoptotic body formation; loss of mitochondrial transmembrane potential, the release of cytochrome c from mitochondria into the cytosol, and the down-regulation of bcl-2; and the activation of caspases 8, 9, and 3, and the efficient inhibition of the apoptotic process by caspases inhibitors . This apoptosis appears not to be associated with Fas/Fas ligand because the expressions of these proteins were unchanged . Apoptotic KG-1 cells showed a high concentration of oh(8)Gua in DNA . Moreover, the increased concentration of oh(8)Gua in DNA, and the apoptotic process were not suppressed by the antioxidant, N-acetylcysteine, and thus the process is independent of reactive oxygen species . Of the 18 cancer cell lines treated with oh(8)dG, 3 cell lines (H9, CEM-CM3, and Molt-4) were found to be committed to apoptosis, and all of these showed very low OGG1 activity and a marked increase in the concentration of oh(8)Gua in DNA . These observations indicate that in addition to its mutagenic action, oh(8)Gua in DNA disturbs cell viability by inducing apoptosis.

In Silico Biol, 2002, 2(4), 467 - 84
MMT--a pathway modeling tool for data from rapid sampling experiments; Hurlebaus J et al.; The identification of metabolic regulation is a major concern in metabolic engineering . Metabolic regulation phenomena depend on intracellular compounds such as enzymes, metabolites and cofactors . A complete understanding of metabolic regulation requires quantitative information about these compounds under in vivo conditions . This quantitative knowledge in combination with the known network of metabolic pathways allows the construction of mathematical models that describe the dynamic changes in metabolite concentrations over time . Rapid sampling combined with pulse experiments is a useful tool for the identification of metabolic regulation owing to the transient data they provide . Enzymatic tests in combination with ESI-LC-MS (Electrospray Ionization Liquid Chromatographic Tandem Mass Spectrometry) and HPLC measurements have been used to identify up to 30 metabolites and nucleotides from rapid sampling experiments . A metabolic modeling tool (MMT) that is built on a relational database was developed specifically for analysis of rapid sampling experiments . The tool allows to construct complex pathway models with information stored in the relational database . Parameter fitting and simulation algorithms for the resulting system of Ordinary Differential Equations (ODEs) are part of MMT . Additionally explicit sensitivity functions are calculated . The integration of all necessary algorithms in one tool allows fast model analysis and comparison . Complex models have been developed to describe the central metabolic pathways of Escherichia coli during a glucose pulse experiment.

Biotechnol Appl Biochem, 2003 Jun, 37(Pt 3), 235 - 44
Optimizing alkaline lysis for DNA plasmid recovery; Clemson M et al.; Optimization of the alkaline lysis (P2) and neutralization (N3) steps in the recovery of DNA plasmids was pursued . Experiments were conducted at the test-tube and 5-litre scales with 3 kb (pUC18) and 20 kb (pQR150) plasmids . The scale and degree of mixing/shear did not affect the optimum yield of supercoiled plasmid during the P2 step, but did effect the time required for the optimum to be achieved . This optimum time for P2 at the large scale was longer (8-9 min), especially when a low-shear impeller was used . Also, when the yield of supercoiled plasmid reached a maximum during the P2 step, the purity (percentage of plasmids in the supercoiled form) simultaneously reached a minimum . As the duration of the N3 step increased from 1 to 6 min, the yield of the supercoiled plasmids remained fairly constant, provided that a lowshear impeller was used . The neutralized (post-N3) plasmid solution was shear-sensitive; however, mixing with a Rushton turbine in a tank (maximum energy dissipation rate in the mixing tank, epsilon (max), 12 m(2)/s(3); mixing-tank power consumption/volume of mixing tank, 2.0 W/m(3)) for 5-10 min resulted in a slight decrease in supercoiled plasmid and a notable increase in genomic DNA concentrations . The loss of the larger 20 kb plasmid (20%) was more than for the 3 kb plasmid . Finally, preparing the cells for alkaline lysis with lysozyme or low-pressure homogenization did not increase the plasmid yield . Furthermore, the homogenizer broke up the genomic DNA into fragments that followed through the entire Qiagen prep with the plasmids as impurities.

Mol Biotechnol, 2003 Jan, 23(1), 29 - 36
Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments; Jamsai D et al.; We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection . The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes . In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system . Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences . We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus . We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones . Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.

Vestn Ross Akad Med Nauk, 2002, (12), 39 - 41
{Protein of Escherichia coli interacting specifically with human low density lipoproteins}; Runova OL et al.; Escherichia coli 48 kDa protein interacting specifically with human low-density lipoproteins is described . The dissociation constant of this highly specific interaction was found to be equal to 4 mkg LDL per 1 ml or 7.3 x 10 M, which is comparable with the dissociation constant of the complex formed by LDL and human LDL receptor . A protocol for purifying the E . Coli binding protein was developed and antibodies against this purified protein were raised . The absence of sequences with homology to the ligand-binding repeats of the human LDL receptor in E . Coli proteome was shown by computer analysis of E . Coli genome . A conclusion was made that binding of the human LDL with specific E . Coli protein is thus mediated by other sequences and by another mechanism different from that, which occurs in human cells during the interaction of lipoproteins with their specific receptor . The establishment of specific interaction between E . Coli protein and human LDL can turn out to be useful in the future for purifying lipoproteins of a specific class and for administering plasmapheresis in patients with severe hyperlipoproteinemia.

J Virol, 2003 Mar, 77(6), 3487 - 94
A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge; Saini M et al.; Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection . Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies . Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs) . The envelope (E) protein of JEV contains the virus-neutralization epitopes . Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods . The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation . Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies . A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies . Importantly, these antibodies were obtained without the use of an adjuvant . The immunized mice showed significant protection against a lethal JEV challenge.

J Virol, 2003 Mar, 77(6), 3477 - 86
Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication; Zhang S et al.; DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR . The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length . Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type . A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent . There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini . Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer . However, the end of the insertion at nucleotide 142 was frequently preserved . The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells . In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters . In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations . Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter . Analysis by Southern blotting and quantification using dot blots showed that approximately 10(3) copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA . Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment . These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.

J Virol, 2003 Mar, 77(6), 3430 - 40
Autographa californica nucleopolyhedrovirus orf69 encodes an RNA cap (nucleoside-2'-O)-methyltransferase; Wu X et al.; The AcNPV orf69 gene encodes a protein that contains an S-adenosylmethionine (AdoMet)-dependent methyltransferase signature motif . More significantly, ORF69 shows high conservation at residues diagnostic for (nucleoside 2'-O)-methyltransferase activity . To analyze the function of this protein, which was renamed MTase1, it was overexpressed in Escherichia coli and purified to homogeneity . Photo cross-linking experiments showed that MTase1 bound AdoMet, and functional assays demonstrated cap 0-dependent methyltransferase activity . In vivo expression assays in insect cells showed that MTase1 was synthesized during the late phase of infection and that its expression was dependent on viral DNA replication . Primer extension analysis identified a late promoter motif, ATAAG, at the transcription start site . A mutant virus was constructed by inserting the lacZ gene into the coding region of mtase1 . Immunoblot analysis confirmed that MTase1 was not synthesized in these cells, and single-step growth curves revealed that the rate of virus replication in tissue culture was not affected by the absence of MTase1.

J Biol Chem, 2003 May 9, 278(19), 17178 - 84 Epub 2003 Feb 27.
A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5; Kumar S et al.; The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5 . 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase . Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation . At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m) . Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity . Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant . All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat) . Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants . Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation . Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored . In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.

Biophys J, 2003 Mar, 84(3), 1756 - 64
The lateral diffusion of selectively aggregated peptides in giant unilamellar vesicles; Lee CC et al.; We have systematically investigated the effect of aggregation of a transmembrane peptide on its diffusion in dimyristoylphosphatidylcholine and in palmitoyloleoylphosphatidylcholine model membranes . The hydrophobic segment of the b subunit from E . coli F(1)F(0)-ATP synthase was modified with a histidine tag at the carbonyl terminus and was aggregated selectively by using a series of multivalent, dendritic chelating agents with nitrilotriacetic acid functional groups . Peptide complexes ranging from monomers to hexamers were formed and studied in giant unilamellar vesicles . The rate of diffusion for the transmembrane peptide complexes were found to depend on the size of the complex . The results agree with predictions from the free area model for monomers and dimers, and the hydrodynamic continuum model for tetramers, pentamers, and hexamers . Comparisons with diffusion of lipids confirm that the diffusion of a transmembrane peptide is enhanced by coupling of density fluctuations between the two monolayers.

Biophys J, 2003 Mar, 84(3), 1606 - 15
Fluctuations and slow variables in genetic networks; Bundschuh R et al.; Computer simulations of large genetic networks are often extremely time consuming because, in addition to the biologically interesting translation and transcription reactions, many less interesting reactions like DNA binding and dimerizations have to be simulated . It is desirable to use the fact that the latter occur on much faster timescales than the former to eliminate the fast and uninteresting reactions and to obtain effective models of the slow reactions only . We use three examples of self-regulatory networks to show that the usual reduction methods where one obtains a system of equations of the Hill type fail to capture the fluctuations that these networks exhibit due to the small number of molecules; moreover, they may even miss describing the behavior of the average number of proteins . We identify the inclusion of fast-varying variables in the effective description as the cause for the failure of the traditional schemes . We suggest a different effective description, which entails the introduction of an additional species, not present in the original networks, that is slowly varying . We show that this description allows for a very efficient simulation of the reduced system while retaining the correct fluctuations and behavior of the full system . This approach ought to be applicable to a wide range of genetic networks.

Blood, 2003 Jun 15, 101(12), 4823 - 7 Epub 2003 Feb 27.
Aggravation of endotoxin-induced disseminated intravascular coagulation and cytokine activation in heterozygous protein-C-deficient mice; Levi M et al.; In the pathogenesis of sepsis and disseminated intravascular coagulation (DIC), dysfunctional anticoagulant pathways are important . The function of the protein C system in DIC is impaired because of low levels of protein C and down-regulation of thrombomodulin . The administration of (activated) protein C results in an improved outcome in experimental and clinical studies of DIC . It is unknown whether congenital deficiencies in the protein C system are associated with more severe DIC . The aim of the present study was to investigate the effect of a heterozygous deficiency of protein C on experimental DIC in mice . Mice with single-allele targeted disruption of the protein C gene (PC+/-) mice and wild-type littermates (PC+/+) were injected with Escherichia coli endotoxin (50 mg/kg) intraperitoneally . PC+/-mice had more severe DIC, as evidenced by a greater decrease in fibrinogen level and a larger drop in platelet count . Histologic examination showed more fibrin deposition in lungs, kidneys, and liver in mice with a heterozygous deficiency of protein C . Interestingly, PC+/- mice had significantly higher levels of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta, indicating an interaction between the protein C system and the inflammatory response . Survival was lower at 12 and 24 hours after endotoxin in the PC+/- mice . These results confirm the important role of the protein C system in the coagulative-inflammatory response on endotoxemia and may suggest that congenital deficiencies in the protein C system are associated with more severe DIC and adverse outcome in sepsis.

