Protein Expr Purif, 2003 Feb, 27(2), 253 - 8 Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli; Liu YF et al.; The gene encoding a putative mature antitumor-analgesic peptide (AGAP) from the venom of the Chinese scorpion Buthus martensii Karsch was obtained by polymerase chain reaction (PCR) according to its cDNA sequence and expressed in Escherichia coli . While most of the recombinant AGAP was expressed in the form of insoluble inclusion body . The recombinant AGAP was purified to homogeneity by metal chelating affinity chromatography . Pharmaceutical tests showed that the recombinant AGAP has both analgesic and antitumor activities on mice . Protein Expr Purif, 2003 Feb, 27(2), 202 - 9 Expression and purification of the anticomplementary peptide Sh-CRIT-ed1 (formerly Sh-TOR-ed1) as a tetramultimer in Escherichia coli; Oh KS et al.; Many complement inhibitors found in plants and other organisms have been recognized as an antiinflammatory drug . Sh-CRIT-ed1 is a complement inhibitory peptide, present on the Schistosoma parasite surface . In the present study, we expressed chemically synthesized oligonucleotides encoding Sh-CRIT-ed1 with an additional hexahistidine codon at the C-terminus and purified in Escherichia coli BL21 . The cloned gene, which was multimerized four times in pBlue-script II KS (+) at the isoschizomer sites (BamHI, BglII), was named Sh4, and expressed in E . coli BL21 harboring pGEX-KG . The fusion protein (GST-Sh4) was purified with high yield successively by affinity chromatographies of glutathione-Sepharose 4B and Ni-NTA-agarose . Recombinant Sh-CRIT-ed1 was obtained readily by thrombin digestion and CNBr cleavage of GST-Sh4, and the yield was 9.03 mg from 1-liter culture of E . coli BL21 harboring pGEX-Sh4 . The recombinant Sh-CRIT-ed1 showed strong anticomplementary activity (IC(50) = 6.02 microM) by complement haemolysis assay . J Chromatogr A, 2003 Feb 7, 986(2), 291 - 6 High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection; Han YS et al.; A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed . In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v) . The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative . A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose . This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E . coli overexpressing dxs gene . It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present. Toxicol Pathol, 2003 Jan-Feb, 31(1), 14 - 21 Safety evaluation of recombinant staphylokinase in rhesus monkeys; Lu Q et al.; Recombinant staphylokinase (rSTAR) is a profibrinolytic agent of bacterial origin . The objective of this study was to assess the toxicity of rSTAR administered with bolus intravenous infusion in rhesus monkeys (2/sex/group) at the dosages of 0, 4, 14, and 49 mg/kg/day for 2 weeks . The clinical signs were thickening of the skin in all animals and mild hematoma formation in three dosage groups at the injection sites . There were no effects on body weight, absolute or relative organ weights, ophthalmology, or electrocardiogram . Urinalysis indicated that 2 monkeys in 14 or 49 mg/kg/day group developed proteinuria and mild hematuria . Increases in serum BUN levels (14 and 49 mg/kg/day), ALT activity, and bilirubin levels (49 mg/kg/day), and decreases in red blood cell counts, hemoglobin concentrations and Hct values (49 mg/kg/day) were observed at week 2 . Significant prolongtion of APTT, PT, and TT (14 and 49 mg/kg/day), and decreases in circulating plasminogen levels (3 treatment groups) were noted . Dose-dependent increases in the titers of anti-rSTAR antibodies and neutralizing rSTAR activity were observed in the three treated groups . Increased neutralizing rSTAR activity diminished the phamacologic effects of rSTAR (ie, prolonged APTT, PT, and TT approaching baseline levels at week 2) . Histopathological findings included hemorrhage, and perivascular inflammatory cell infiltration at the injection sites, heptocellular degeneration characterized as cytoplasmic eosinophilia, vacuolation and condensed nuclei (49 mg/kg/day), effusion of RBCs and plasma within some Bowman's capsules and hyaline casts within the lumen of some renal tubules in the kidneys (14 and 49 mg/day/kg), and mild to moderate megakaryocyte hypoplasia with varying levels of pyknotic nuclei at all dose levels . Immune deposits in glomeruli in the kidneys from the three treated groups were detected . These changes were reversible following a 4-week recovery period . In the present preclinical evaluation of toxicity in monkeys, rSTAR is well toleratte at doses up to 49 mg/kg/day . The toxic target organs are the liver, kidney, and bone marrow. Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2735 - 8 Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein; Fujita C et al.; Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein . SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E . coli CF8295 (delta relA, delta spoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein. Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2719 - 22 Molecular characterization of the gene encoding rice allene oxide synthase and its expression; Ha SB et al.; The gene encoding rice allene oxide synthase, OsAOS, was intronless and had nucleotide sequences with the high GC content of 67% . Deduced amino acid sequences had very high similarity with other AOS proteins, in particular 74% similarity to barley, characterized by the conserved motifs of P450 cytochrome of the CYP74A family . Purified recombinant rice AOS protein expressed in Escherichia coli converted 13-hydroperoxylinolenic acid to allene oxide . Several restriction enzyme digestions and Southern analysis showed that OsAOS was likely to have two copies in its genome . The basal level of OsAOS expression was detected in various tissues and the transcription level was increased by jasmonate treatment. Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2600 - 5 Cloning and nucleotide sequence of the glutamate decarboxylase-encoding gene gadA from Aspergillus oryzae; Kato Y et al.; We cloned a genomic DNA encoding the glutamate decarboxylase (GAD) from Aspergillus oryzae using a 200-bp DNA fragment as the probe . This DNA fragment was amplified by the reverse transcription polymerase chain reaction with mRNA of A . oryzae as the template and degenerate primers designed from the conserved amino acid sequence of Escherichia coli GAD and Arabidopsis thaliana GAD . Nucleotide sequencing analysis showed that the cloned gene (designated gadA) encoded 514 amino acid residues and contained three introns . Southern hybridization showed that the gadA gene was on a 6.0-kb SacI fragment and that there was a single copy in the A . oryzae chromosome . The cloned gene was functional, because one transformant of A . oryzae containing multiple copies of the gadA gene had 10-fold the GAD activity and a 12-fold increase in gamma-aminobutyric acid production compared with the control strain. J Basic Microbiol, 2003, 43(1), 28 - 35 A novel membrane glycoprotein of Escherichia coli; Kumar M et al.; A novel glycoprotein (Gp45) has been isolated and purified from Escherichia coli . To our knowledge, Gp45 is the third glycoprotein isolated from E . coli membrane and it is the second in the non-pathogenic strain of the organism . For the isolation of Gp45, cell extract or membrane fraction was treated with sodium deoxycholate for 4 h and precipitated with trichloroacetic acid (TCA) . The supernatant fraction of TCA containing the Gp45 was further purified on DEAE-Sephadex A-25 . SDS-PAGE showed a single band at 45 kDa position that stained with periodic-Schiff reagent . It contained 60% carbohydrate and 40% protein content . The monosaccharide composition also substantiated the characteristics of the glycoprotein . The E . coli grown in presence of (14)C-glucosamine further confirmed the localization and biosynthesis of this glycoprotein on the membrane during the growth phase . Bacitracin, a general inhibitor of the glycosylation, inhibited its biosynthesis. Theor Appl Genet, 2003 Feb, 106(4), 629 - 35 Epub 2002 Oct 19. The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume; Clarke BC et al.; Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes . In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits . Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, epsilon hordein, which plays a role in beer foam stability in the brewing industry . Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins . It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour . Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour . Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking. Theor Appl Genet, 2003 Feb, 106(4), 620 - 8 Epub 2002 Dec 06. Isolation and expression analysis of salt stress-associated ESTs from contrasting rice cultivars using a PCR-based subtraction method; Sahi C et al.; Salt stress adversely affects the growth of rice plants . To understand the molecular basis of salt-stress response, four subtracted cDNA libraries were constructed employing specific NaCl-stressed tissues from salt-tolerant (CSR 27 and Pokkali) and salt-sensitive (Pusa basmati 1) rice cultivars . An efficient PCR-based cDNA subtraction method was employed for the isolation of the salt-stress responsive cDNA clones . In all, 1,266 cDNA clones were isolated in the course of this study, out of which 85 clones were end-sequenced . Database search of the sequenced clones showed that 22 clones were homologous to genes that have earlier been implicated in stress response, 34 clones were novel with respect to their function and six clones showed no homology to sequences in any of the public database . Northern analysis showed that the transcript expression pattern of selected clones was variable amongst the cultivars tested with respect to stress-regulation. J Biomed Sci, 2003 Mar-Apr, 10(2), 276