J Immunol Methods, 2003 Mar 1, 274(1-2), 233 - 44
A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions; Soltes G et al.; Phage display technology (PDT) is a powerful method for isolating functional gene products such as antigen-specific monoclonal antibodies (mAbs) . To improve the effectiveness of PDT, we sought to optimize display of Fab-g3p (antibody fragment fused with viral gene 3 protein) on phagemid virions and to optimize the yield of such phage . To do so, we constructed a novel helper phage, Phaberge, having a conditional deficiency in g3p production . Unlike most other published g3p-deficient helper phage, Phaberge is produced at high levels, 10(11) PFU/ml . As compared to g3p-sufficient helper phage, Phaberge caused a 5-20-fold increase in display level . Another novel feature is that Phaberge only packages insert-containing, not insert-less, phagemid into infectious virions . This should prove useful in preserving quality of phagemid libraries during propagation.In addition, other parameters were also found to affect production of phagemid virions . In particular, the choice of bacterial host cell, phagemid construct and growth temperature had a substantial impact on display levels, but generally no effect on number of phagemid virions produced.In short, we have established a set of parameters that improve production and quality of phagemid virions which we expect to facilitate the isolation of mAbs or other gene products by PDT.

Insect Biochem Mol Biol, 2003 Mar, 33(3), 345 - 54
A potential role for phenylalanine hydroxylase in mosquito immune responses; Johnson JK et al.; In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites . The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine . The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH) . In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti . A 1.6 kb A . aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system . In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues . Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes . Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples . Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes . Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Zhonghua Yi Xue Za Zhi, 2002 Nov 25, 82(22), 1541 - 5
{Construction, bioactivity identification and structural characteristics analysis of a novel recombinant immunosuppression protein B7-2-L-PE40KDEL}; Yuan Z et al.; OBJECTIVE: To construct a novel recombinant B7-2-L- PE40KDEL fusion protein used to selectively kill T cells expressing high levels of CD28 so as to induce immune tolerance and prevent graft versus host disease (GVHD) and host versus graft disease (HVGD) . METHODS: The cDNA encoding human B7-2 was ligated with cDNA encoding PE40KDEL by using sequence overlapping extension (SOE) techniques . The gene of interest was subcloned into a high output expression vector pRSETA and transformed into E . coli cells . Its molecular structural characteristics, such as flexibility, antigenicity, hydrophilicity, and epitope were analyzed . The purification protocol of expressed protein was established and its cytotoxicity to selectively kill T cells expressing high levels of CD28 was measured by MTT method . RESULTS: B7-2-L-PE40KDEL fusion protein was expressed at high levels in E . coli cells and the purified product attained over 95% of purity . The structural characteristics of B7-2-L-PE40KDEL were not significantly changed in comparison with B7-2 and PE40KDEL . In cytotoxicity assay, B7-2-L-PE40KDEL fusion protein specifically killed Jurkat cells which express high level CD28 receptor and was non-cytotoxic to CD28 receptor-negative cell line Raji . CONCLUSION: B7-2-L-PE40KDEL novel fusion protein can selectively kill the T cells which express CD28 receptor and may become a kind of new effective drug for inducing T cell immune tolerance and preventing GVHD and HVGD.

Plant J, 2003 Mar, 33(5), 923 - 37
CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis; Chen S et al.; Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides . We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana . Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates . In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines . CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns . CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques . A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds . The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected . Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes . This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.

Plant J, 2003 Mar, 33(5), 841 - 51
Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species; Sakamoto A et al.; The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident . However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS) . Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules . Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms . The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein . The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound . We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant . Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity . The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages . The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.

Neurochem Res, 2003 Feb, 28(2), 215 - 23
White matter injury following systemic endotoxemia or asphyxia in the fetal sheep; Mallard C et al.; White matter injury is the most frequently observed brain lesion in preterm infants . The etiology remains unclear, however, both cerebral hypoperfusion and intrauterine infections have been suggested as risk factors . We compared the neuropathological outcome, including the effect on oligodendrocytes, astrocytes, and microglia, following either systemic asphyxia or endotoxemia in fetal sheep at midgestation . Fetal sheep were subjected to either 25 minutes of umbilical cord occlusion or systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (LPS O111:B4, 100 ng/kg, IV) . Periventricular white matter lesions were observed in 2 of 6 asphyxiated fetuses, whereas the remaining animals showed diffuse injury throughout the subcortical white matter and neuronal necrosis in subcortical regions, including the striatum and hippocampus . LPS-treatment resulted in focal inflammatory infiltrates and cystic lesions in periventricular white matter in 2 of 5 animals, but with no neuron specific injury . Both experimental paradigms resulted in microglia activation in the white matter, damaged astrocytes, and loss of oligodendrocytes . These results show that the white matter at midgestation is sensitive to injury following both systemic asphyxia and endotoxemia . Asphyxia induced lesions in both white and subcortical grey matter in association with microglia activation, and endotoxemia resulted in selective white matter damage and inflammation.

Inflamm Res, 2003 Jan, 52(1), 1 - 7
Effect of fangchinoline in murine models of multiple organ dysfunction syndrome and septic shock; Hristova M et al.; OBJECTIVE AND DESIGN: To study the effect of the alkaloid fangchinoline on zymosan-induced multiple organ dysfunction syndrome (MODS) and Escherichia coli (E . coli)-induced septic shock . MATERIAL: Male ICR mice were used . Macrophages were isolated from peritoneal cavity for in vitro study . Treatment: Fangchinoline was administered i.p . at a dose of 1 or 5 mg/kg into the mice . METHODS: MODS was induced by intraperitoneal (i.p.) injection of zymosan at a dose 1.0 or 0.8/g b.w . E . coli-induced septic shock was provoked by i.p . inoculation of 5 x 10(8) bacterial cells into mice . TNF-alpha in serum and supernatants from peritoneal macrophages was detected by the use of L-929 cell cytotoxic assay . Alternative pathway (AP) complement activity was determined by hemolytic assay . RESULTS: Fangchinoline increased the survival rate in lethal MODS and septic shock . The alkaloid prevented the loss of body weight and liver enlargement in MODS and suppressed serum tumor necrosis factor-alpha (TNF-alpha) accumulation in MODS and septic shock . CONCLUSIONS: The result suggest that fangchinoline due mainly to its ability to downregulate TNF-alpha production might have protective effect in murine models of zymosan-induced MODS and E . coli-induced septic shock.

Microbiol Res, 2003, 158(1), 19 - 27
Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources; Mahishi LH et al.; Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB . Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E . coli were studied . Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation . No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source . Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources . The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources . Scanning electron microscopy of the PHB granules isolated from recombinant E . coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.

Microbiol Res, 2003, 158(1), 7 - 17
An alternative model of the twin arginine translocation system; Bruser T et al.; The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes . Currently it is supposed that Tat substrates bind directly to Tat translocon components before a ApH-driven translocation occurs . In this review, an alternative model is presented which proposes that membrane integration could precede Tat-dependent translocation . This idea is mainly supported by the recent observations of Tat-independent membrane insertion of Tat substrates in vivo and in vitro . Membrane insertion may allow i) a quality control of the folded state by membrane bound proteases like FtsH, ii) the recognition of the membrane spanning signal peptide by Tat system components, and iii) a pulling mechanism of translocation . In some cases of folded Tat substrates, the membrane targeting process may require ATP-dependent N-terminal unfolding-steps.

Fa Yi Xue Za Zhi, 2002 Aug, 18(3), 140 - 3
{Molecular cloning of recombinant fibronectin EDA and EDB fusion protein}; Xue AM et al.; OBJECTIVE: Construct a recombinant plasmid pET28a-EDA-EDB, prepare the fusion EDA-EDB protein . METHODS: For the production of recombinant fibronectin EDA-EDB in Escherichia coli, the EDA and EDB segments were separated from pGEM2-EDA/EDB and recomposed with two additional amino acids, then cloned into the expression vector pET28a . pET system to express EDA-EDB fusion protein and 6 x His/Ni-NTA system to purify it in a single step were used . Western blotting confirmed the purified protein . RESULTS: The EDA and EDB segments were ligated and inserted into pET28a vector . EDA-EDB fusion protein was highly expressed in Escherichia coli BL21 (DE3) . Afterwards, it was purified by Ni-NTA resin and verified by western blotting . CONCLUSION: EDA-EDB fusion protein can be expressed in pET system and purified by 6 x His/Ni-NTA system.

Vopr Virusol, 2003 Jan-Feb, 48(1), 21 - 4
{In vitro synthesis of immunoglobulins caused by an inactivated Ebola virus}; Tuzova MN et al.; An in vitro model Ebola infection was used to study the humoral response of human mononuclear cells to stimulation by purified inactivated Ebola virus antigen . Inactivated Ebola virus was cocultivated with human mononuclear cells in the presence or absence of B-cell mitogen LPS E . coli: B5 . An increase in the rate of synthesis of immunoglobulins (both IgG and, to a less extent, other classes) was observed . The Ebola virus proteins were suggested to exert no suppression effect on B-cells . The IgM/IgG synthesis was evaluated by EIA in supernatants after 7 days of cultivation . It was concluded that Ebola fever is accompanied by active humoral immune response, which provides a promising basis for further search of the methods of treatment of this disease.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2002 Oct, 20(5), 313 - 5
{The cloning and sequencing of H-2Kk gene cDNA of 615 mice}; Li L et al.; OBJECTIVE: The purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy . METHODS: The 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR) . The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E . coli JM109 was transformed with the ligated product . The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants . The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method . RESULTS: The sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other . CONCLUSION: The authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.

Arch Virol, 2003 Mar, 148(3), 435 - 48
CpG immuno-stimulatory motifs enhance humoral immune responses against hepatitis C virus core protein after DNA-based immunization; Encke J et al.; Chronic HCV infection is associated with a high morbidity and mortality rate, and currently a prophylactic or therapeutic vaccine is not available . DNA-based immunization is a powerful method to generate cellular and humoral immune responses . However, DNA immunization against HCV core results only in a weak humoral immune response demonstrated in several studies . Therefore, co-immunization with a novel adjuvant may enhance such potentially important immune responses . We examined whether unmethylated CpG motifs in the form of oligodeoxynucleotides (ODN) or E . coli DNA can act as adjuvants for a DNA vaccination approach, since CpG motifs have been shown to stimulate the innate immune system as well as B and T cell immune reactivity . The present study demonstrates that CpG motifs enhance in vivo antibody levels after DNA immunization against HCV core . However, despite some in vitro activity of CpG motifs, no enhancement of T cell responses in vivo was observed after immunization with HCV plasmid DNA and CpG motifs in mice . Our results suggest that co-immunization with CpG-ODN may strengthen humoral immune responses but show no potential effect as an adjuvant to induce cellular immunity against HCV core.

Oncogene, 2003 Feb 27, 22(8), 1124 - 34
Role for RhoB and PRK in the suppression of epithelial cell transformation by farnesyltransferase inhibitors; Zeng PY et al.; Recent genetic investigations have established that RhoB gain-of-function is sufficient to mediate the antitransforming effects of farnesyltransferase inhibitors (FTIs) in H-Ras-transformed fibroblast systems . In this study, we addressed the breadth and mechanism of RhoB action in epithelial cells transformed by oncoproteins which are themselves insensitive to FTI inactivation . Rat intestinal epithelial (RIE) cells transformed by activated K-Ras or Rac1 were highly sensitive to FTI-induced actin reorganization and growth inhibition, despite the inability of FTI to block prenylation of either K-Ras or Rac1 . Ectopic expression of the geranylgeranylated RhoB isoform elicited in cells by FTI treatment phenocopied these effects . Analysis of RhoB effector domain mutants pointed to a role for PRK, a Rho effector kinase implicated in the physiological function of RhoB in intracellular receptor trafficking, and these findings were supported further by experiments in a fibroblast system . We propose that FTIs recruit the antioncogenic RhoB protein in the guise of RhoB-GG to interfere with signaling by pro-oncogenic Rho proteins, possibly by sequestering common exchange factors or effectors such as PRK that are important for cell transformation.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2243 - 8 Epub 2003 Feb 26.
Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase; Sharma V et al.; In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane . SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation . Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis . Each subunit of the homodimer contains a "motor" domain and a translocation domain . The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein . Knowledge of this structure provides a framework for further elucidation of the translocation process.

J Biol Chem, 2003 May 9, 278(19), 16642 - 50 Epub 2003 Feb 26.
A phage display-based method for determination of relative affinities of mutants . Application of the actin-binding motifs in thymosin beta 4 and the villin headpiece; Rossenu S et al.; We propose phage display combined with enzyme-linked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein . Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity . Hence this method enables us to calculate the relative affinities of a large number of mutants . As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin . The hexapeptide motifs LKKTET, present in thymosin beta4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed . Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin . In addition, our data show that the second and the fourth amino acid of the thymosin beta4 motif and the first three residues of the villin headpiece motif are most important for actin binding . The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.

J Biol Chem, 2003 Jul 4, 278(27), 24714 - 20 Epub 2003 Feb 26.
Tyrosine phosphorylation of the well packed ephrinB cytoplasmic beta-hairpin for reverse signaling . Structural consequences and binding properties; Song J; Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, thus initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling . Recently, the region corresponding to this 22-residue motif was demonstrated to adopt a well packed beta-hairpin structure with a high conformational stability in the unphosphorylated cytoplasmic subdomain . However, because the binding to Grb4 is phosphorylation-dependent and the hairpin contains three conserved tyrosine residues that may be phosphorylated, the key events remain unknown as to how tyrosine phosphorylation affects the structure of this well packed beta-hairpin and which phosphorylation site is relevant to SH2 domain binding . By characterizing the structural and binding properties of six 22-residue SH2 domain-binding motifs with different phosphorylated sites, the present study reveals that, as shown by circular dichroism and NMR, the unphosphorylated 22-residue motif adopts a well formed beta-hairpin structure in isolation from the ephrinB cytoplasmic subdomain . However, this beta-hairpin is radically abolished by tyrosine phosphorylation, regardless of the relative location and number of Tyr residues . Unexpectedly, the peptides with either Tyr304 or Tyr316 phosphorylated show high affinity binding to SH2 domain, whereas the peptide with Tyr311 phosphorylated has no detectable binding . This implies that ephrinB with Tyr311 phosphorylated might have a currently unidentified binding partner distinct from the Grb4 protein, because Tyr311 is known to be phosphorylated in vivo . Based on the results above, it is thus proposed that the disruption of the tight side-chain packing by tyrosine phosphorylation in the well structured region of a signaling protein may represent a general activation mechanism by which a cryptic binding site is disclosed for new protein-protein interactions.

J Biol Chem, 2003 May 9, 278(19), 16651 - 7 Epub 2003 Feb 26.
Molecular dissection of GTP exchange and hydrolysis within the ternary complex of tubulin heterodimers and Op18/stathmin family members; Brannstrom K et al.; The ubiquitous Op18 and the neural RB3 and SCG10 proteins are members of the oncoprotein18/stathmin family of microtubule regulators . These proteins bind two tubulin heterodimers via two imperfect helical repeats to form a complex of heterodimers aligned head-to-tail . Here we have analyzed GTP exchange and GTP hydrolysis at the exchangeable GTP-binding site (E-site) of tubulin heterodimers in complex with Op18, RB3, or SCG10 . These proteins stimulate a low and indistinguishable rate of GTP hydrolysis, and our results show that GTP exchange is blocked at both E-sites of the ternary complex, whereas GTP hydrolysis only occurs at one of the two E-sites . Results from mutational analysis of clusters of hydrophobic residues within the first helical repeat of Op18 suggest that GTP is hydrolyzed at the E-site that is interfaced between the head-to-tail arranged heterodimers, which is consistent with predicted GTPase productive interactions between the two tubulin heterodimers . Our mutational analysis has also indicated that Op18/stathmin family members actively restrain the otherwise potent GTPase productive interactions that are generated by longitudinal interactions within protofilaments . We conclude that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.

Biol Reprod, 2003 May, 68(5), 1525 - 37 Epub 2002 Nov 27.
SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa; Mandal A et al.; We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm . C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds . Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively . The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88 . The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm . As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity . A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1 . SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line . The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2 . Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

Biol Reprod, 2003 May, 68(5), 1695 - 702 Epub 2002 Dec 11.
Fetal responses to maternal and intra-amniotic lipopolysaccharide administration in sheep; Grigsby PL et al.; A link between intrauterine infection and premature labor is widely accepted, yet the fetal inflammatory responses to such infections are not well understood . Our aim was to use a sheep model in which an inflammatory state was induced by lipopolysaccharide (LPS) administration during pregnancy to the maternal systemic, intra-amniotic or extra-amniotic compartments . Fetal and maternal blood gases and uterine electromyographic activity along with fetal and maternal circulating concentrations of prostaglandins PGE2 and PGFM, cortisol, and interleukin-6 were determined . Maternal systemic LPS treatment resulted in mild maternal hypoxemia, a rise in temperature, greater fetal hypoxemia, and a marked rise in fetal cortisol and PGE2 concentrations that persisted for 48 h . Intra-amniotic administration of LPS at doses higher than those used systemically caused an increase in fetal cortisol and PGE2 concentrations as well as a rise in uterine activity, but these were lesser in magnitude . Extra-amniotic LPS administration caused no overt fetal or maternal inflammatory responses . We conclude that maternal LPS treatment markedly elevated fetal cortisol and PGE2 concentrations . This may be a potential protective mechanism that aids the fetus in the event of premature delivery . The attenuated fetal response to intra-amniotic LPS treatment, despite the much higher dose used, may support a role for the amniotic fluid in protecting the fetus from endotoxin exposure during pregnancy.

DNA Repair (Amst), 2003 Apr 2, 2(4), 417 - 26
Polymorphism of genes encoding SOS polymerases in natural populations of Escherichia coli; Bjedov I et al.; High fidelity replicative DNA polymerases can be blocked during DNA replication by various base damages, which represents a potentially lethal event . Escherichia coli possesses three DNA polymerases, PolII, PolIV and PolV, that can continue replication over such lesions in template DNA, thus allowing for cell survival . Genes coding for these enzymes, polB, dinB, and umuCD respectively, belong to the stress-inducible SOS regulon . We have analyzed the patterns of nucleotide sequence variability of genes encoding for three SOS polymerases from E . coli natural isolates in order to identify the nature of selective forces that determine their evolution . The frequency of inferred inter-strain recombination events, and the frequency of synonymous and non-synonymous base substitutions within these genes do not deviate significantly from those observed for the control group composed of 2 genes coding for DNA polymerases PolI and PolIII and 10 metabolic genes . This suggests that the loci coding for SOS polymerases are subject to selective pressure for the maintenance of their function and specificity . The fact that genes coding for translesion-synthesis (TLS) polymerases, particularly dinB and umuC homologs, have been conserved during evolution and the present analysis suggest that their activity is essential for the cellular survival and fitness.

DNA Repair (Amst), 2003 Apr 2, 2(4), 387 - 405
In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination; Junop MS et al.; We have used the recently determined crystal structures of Escherichia coli (E . coli) MutS, MutL and MutH to guide construction of 47 amino-acid substitutions in these proteins and analyzed their behavior in mismatch repair and recombination in vitro and in vivo . We find that the active site of the MutH endonuclease is composed of regions from two separate structural domains and that the C-terminal 5 residues of MutH influence both DNA binding and cleavage . We also find that the non-specific DNA-binding activity of MutL is required for mismatch repair and probably functions after strand cleavage by MutH . Alteration of residues in either the mismatch recognition domain, the ATPase active site, or the domain interfaces linking the two activities can diminish the differential binding of MutS to homoduplex versus heteroduplex and results in the loss of mismatch-specific MutH activation . Finally, every mutation that abolishes mismatch repair is deficient in blocking homeologous recombination, suggesting that mismatch repair and prevention of homeologous recombination use the same MutS-MutL complexes for signaling in E . coli.

Acta Trop, 2003 Feb, 85(2), 133 - 43
Progress in control of hydatidosis using vaccination--a review of formulation and delivery of the vaccine and recommendations for practical use in control programmes; Heath DD et al.; A vaccine to protect sheep, goats, and bovines against hydatid disease caused by the cysts of Echinococcus granulosus is prepared as a recombinant fusion protein expressed in Escherichia coli . Solubilised inclusion bodies are injected, together with Quil A, subcutaneously on two occasions 1 month or more apart, and induce protection against infection which lasts for at least 12 months . A third injection given 6-12 months after the second injection induces a high and long-lasting protection against artificial or natural challenge infections . This review describes work carried out on the formulation, safety and efficacy of the vaccine under laboratory and field conditions, using artificial or natural challenges with E . granulosus eggs, followed by necropsy . Hydatid control programmes based on regular treatment of all dogs with the correct dose of a highly-efficient anthelmintic have sometimes not been successful in Continental environments . Access to dogs is difficult in summer because of the distances to summer pastures, and is often impossible in winter because of snow . A control program using strategic twice-yearly anthelmintic treatment of dogs is likely to be successful provided grazing animals are vaccinated as well . Vaccination as a control tool only requires the veterinarians to visit two times a year, and while the veterinarian is present, the dogs can be treated with anthelmintic for little additional cost . One visit should take place after the autumn kill of animals for winter consumption, and this is a good time to vaccinate animals born in the summer, and also all other animals while they are healthy and immunologically responsive . The other visit should take place in the spring, at which time animals born during winter can be vaccinated . Although a single immunization has been shown to induce a useful degree of protection, where possible it is best to give two initial injections, 1 month apart . If it is possible for veterinarians to stay in the field for 2 months in November/December and March/April, in order to give the two injections, a more rapid onset of full protective immunity will initially be achieved than if the injections are given 6 months apart . A large-scale safety and efficacy trial involving 50,000 and 100,000 lambs in Qinghai and Xinjiang Provinces of China has taken place . Results have confirmed safety and efficacy . In most countries, prevalence of infection increases with age . The vaccine has no effect on established cysts, and therefore, in order to prevent the biomass of Echinococcus spp . from increasing, it might be an effective strategy to begin a control programme by vaccinating all animals . Because many of the older stock will already be infected, they will remain a source of infection for dogs for the average lifetime of the stock . Dogs will still be able to be infected from the older stock, and will continue to infect humans . We advocate that a vaccination programme be accompanied by education about hydatid disease, and anthelmintic treatment of dogs in late autumn and early spring.

FEBS Lett, 2003 Feb 27, 537(1-3), 215 - 21
Role of molecular chaperones in inclusion body formation; Carrio MM et al.; Protein misfolding and aggregation are linked to several degenerative diseases and are responsible for the formation of bacterial inclusion bodies . Roles of molecular chaperones in promoting protein deposition have been speculated but not proven in vivo . We have investigated the involvement of individual chaperones in inclusion body formation by producing the misfolding-prone but partially soluble VP1LAC protein in chaperone null bacterial strains . Unexpectedly, the absence of a functional GroEL significantly reduced aggregation and favoured the incidence of the soluble protein form, from 4 to 35% of the total VP1LAC protein . On the other hand, no regular inclusion bodies were then formed but more abundant small aggregates up to 0.05 microm(3) . Contrarily, in a DnaK(-) background, the amount of inclusion body protein was 2.5-fold higher than in the wild-type strain and the average volume of the inclusion bodies increased from 0.25 to 0.38 microm(3) . Also in the absence of DnaK, the minor fraction of soluble protein appears as highly proteolytically stable, suggesting an inverse connection between proteolysis and aggregation managed by this chaperone . In summary, GroEL and DnaK appear as major antagonist controllers of inclusion body formation by promoting and preventing, respectively, the aggregation of misfolded polypeptides . GroEL might have, in addition, a key role in driving the protein transit from the soluble to the insoluble cell fraction and also in the opposite direction . Although chaperones ClpB, ClpA, IbpA and IbpB also participate in these processes, the impact of the respective null mutations on bacterial inclusion body formation is much more moderate.

FEBS Lett, 2003 Feb 27, 537(1-3), 157 - 60
Nicotinamide adenine dinucleotide stimulates oligomerization, interaction with adenovirus E1A and an intrinsic dehydrogenase activity of CtBP; Balasubramanian P et al.; The C-terminal region of adenovirus E1A interacts with the transcriptional corepressor, CtBP . The mechanism of transcriptional regulation by CtBP is not known . CtBP shares a significant homology with NAD(+)-dependent D2-hydroxy acid dehydrogenases . CtBP binds to NAD(+) and NADH . Both forms of the dinucleotide stimulate oligomerization of native CtBP and enhance complex formation with E1A . CtBP also has a slow dehydrogenase activity . Interaction of CtBP with E1A reduces the dehydrogenase activity . Our results raise the possibility that the oxidation/reduction reactions of CtBP may regulate transcription . Thus, CtBP is a unique transcriptional regulator with an enzymatic activity similar to metabolic dehydrogenases . The levels of intracellular nicotinamide adenine dinucleotide may modulate transcriptional activity of CtBP.

FEBS Lett, 2003 Feb 27, 537(1-3), 63 - 7
Oxidized glutathione stimulated the amyloid formation of alpha-synuclein; Paik SR et al.; alpha-Synuclein is the major filamentous constituent of Lewy bodies found in Parkinson's disease (PD) . The amyloid formation of alpha-synuclein was significantly facilitated by oxidized glutathione (GSSG) as the lag period of the aggregation kinetics was shortened by 2.5-fold from its absence . Reduced glutathione (GSH), on the other hand, did not influence the lag phase although it increased the final amyloid formation . The GSSG stimulation was specific for not only alpha-synuclein but also its intactness . The preferred GSSG interaction of alpha-synuclein to GSH was also demonstrated with dissociation constants of 0.53 and 43.5 mM, respectively . It is suggested that the oxidative stress favoring the GSSG generation from GSH could result in the augmented amyloid formation of alpha-synuclein, which ought to be related to the pathogenesis of PD.

FEBS Lett, 2003 Feb 27, 537(1-3), 47 - 52
The pp60c-Src inhibitor PP1 is non-competitive against ATP; Karni R et al.; Glutathione-S-transferase (GST)-pp60(c-Src) (GST-Src) expressed in Escherichia coli is as catalytically active as purified, activated pp60(c-Src) protein derived from human platelets . We utilized the bacterially expressed enzyme, together with information about the structures of Src family kinases in complex with their inhibitors PP1 and PP2, to modify PP1 in a quest for improved inhibitors . Despite the detailed structural information on Hck-PP1 and Lck-PP2 complexes, which shows that PP1 and PP2 bind to the adenosine triphosphate (ATP) pocket, we were unable to improve the affinity between modified PP1 and Src . Puzzled, we examined in detail the mechanism by which PP1 inhibits the kinase activity of Src . Here we report that PP1 is non-competitive with ATP for the inhibition of Src, at variance with what is currently accepted, and is a 'mixed competitive inhibitor' vis-a-vis the substrate . These findings shed new light on the mechanism whereby PP1-like molecules inhibit Src . Examination of the homology between the kinase domain of Src and those of Hck and Lck reveals significant differences outside the ATP binding pocket, whereas they are identical within the ATP binding domain . These results suggest that PP1 may be a leading compound for ATP non-competitive inhibitors of Src family kinases . Since Src in its active form is the hallmark of numerous cancers, understanding how PP1 inhibits activated Src will aid in the discovery of potent and selective Src kinase inhibitors.

FEBS Lett, 2003 Feb 27, 537(1-3), 42 - 6
Identification of key regions within the Escherichia coli TatAB subunits; Barrett CM et al.; The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane . In Escherichia coli and most other species, three important tat genes have been identified but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear . In this report we have used site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits . We find that an apparent 'hinge' region between the transmembrane (TM) span and an adjacent amphipathic region is important in both proteins, in that substitution of turn-inducing residues inhibits the export of a natural Tat substrate . Surprisingly, large-scale mutagenesis of the conserved amphipathic regions of TatA and TatB leads only to minor effects on Tat-dependent export suggesting that this particular feature is not central to the translocation mechanism . This domain is, however, critical for the translocation process and we identify Gly/Pro residues in these regions of TatA/B that are essential for efficient export.

Steroids, 2003 Feb, 68(2), 205 - 9
Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response; Kagawa N et al.; CYP19 (P450arom) catalyzes the aromatization reaction of C19 steroids leading to estrogens . While readily expressed in insect cells, the human P450arom has been a difficult P450 to express in Escherichia coli at useful levels . In the present study, we replaced the N-terminal sequence in human CYP19 with the corresponding sequences of other microsomal P450s (CYP2C11 and CYP17) that are efficiently expressed in E . coli . Although the N-terminal replacement alone was not sufficient for the expression, human P450arom was successfully expressed up to the level of 240nmol/l culture by the combination of the N-terminal replacement and the induction of cold stress response by 1 microg/ml chloramphenicol . Membrane fractions containing the expressed P450arom catalyzed aromatization of androstenedione with a specific activity of 4.9 nmol/min/nmol P450 . Our results are important to provide large quantities of human P450arom as an active form for structure-function studies.

Anal Biochem, 2003 Feb 15, 313(2), 301 - 6
A spectrophotometric method to quantify linear DNA; Samuel M et al.; A spectrophotometric method for quantification of linear DNA is described . The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease . Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg) . The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound . Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements . However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme . The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA . The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase . NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm . This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

Anal Biochem, 2003 Feb 15, 313(2), 187 - 95
The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue; Bertsch M et al.; Hydrophobic sites on the surface of protein molecules are thought to have important functional roles . The identification of such sites can provide information about the function and mode of interaction with other cellular components . While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c(') . Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants . Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants . The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes . The absorption spectrum of BPB is also observed to be polarity sensitive . A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy . An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment . The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

Clin Exp Immunol, 2003 Mar, 131(3), 477 - 83
Monocyte-derived RANTES is intrinsically elevated in periodontal disease while MCP-1 levels are related to inflammation and are inversely correlated with IL-12 levels; Fokkema SJ et al.; Bacteria colonizing tooth surfaces are essential in the induction of an inflammatory response in the periodontal tissues, but do not cause periodontitis in everyone, implicating differences in the host immune response . These possible differences were studied using lipopolysaccharide (LPS)-stimulated whole blood cell cultures (WBCC), which revealed a down regulation of monocyte derived interleukin-12 (IL-12p70) in untreated periodontitis patients and an up regulation after therapy . IL-12p70 is a crucial factor in the differentiation of Th1 cell responses . Since CC chemokines are able to influence the T cell differentiation via cytokine secretion in antigen-presenting cells, the production of CC chemokines in periodontitis was evaluated . Therefore WBCC were stimulated with LPS from Escherichia coli for 18 h and the levels of IL-12p70 and CC chemokines were measured in the supernatants by ELISA . Untreated periodontitis patients released 2 fold more RANTES (regulated on activation normal T cell expressed and secreted) (P = 0.01) and lower levels of IL-12p70 in comparison to controls (P < 0.05) . A trend towards higher levels of macrophage chemoattractant protein-1 (MCP-1) (P = 0.07) was also seen in untreated periodontitis patients; while similar levels of monocyte derived chemokine (MDC) and macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha and -1 beta) were found . After periodontal therapy no changes were seen with regard to MDC, MIP-1 alpha, MIP-1 beta and RANTES, whereas the MCP-1 levels decreased (P < 0.05) and the IL-12p70 levels strongly increased (P < 0.01) . The data showed a consistent inverse correlation between the levels of MCP-1 and IL-12p70, and their proportional changes after therapy correlated with the clinical inflammatory response after therapy . This indicates that the disease state regulates the release of IL-12p70 and MCP-1 in E . coli LPS-stimulated WBCC . In contrast, the persistent augmented levels of RANTES after therapy are suggestive for an intrinsic behaviour.

J Neurosci Res, 2003 Mar 15, 71(6), 777 - 84
Expression and properties of the recombinant murine Golli-myelin basic protein isoform J37; Kaur J et al.; A recombinant form of the murine Golli-myelin basic protein (MBP) isoform J37 (rmJ37) has been expressed in Escherichia coli and isolated to 95% purity via metal chelation and ion exchange chromatography . The protein did not aggregate lipid vesicles containing acidic phospholipids, unlike the 18.5 kDa isoform of MBP . This result is consistent with J37 having a functional role prior to the assembly of compact myelin . Circular dichroic spectroscopy showed that rmJ37 had a large proportion of random coil in aqueous solution but gained alpha-helix and beta-sheet in the presence of monosialoganglioside G(M1) and PI(4)P . Thus, like "classic" MBP, J37 is intrinsically unstructured, and its conformation depends on its environment and bound ligands . Analyses of the amino acid sequence of rmJ37 predicted an N-terminal calmodulin (CaM)-binding site . It was determined via a gel-shift assay and fluorescence spectroscopy that rmJ37 and CaM interacted in a 1:1 ratio in a Ca(2+)-dependent manner . However, the interaction was weak compared with 18.5 kDa MBP .

J Pharmacol Exp Ther, 2003 Mar, 304(3), 1280 - 4
A long-acting suicide gene toxin, 6-methylpurine, inhibits slow growing tumors after a single administration; Gadi VK et al.; We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-methylpurine in tumor cells or direct intratumoral injection of 6-methylpurine itself . A single i.p . injection in mice of the prodrug 9-beta-D-{2-deoxyribofuranosyl}-6-methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma . Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E . coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past . A prolonged retention time of 6-methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of <1 h) . By high-pressure liquid chromatography, metabolites of {(3)H}MeP-dR were 5- to 6-fold higher in tumors expressing E . coli PNP . These experiments point to new endpoints for monitoring E . coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin . The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E . coli PNP in the past.

J Pharmacol Exp Ther, 2003 Mar, 304(3), 1120 - 8
Selective tryptic cleavage at the tethered ligand site of the amino terminal domain of proteinase-activated receptor-2 in intact cells; Al-Ani B et al.; In intact cells, trypsin activates proteinase-activated receptor-2 (PAR(2)) by hydrolysis at residues R(36)/S(37) (amino acids are abbreviated by their one-letter code), revealing an active tethered ligand sequence . We sought to determine whether in intact cells, the tryptic cleavage/activation of PAR(2) might also be accompanied by hydrolysis at other potential N-terminal cleavage sites, like residues K(34), R(41), K(51), and K(72), as implied by the tryptic cleavage in vitro at these residues of Escherichia coli-expressed human N-terminal PAR(2)R(31)-P(79) . To this end, four PAR(2) mutants with altered tryptic cleavage sites were prepared (PAR(2)R(36)A, PAR(2)S(37)P, PAR(2)R(41)A, and PAR(2)R(36)AR(41)A), expressed in Kirsten virus-transformed rat kidney cells and were evaluated together with the wild-type PAR(2)-expressing cells for 1) activation (Ca(2+) signaling) by trypsin and the receptor-activating peptide SLIGRL-NH(2) (SL-NH(2)) and 2) the tryptic release of two antigenic receptor determinants, one N-terminal to the R(36)/S(37) cleavage/activation site detected by SLAW-A antibody and the second (detected by antibody, B5), N-terminal to residues K(51), K(72) . None of the mutants resistant to cleavage at R(36) were activated by trypsin, yet all retained reactivity to B5 and all were activated by SL-NH(2) . In contrast, trypsin activated both wild-type and PAR(2)R(41)A, leading to a disappearance of SLAW-A but not B5 reactivity . We conclude that, as opposed to the E . coli-expressed PAR(2) N-terminal polypeptide, PAR(2) expressed in intact cells displays selective tryptic cleavage at the R(36)/S(37) activation site, without cleaving downstream . Thus, in intact cells, trypsin activation does not concurrently "disarm" rat PAR(2), but leaves the "tethered ligand" persistently attached to the body of the receptor.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 1169 - 72
A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland; Perreten V et al.; A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized . Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E . coli . Two copies of the insertion element IS15Delta/26 flanked the region containing sul3 . The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E . coli isolates from pigs in Switzerland.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 1052 - 61
Structure-based design and engineering of a nontoxic recombinant pokeweed antiviral protein with potent anti-human immunodeficiency virus activity; Uckun FM et al.; A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102((151)AA(152)) and FLP-105((191)AA(192)) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities . FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo . These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP . They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS . Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1 . The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 1037 - 46
Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance; Gross CH et al.; DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA . Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP . PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis . Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme . Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis . Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities . All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected . Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E . coli gyrB mutant, and the activities correlated well with the in vitro activities . We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay . Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E . coli.

Biochem Biophys Res Commun, 2003 Mar 7, 302(2), 311 - 5
Protein minimization: characterization of the synthetic cyclic dodecapeptide corresponding to the reactive site region of the oil rape trypsin inhibitor type-III; Trovato M et al.; The design of minimal units required for enzyme inhibition is a major field of interest in structural biology and biotechnology . The successful design of the cyclic dodecapeptide corresponding to the Phe17-Val28 reactive site amino acid sequence of the low-molecular-mass trypsin inhibitor RTI-III from Brassica napus (micro-RTI-III) and of the recombinant murine dihydrofolate reductase-(DHFR-)micro-RTI-III fusion protein (DHFR-micro-RTI-III) is reported here . Micro-RTI-III was synthesized using a stepwise solid-phase approach based on the standard Fmoc chemistry, purified by RP-HPLC, and oxidatively refolded . DHFR-micro-RTI-III was expressed in Escherichia coli, purified by metal-chelate affinity chromatography, and oxidatively refolded . The affinity of micro-RTI-III for bovine trypsin (K(d)=1.6x10(-9)M) is similar to that determined for DHFR-micro-RTI-III (K(d)=6.3x10(-10)M) and native RTI-III (K(d)=2.9x10(-10)M), at pH 8.2 and 22.0 degrees C . Remarkably, micro-RTI-III protects the DHFR domain of DHFR-micro-RTI-III from trypsin digestion . Micro-RTI-III is a new minimal trypsin inhibitor and may be regarded as a tool in protein structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.

Biochem Biophys Res Commun, 2003 Mar 7, 302(2), 284 - 9
New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli; Spector D et al.; It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage . MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins . However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins . Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E . coli . Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity . The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E . coli . In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.

Biochem Biophys Res Commun, 2003 Mar 7, 302(2), 246 - 52
Insertion mutagenesis of Escherichiacoli GroEL; Amatore D et al.; To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in . Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367 . Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively . Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding . Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed . A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90 . By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible . Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.

Chem Biol Interact, 2003 Feb 1, 143-144, 533 - 42
Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli; Nidetzky B et al.; The plasmid-encoded gene of xylitol dehydrogenase from the yeast Galactocandida mastotermitis was expressed in Escherichia coli at 25 degrees C . Recombinant enzyme was isolated in 70% yield using two steps of biomimetic affinity chromatography with the dye ligand Procion Red HE3B immobilized onto Sepharose 4B-CL . Similar to natural enzyme, recombinant xylitol dehydrogenase is a functional homotetramer with a stoichiometric content of catalytic zinc in each 37-kDa subunit . Though lacking bound Mg(2+) found in xylitol dehydrogenase isolated from yeast cell extracts, the recombinant enzyme is as active and stable as the native enzyme . Stereospecificity of enzymic hydrogen transfer from NADH has been determined by 1H-NMR and is 4-pro-R . A detailed steady-state kinetic analysis has been carried out for the enzymic reaction, polyol+NAD(+)<-->ketose+NADH+H(+), at pH 7.5 and 25 degrees C using xylitol and D-xylulose, the physiological polyol-ketose pair, as well as D-sorbitol and D-fructose . Primary deuterium kinetic isotope effects on steady-state kinetic parameters for oxidation of D-sorbitol and reduction of D-fructose have been measured at pH 7.5 . Combined results of initial-rate analysis and isotope effect studies suggest that the enzyme utilizes a preferentially ordered kinetic mechanism in which NAD(+) binds before D-sorbitol and D-fructose is released before NADH . Dissociation of NADH appears to be the main rate-limiting step for D-sorbitol oxidation under substrate-saturated reaction conditions.

Chem Biol Interact, 2003 Feb 1, 143-144, 523 - 32
Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant; Klimacek M et al.; Xylose reductase (XR) from Candida tenuis (CtXR) is a structurally characterized member of family 2 of the aldo-keto reductase (AKR) superfamily of proteins, its family designation being AKR2B5 . The enzyme is composed of two identical subunits that contact each other in a largely hydrated, predominantly polar interface . An important question not clearly answered by CtXR structure pertains to the relationship of oligomerization and enzyme activity . In an effort to destabilize the wild-type dimer, the most important secondary structural element of the CtXR interface, the alpha5 helix, was altered by site-directed mutagenesis . Ala-173 and Leu-174 were replaced individually by arginine, and Arg-180 was changed into alanine . A173R and L174R mutants did not fold properly during recombinant protein production in Escherichia coli and could not be isolated . Like the wild type, the R180A mutant is a dimer in solution which does not dissociate into subunits under mild urea conditions (</=2 M) . Catalytic efficiency (k(cat)/K(xylose)) and turnover number (k(cat)) of the R180A mutant for NADH-dependent reduction of D-xylose are both approximately 2.5-fold decreased compared to corresponding kinetic parameters of the wild type . Differences in kinetic isotope effects for the mutant (Dk(cat)=1.0; Dk(cat)/K(xylose)=1.9) and the wild type (Dk(cat)=1.5; Dk(cat)/K(xylose)=2.8) suggest subtle changes in catalytic function as result of the mutation . Therefore, altering interactions at the dimer interface may have long range effects that were not predictable from the X-ray structure.

Chem Biol Interact, 2003 Feb 1, 143-144, 493 - 501
Significance of individual amino acid residues for coenzyme and substrate specificity of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus; Kristan K et al.; 17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is a NADPH dependent member of the short-chain dehydrogenase reductase (SDR) superfamily . Recently, we prepared a homology-built structural model of 17beta-HSDcl using the known three-dimensional structure of homologous 1,3,8-trihydroxynaphthalene reductase from the fungus Magnaporthe grisea . This model structure directed our studies of structure-function relationship of the fungal 17beta-HSD, as one of the model enzymes of the SDR superfamily . In this work, we investigated the significance of individual amino acid residues for coenzyme and substrate specificity . We performed site directed mutagenesis of R28, a basic residue conserved in most NADPH dependent SDR structures; T200, found only in Streptomyces hydrogenans 3alpha,20beta-HSD and Drosophila alcohol dehydrogenases; and H230, a residue corresponding to the substrate specificity important H221 in human 17beta-HSD type 1 . All recombinant proteins were expressed in Escherichia coli and purified to homogeneity . Kinetic evaluation of individual mutations was performed by analysis of progress curves of interconversions between 4-estrene-3,17-dione and 4-estrene-17beta-ol-3-one, in the presence of NADPH and NADP(+); according to the Theorell-Chance reaction mechanism . The results demonstrate the role of the selected amino acid residues; R28 seems to interact with the NADPH 2'-phosphate group; T200 may be involved in binding and dissociation of NADPH/NADP(+); while H230 and the neighboring A231 appears not to be responsible for substrate specificity of 17beta-HSDcl.

Chem Biol Interact, 2003 Feb 1, 143-144, 425 - 33
Characterization and recombinant expression of the translational repressor RepB of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase in Comamonas testosteroni; Xiong G et al.; 3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds . The gene of 3alpha-HSD/CR (hsdA) was cloned and characterized by our group . We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression . To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state . Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level . The addition of testosterone to the culture medium led to a derepression . Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA, but has no effect on hsdA, with which it happens to partly overlap . The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA . In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3alpha-HSD/CR.

Chem Biol Interact, 2003 Feb 1, 143-144, 239 - 45
Tetrameric NAD-dependent alcohol dehydrogenase; Karlsson A et al.; Three-dimensional structures of the ethanol-induced, tetrameric alcohol dehydrogenase from Escherichia coli have recently been determined in the absence and presence of NAD . The structure of the E . coli enzyme is similar to those of the dimeric mammalian alcohol dehydrogenases, but it has a deletion of 21 residues located at the surface of the catalytic domain . The catalytic zinc ions have two different types of coordination, which are also observed in the class III dimeric mammalian alcohol dehydrogenase . Comparison of the structures provide new insights into the relationship between tetrameric and dimeric alcohol dehydrogenases and provide a link to the structure of the tetrameric yeast alcohol dehydrogenase.

J Liposome Res, 2002, 12(3), 271 - 83
Enhanced protein loading into liposomes by the multiple crossflow injection technique; Wagner A et al.; Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity . Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method . Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration . Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase . The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures . Additionally, scalability studies demonstrate reproducibility independent of the preparation volume . Vesicle size distribution and encapsulation efficiency remain constant . Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.

J Vet Pharmacol Ther, 2003 Feb, 26(1), 65 - 9
Influence of Escherichia coli endotoxin-induced fever on the pharmacokinetic behavior of marbofloxacin after intravenous administration in goats; Waxman S et al.; The pharmacokinetic behavior of marbofloxacin was studied in seven healthy goats and in the same goats with induced fever after single-dose intravenous (i.v.) administration of 2 mg/kg b.w . Fever was induced by the administration of Escherichia coli endotoxin . Drug concentration in plasma was determined by high-performance liquid chromatography (HPLC) . Drug distribution was somehow altered by fever as febrile goats showed a volume of distribution at steady-state (Vss = 0.72 +/- 0.15 L/kg) lower than normal goats (Vss = 1.19 +/- 0.33 L/kg) . The elimination of the drug was also modified . Total plasma clearance (Cl) decreased from 0.24 +/- 0.12 L/kg/h in healthy animals to 0.13 +/- 0.05 L/kg/h in animals with endotoxin-induced fever, which is related to an increase in the area under the plasma concentration-time curve (AUC) . Consequently, mean residence time (MRT) was also slightly increased in sick animals (MRT = 5.28 +/- 00.99 and 6.09 +/- 01.45 h, in healthy and febrile animals, respectively).

Mol Microbiol, 2003 Mar, 47(5), 1317 - 28
Trigger Factor and DnaK possess overlapping substrate pools and binding specificities; Deuerling E et al.; Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli . Here, we show that DnaK and TF share a common substrate pool in vivo . In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C . A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent . Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins . Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries . Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK . In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF . In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK . In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Eur J Biochem, 2003 Mar, 270(5), 1000 - 5
Nucleotide excision repair rates in rat tissues; Gospodinov A et al.; We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts . After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells . Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts . After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts . Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen . When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.

Eur J Biochem, 2003 Mar, 270(5), 929 - 38
Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase; Solstad T et al.; Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues {Solstad, T . and Flatmark, T . (2000) Eur . J . Biochem.267, 6302-6310} . Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues . First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E . coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da . Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77) . Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days . The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C . Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network . Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties . The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.

Eur J Biochem, 2003 Mar, 270(5), 921 - 8
Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts; Shimaoka T et al.; Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor . We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli . Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH . The Km value for DHA and the summed Km value for GSH were determined to be 53 +/- 12 micro m and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s-1 . Incubation of 10 microm DHAR with 1 mm DHA and 10 microm GSH resulted in stable binding of GSH to the enzyme . Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate . Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA . The mechanism of catalysis of spinach chloroplast DHAR is proposed.

Eur J Biochem, 2003 Mar, 270(5), 880 - 91
Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS; Fischer E et al.; We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities . This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from {1-13C} and {U-13C}glucose experiments . Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism . Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety . Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E . coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle . Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction . Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism . Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.

Biochem J, 2003 Jun 1, 372(Pt 2), 625 - 30
Cobalt activation of Escherichia coli 5'-nucleotidase is due to zinc ion displacement at only one of two metal-ion-binding sites; McMillen L et al.; Escherichia coli 5'-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co(2+) . Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E . coli 5'-nucleotidase (Asp(41), His(43), Asp(84), His(117), Glu(118), His(217) and His(252)) were selected for modification using site-directed mutagenesis of the cloned ushA gene . On the basis of comparative studies between the resultant mutant proteins and the wild-type enzyme, a model is proposed for E . coli 5'-nucleotidase in which a Co(2+) ion may displace the Zn(2+) ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn(2+) ion already present . The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp(84), Asn(116), His(217) and His(252), leading to the observed increase in 5'-nucleotidase activity.

J Am Chem Soc, 2003 Mar 5, 125(9), 2541 - 5
Slow diffusion of macromolecular assemblies by a new pulsed field gradient NMR method; Ferrage F et al.; The translational diffusion coefficient of an integral membrane protein/surfactant complex has been measured using a novel pulsed field gradient NMR method . In this new approach, the information about the localization of the molecules is temporarily stored in the form of longitudinal magnetization of isotopes with long spin-lattice relaxation times . This allows one to increase the duration of the diffusion interval by about 1 order of magnitude . Unlike standard proton NMR methods using pulsed field gradients and stimulated echoes, the new method can be applied to macromolecular assemblies with diffusion coefficients well below 10(-10) m(2) s(-1), corresponding to masses in excess of 25 kDa in aqueous solution at room temperature . The method was illustrated by application to a water-soluble complex of tOmpA, the hydrophobic transmembrane domain of bacterial outer membrane protein A, with the detergent octyl-tetraoxyethylene (C(8)E(4); overall mass of complex approximately 45 kDa) . The diffusion coefficient was found to be D = (4.99 +/- 0.07) x 10(-11) m(2) s(-1), consistent with measurements by size exclusion chromatography and by ultracentrifugation . The method has also been applied to a solution of recombinant human tRNA(3)(Lys), which has a molecular mass of 24 kDa, and the diffusion coefficient D = (1.05 +/- 0.015) x 10(-10) m(2) s(-1).

Appl Biochem Biotechnol, 2003 Feb, 104(2), 97 - 104
Beta-D-galactopyranosyl azide: its one-step quantitative synthesis using E461G-beta-galactosidase (Escherichia coli) and a demonstration of its potential as a reagent for molecular biology; Rob B et al.; A simple one-step synthesis of beta-D-galactopyranosyl azide from o-nitrophenyl-beta-D-galactopyranoside and azide catalyzed by E461G-beta-galactosidase is described . The synthesis is quantitative in the presence of excess azide and only the beta anomer is produced . The product was purified (71% yield) from the other reaction components by extraction with ethyl acetate, silica gel chromatography, and crystallization . The purity was verified by GLC, TLC, and NMR . Thus, E461G-beta-galactosidase is able to specifically and quantitatively form beta-D-galactopyranosyl-azide . The purified beta-D-galactopyranosyl azide inhibited the growth of Escherichia coli that express beta-galactosidase but not of E . coli that do not . Growth is stopped because beta-galactosidase catalyzes the hydrolysis of the beta-galactopyranosyl-azide, and the azide that is produced inhibits cell growth . This selective inhibition of growth has potential application in molecular biology screening.

Pac Symp Biocomput . 2003;:327-38.
Identification of regulatory binding sites using minimum spanning trees; Olman V et al.; Recognition of protein-binding sites from the upstream regions of genes is a highly important and unsolved problem . In this paper, we present a new approach for studying this challenging issue . We formulate the binding-site recognition problem as a cluster identification problem, i.e., to identify clusters in a data set that exhibit significantly different features (e.g., density) than the overall background of the data set . We have developed a general framework for solving such a cluster identification problem . The foundation of the framework is a rigorous relationship between data clusters and subtrees of a minimum spanning tree (MST) representation of a data set . We have proposed a formal and general definition of clusters, and have demonstrated that a cluster is always represented as a connected component of the MST, and further it corresponds to a substring of a linear representation of the MST . Hence a cluster identification problem is reduced to a problem of finding substrings with certain features, for which algorithms have been developed . We have applied this MST-based cluster identification algorithm to a number of binding site identification problems . The results are highly encouraging.

Pac Symp Biocomput . 2003;:303-14.
MAP: searching large genome databases; Kahveci T et al.; A number of biological applications require comparison of large genome strings . Current techniques suffer from both disk I/O and computational cost because of extensive memory requirements and large candidate sets . We propose an efficient technique for alignment of large genome strings . Our technique precomputes the associations between the database strings and the query string . These associations are used to prune the database-query substring pairs that do not contain similar regions . We use a hash table to compare the unpruned regions of the query and database strings . The cost of the ensuing search is determined by how the hash table is constructed . We present a dynamic strategy that optimizes the random disk I/O needed for accessing the hash table . It also provides the user a coarse grain visualization of the similarity pattern quickly before the actual search . The experimental results show that our technique aligns genome strings up to 97 times faster than BLAST.

Plant Mol Biol, 2003 Jan, 51(1), 119 - 33
Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase; Faldt J et al.; Picea abies (L.) Karst . (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens . In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002) . Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts . The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens . In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level . Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce . In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E . coli and functionally characterized in vitro . The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase . The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product) . (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae.

Plant Mol Biol, 2003 Jan, 51(2), 183 - 9
Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion; Park SY et al.; During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract . In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells . We reported two molecules involved in this adhesion event . One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin . SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere . The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style . For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.

Acta Pol Pharm, 2002 Sep-Oct, 59(5), 379 - 86
Search for new non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-1 RT); Brzezinska E et al.; The derivatives of dibenzoxazocinone, dibenzoxadiazocine, dibenzoxadiazonine, and benzodiazepine systems were synthesized as potential lead compounds for inhibitors of HIV-1 reverse transcriptase . The suggested structures were derived from analysis of the described spatial and physicochemical requirements for HIV-1 RT inhibitors . All of the evaluated compounds (apart from the oxadiazocine derivatives) showed a week inhibitory activity on recombinant HIV-1 RT from Escherichia coli.

Comp Immunol Microbiol Infect Dis, 2003 Jan, 26(1), 55 - 63
Serotypes of Escherichia coli isolated from healthy infants in Berlin, Germany and Melbourne, Australia; Bettelheim KA et al.; The characterization of Escherichia coli strains isolated from healthy infants under one year of age with respect to O:H serotype, K1 and K5 antigens in two disparate parts of the developed world was the purpose of this investigation . A total of 450 strains were examined, 264 from Berlin and 186 from Melbourne . Of all the 220 different O:H serotypes found, 179 were only isolated once, 90 in Berlin and 89 in Melbourne . However, 30 of the 41 O:H serotypes (73.2%) found more than once were isolated in both centers . The most commonly identified serotypes were found in both centers and included O1:H-; O1:H7; O2:H2; O2:H4; O2:H7; O4:H5; O6:H-; O6:H1; O15:H1; O18:H7; O25:H1; and 075:H- . Potentially pathogenic serotypes were found in both cities . Enteropathogenic E . coli (EPEC)-associated serotypes (O18:H7; O26:H-; O44:H34; O86:H-; O128:H2) were present in 11 cases and enterohaemorrhagic E . coli (EHEC)-associated types including O26:H11; O128:H2) were present in four cases . The distributions of serotypes found were similar in the two cities, strongly suggesting the wider applicability of these results.

Intervirology, 2002, 45(4-6), 340 - 9
Stop codon insertion restores the particle formation ability of hepatitis B virus core-hantavirus nucleocapsid protein fusions; Kazaks A et al.; In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles . Although the C-terminus of a C-terminally truncated HBc (HBc) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines . Previously, we described a new system to generate HBc mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain {J Gen Virol 1997;78:2049-2053} . Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein . To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of 'non-mosaic' HBc fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein . Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBc particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa . The mosaic particles formed exhibited PUUV-N protein antigenicity . Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1-114, aa 1-213 and aa 340-433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1-114 or aa 1-213 of the N protein . Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated . In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and 'problematic', e.g . hydrophobic, hantavirus sequences.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2237 - 42 Epub 2003 Feb 24.
Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor; Zhu Y et al.; Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout . Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig, Xenopus, zebrafish, and Fugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR . The 13 vertebrate genes identified seem to belong to an unknown gene family . Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named alpha, beta, and gamma) within this gene family . Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains . The transcript sizes of the human alpha, beta, and gamma putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively . Recombinant human alpha, gamma, and mouse beta proteins produced in an Escherichia coli expression system demonstrated high affinity (K(d) = 20-30 nM) saturable binding for progesterone . Further analysis of binding to the gamma-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t(1/2) = 2-8 min) . These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2278 - 83 Epub 2003 Feb 24.
The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54; Bordes P et al.; Members of the protein family called ATPases associated with various cellular activities (AAA(+)) play a crucial role in transforming chemical energy into biological events . AAA(+) proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action . The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA(+) mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the sigma(54)-RNA polymerase . Closed promoter complexes formed by the sigma(54)-RNA polymerase are substrates for the action of PspF . By using a protein fragmentation approach, we identify here at least one sigma(54)-binding surface in the PspF AAA(+) domain . Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one sigma(54)-binding surface . This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between sigma(54) and PspF are transient and could be part of a mechanism used generally by other AAA(+) proteins to regulate activity.

Protein Eng, 2002 Dec, 15(12), 1021 - 4
Novel mutant human fibronectin FIII9-10 domain pair with increased conformational stability and biological activity; van der Walle CF et al.; The ninth and tenth type III domains (FIII9-10) in the central cell binding domain of human fibronectin contain integrin receptor binding sites, including RGD in FIII10 and a synergy site, PHSRN, in FIII9 . The specific amino acids that contribute to cell binding have been identified by the use of wild-type and mutant fragments of human fibronectin containing the FIII9-10 domain pair . At high concentrations FIII9-10 mimics, to a large extent, the biological activity of the full-length fibronectin molecule . However, FIII9 is conformationally unstable, even in the context of the FIII9-10 pair . Here we report the construction of a series of hybrid mouse-human FIII9-10 pairs that confer varying degrees of conformational stability to FIII9 . The conformational stability of the human FIII9 module was increased 2-3-fold by substitution of Leu1408 with Pro . We demonstrate that the biological activity of this mutant is enhanced . The resulting FIII9-10 mutant has good solution properties and will provide a template into which further mutations can be incorporated in order to probe the structure-function relationship of the cell binding module of fibronectin.

J Gen Physiol, 2003 Mar, 121(3), 227 - 44
On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL; Anishkin A et al.; COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear . The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H . Spencer, A.T . Lee, M.T . Barclay, and D.C . Rees . 1998 . SCIENCE: 282:2220-2226) . The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions . Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E . coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle . Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments . The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant . The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model . Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating . Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially . The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls . The data strongly suggest that in contrast to previous gating models (Sukharev, S., M . Betanzos, C.S . Chiang, and H.R . Guy . 2001a . NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations . The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.

Mol Cell Proteomics, 2003 Jan, 2(1), 11 - 8
Sensitivity and specificity of photoaptamer probes; Smith D et al.; The potential of photoaptamers as proteomic probes was investigated . Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2'-deoxyuridine . A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction . The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins . The range of target/non-target specificity varies from 100- to >10(6)-fold with most values >10(4)-fold . The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction . Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases . The application of photoaptamer technology to proteomics was investigated in microarray format . Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum . The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology.

J Biol Chem, 2003 May 9, 278(19), 17314 - 9 Epub 2003 Feb 24.
A developmentally regulated two-component signal transduction system in Chlamydia; Koo IC et al.; Two-component systems allow bacteria to adapt to changing environmental conditions and may induce developmental changes necessary for survival . Chlamydia trachomatis alternates between two distinct developmental forms, each optimized for survival in a separate niche . Transcriptional regulation of development is not understood . The C . trachomatis genome sequence revealed a single pair of genes (ctcB-ctcC) predicted to encode proteins with sequence conservation to bacterial two-component systems . Sequence analysis revealed that the sensor kinase, CtcB, possessed an energy-sensing PAS domain and phosphorylation site . The response regulator, CtcC, had homology to varsigma(54) activators, possessing conserved receiver and ATPase domains and phosphorylation site, but lacked the C-terminal DNA-binding domain . ctcB and ctcC were expressed late in the developmental cycle, and both proteins were detected in EB lysates . Recombinant CtcB and CtcC were purified from denatured Escherichia coli inclusion bodies and refolded . CtcC was found to aggregate as dimers and tetramers in solution . In vitro phosphorylation assays showed that CtcB autophosphorylated in the presence of Mg(2+), Mn(2+), and Fe(2+) and transferred the phosphoryl group in the presence of CtcC . Collectively, these results show that CtcB and CtcC function as a two-component system and are likely responsible for transcriptional regulation by varsigma(54) holoenzyme during late-stage chlamydial development.

Water Res, 2003 Apr, 37(7), 1557 - 70
Disinfection efficacy of organic chloramines; Donnermair MM et al.; The disinfection efficacies of model organic chloramines were investigated . Twenty amino acids and two nucleic acid bases were chlorinated separately with sodium hypochlorite at a Cl:N molar ratio of 0.4:1, and were then used to treat an E . coli suspension for 60 min . DPD/FAS titration was carried out to obtain the concentration of the chlorinated nitrogenous organic compounds as a function of time . In addition, membrane introduction mass spectrometry (MIMS) was used to quantify inorganic chloramines (mono-, di-, and trichloramine) . The results of these experiments showed that the organic chloramines examined in this research had little or no effect on the viability of E . coli . MIMS analyses demonstrated that there was no quantifiable formation of inorganic chloramines when the organic nitrogen compounds were chlorinated .

Biochemistry, 2003 Mar 4, 42(8), 2449 - 55
Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes; Kroeger KM et al.; 2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents . The lesion undergoes beta-elimination to form a second metastable electrophilic product (4) . In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor . 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested . One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme . Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4) . The observed results are ascribable to characteristics of the enzymes and the lesions . These enzymes are also important for the removal of oxidative base lesions . These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.

Hua Xi Yi Ke Da Xue Xue Bao, 2001 Jun, 32(2), 163 - 6
{DNA sequence analysis and expression of the recombinant plasmid pBX1 from Borrelia burgdorferi B31 strain}; Xie Y et al.; OBJECTIVE: This study was to provide the target antigen for the development of a Lyme disease vaccine and serodiagnosis reagent . METHODS: We used the automatic DNA sequencing machine (Model 377) to detect the nucleotide sequence of the inserted part of the recombinant plasmid pBX1 from Borrelia burgdorferi B31 strain . The restriction enzyme map of the inserted part of pBX1 was analysed by using computer software . The expressed product of pBX1 in E . coli XLI-Blue MRF was analysed by using SDS-PAGE and western-blotting . RESULTS: 1 . DNA sequencing showed that pBX1 contained a 477bp inserted gene fragment, and when it was compared with the published sequence of the specific region of the gene of the 83 kd antigen protein from Borrelia burgdorferi B31 strain, only one amino acid codon was different . 2 . The restriction enzyme map of the inserted part of pBX1 was successfully constructed . 3 . The recombinant plasmid pBX1 expressed a 29 kd fusion protein in E . coli XL1-Blue MRF' after induced with IPTG . The recombinant fusion protein could be recongnized by rabbit polyclonal antiserum against Borrelia burgdorferi B31 strain . CONCLUSION: A recombinant plasmid which contains the gene fragment encoding the specific region of the 83 kd antigen protein from Borrelia burgdorferi B31 strain has been successfully constructed . The recombinant plasmid can stably express 29 kd fusion protein in E . coli XL1-Blue MRF' . These results could serve as a base of further studies on the usefulness of the fusion protein in serodiagnosis and vaccine for Lyme disease.

Org Lett, 2002 Oct 31, 4(22), 3867 - 9
5-amino-2'-deoxyuridine, a novel thymidine analogue for high-resolution footprinting of protein-DNA complexes; Storek MJ et al.; {formula: see text} 5-Amino-2'-deoxyuridine 5'-triphosphate, an analogue of deoxythymidine triphosphate, was synthesized and found to be a substrate of Taq DNA polymerase . The DNA-borne analogue underwent selective chemical reaction with permanganate . The use of 5-amino-dU as an interference probe was validated using the Ada protein/ada promoter complex . The performance of 5-amino-dU in interference footprints is similar to that of the previously described analogue 5-hydroxy-dU, but the former is incorporated more readily into DNA during enzymatic polymerization.

Hua Xi Yi Ke Da Xue Xue Bao, 2002 Jan, 33(1), 35 - 9
{Construction and identification of recombinant shuttle-plasmid with ESAT-6 from Mycobacterium tuberculosis}; Chen W et al.; OBJECTIVE: To construct a recombinant BCG secretively expressing ESAT-6 of Mycobacterium tuberculosis . METHODS: alpha-antigen(alpha-Ag) signal sequence and esat-6 gene were amplified from the genome of Bacille Calmette-Guerin (BCG) and Mycobacterium tuberculosis by PCR respectively . esat-6 gene was cloned in E . coli-BCG shuttle-plasmid pMV261 to get pME . Then a new recombinant plasmid pSME was constructed by inserting BCG alpha-Ag signal sequence into pME . RESULTS: The cloned genes alpha-Ag signal sequence and esat-6 were correctly inserted into the vector pMV261, which was confirmed by restriction endonuclease digestion and PCR amplification of pSME . CONCLUSION: pSME was expected to secretively express ESAT-6 of Mycobacterium tuberculosis in BCG . This study provides the possibility of further researches on the development of new anti-tuberculosis vaccine.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2363 - 8 Epub 2003 Feb 21.
Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation; Xiao B et al.; The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis . Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies . Many of the functions of pRb are mediated by its regulation of the E2F transcription factors . To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein . The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein . The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor . The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both . We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own . In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE . Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3149 - 54 Epub 2003 Feb 21.
Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis; Cheng YQ et al.; Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a beta-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein . Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain . The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG . Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis . Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules . These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to "AT-less" multifunctional type I PKS proteins for polyketide biosynthesis . This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

J Biol Chem, 2003 May 2, 278(18), 16372 - 80 Epub 2003 Feb 20.
C-terminal deletions of the Escherichia coli RecA protein . Characterization of in vivo and in vitro effects; Lusetti SL et al.; A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified . In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C . In vitro, the deletions enhance binding to duplex DNA as previously observed . We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346 . In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions . The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction . However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9 . The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein . These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

J Biol Chem, 2003 May 2, 278(18), 16389 - 96 Epub 2003 Feb 20.
The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein; Eggler AL et al.; The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E . coli ssDNA-binding protein (SSB) . The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions . The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein . The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein . Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA . However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation . RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure . Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A . The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA . The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.

J Biol Chem, 2003 May 9, 278(19), 17114 - 20 Epub 2003 Feb 20.
Platelet-derived growth factor (PDGF)-C, a PDGF family member with a vascular endothelial growth factor-like structure; Reigstad LJ et al.; Platelet-derived growth factor (PDGF)-C is a novel member of the PDGF family that binds to PDGF alphaalpha and alphabeta receptors . The growth factor domain of PDGF-C (GFD-PDGF-C) was expressed in high yields in Escherichia coli and was purified and refolded from inclusion bodies obtaining a biologically active growth factor with dimeric structure . The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains three intramonomeric disulfide bonds, which is in accordance with GFD-PDGF-C being a member of the cystine knot superfamily of growth factors . The recombinant GFD-PDGF-C was characterized by CD, fluorescence, NMR, and infrared spectroscopy . Together, our data indicate that GFD-PDGF-C is a highly thermostable protein that contains mostly beta-sheet secondary structure and some (6%) alpha-helix structure . The structural model of PDGF-C, obtained by homology-based molecular modeling using the structural representatives of this family of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to PDGF-B . The modeled structure can give further insights into the function and specificity of this molecule.

Chem Phys Lipids, 2003 Jan, 122(1-2), 185 - 90
Acyl chains are responsible for the irreversibility in the Escherichia coli alpha-hemolysin binding to membranes; Herlax V et al.; Hemolysin (HlyA) is an extracellular protein secreted by uropathogenic strains of Escherichia coli . The mature HlyA is able to bind to mammalian target cell membranes including those of the immune system, causing lysis . The lytic activity is absolutely dependent upon the Hlyc-dependent acylation of Prohemolysin . In this paper we show, through Trp fluorescence studies and denaturation in Guanidine hydrochloride, that the acylation is responsible for the loose conformation of the active protein, necessary to transform it from soluble to membrane-bound form . Previous studies showed that toxin binding to the bilayers occurs in, at least two ways, a reversible adsorption and irreversible insertion . We demonstrated that the irreversibility is due to the acyl chains in the HlyA, as shown by the protein transfer from multilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) to large unilamellar vesicles containing POPC-doxyl as protein fluorescence quencher.

Protein Expr Purif, 2003 Feb, 27(2), 384 - 90
One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography; Glynou K et al.; A hexahistidine tag was fused to the N-terminus of apoaequorin . A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells . Apoaequorin was overexpressed under the control of tac promoter . It was found, however, that most of the protein existed in the form of inclusion bodies . Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose . The purity, as determined by SDS-PAGE analysis, was greater than 80% . The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture . The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin . The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude . As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8 .

Protein Expr Purif, 2003 Feb, 27(2), 365 - 74
Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli; McNulty DE et al.; In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E . coli mRNAs or 9.8% when an arginine residue is encoded for . When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity . The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein . When expressed in E . coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted . Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches . Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster . Translation proceeded in the missense frame to the next termination codon . In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG . Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold . We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E . coli, allowing for efficient and accurate translation of heterologous gene products .

Protein Expr Purif, 2003 Feb, 27(2), 338 - 45
Expression, purification, and characterization of metallothionein-A from rainbow trout; Vergani L et al.; Recombinant metallothionein A (MT-A) from rainbow trout has been successfully produced in milligram quantities in Escherichia coli . cDNA has been subcloned into pGEX-6P.1 vector, in-frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail . Purification to electrophoretic homogeneity has been obtained by affinity chromatography using GSH-Sepharose . After enzymatic cleavage of GST tail, the MT-A moiety shows a molecular weight, corresponding to the expected one (6630 Da) . The final yield of the entire expression and purification process was about 5 mg of pure metallothionein per liter of bacterial culture . The effects of different reducing and alkylating agents have been evaluated at the level of the formation of higher molecular weight aggregates . To investigate the metal-binding ability of the recombinant MT-A, we carried out a spectrophotometrical titration with cadmium ions . Finally, we checked the metal dissociation by recording the UV absorbance of the protein as a function of the environmental pH .

Protein Expr Purif, 2003 Feb, 27(2), 313 - 8
Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein; Monteiro RA et al.; The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression . The C-terminal domain of the H . seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein . Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H . seropedicae nifB promoter region in vitro . In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K . pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription .

Protein Expr Purif, 2003 Feb, 27(2), 272 - 8
Expression, oxidative refolding, and characterization of six-histidine-tagged recombinant human LECT2, a 16-kDa chemotactic protein with three disulfide bonds; Ito M et al.; Human LECT2 is a 16-kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulfide bonds . Here, we present the oxidative refolding of (His)(6)-LECT2, an N-terminally (His)(6)-tagged recombinant protein of human LECT2 . (His)(6)-LECT2 was overproduced in Escherichia coli in the form of insoluble aggregates, solubilized with 8 M urea in the presence of 10 mM DTT, and purified and refolded on Ni-NTA agarose by lowering the urea concentration before the elution . This process, however, gave a mixture of oligomers of (His)(6)-LECT2 as well as the monomer, whose composition was as low as 36% . The oligomers formed as a result of incorrect intermolecular disulfide bonds . After the refolding on Ni-NTA agarose (step 1), the disulfide bonds were shuffled using a glutathione redox buffer (step 2) and the remaining thiols were completely oxidized (step 3) to improve the yield of correctly folded, monomeric (His)(6)-LECT2 . The monomer composition was significantly improved to 81% by the three-step refolding method and the monomer thus obtained was shown to have the same conformation as the authentic LECT2 produced in CHO cells by CD and NMR spectroscopies . The yield of (His)(6)-LECT2 was 1.0 mg/L E . coli culture and was 16 times as high as that in our previous report, in which (His)(6)-LECT2 was purified from the soluble fractions of E . coli cell lysates .

Protein Expr Purif, 2003 Feb, 27(2), 267 - 71
Expression and purification of a biologically active basic fibroblast growth factor fusion protein; Sheng Z et al.; Basic fibroblast growth factor (bFGF) is a potent mitogen of many cell types and plays an important role in angiogenesis . To help identify proteins that bind to bFGF and mediate its intracellular transport and signaling, we overexpressed and purified a bFGF fusion protein in Escherichia coli . The fusion protein consists of bFGF fused to the C-terminus of glutathione S-transferase (GST) . The GST-bFGF fusion protein was purified using SP-Sepharose and glutathione-Sepharose affinity chromatography . The ability of the purified GST-bFGF to stimulate the growth of human umbilical vein endothelial cells (HUVECs) was equivalent to that of purified recombinant 18 kDa bFGF . Copyright 2002 Elsevier Science (USA)

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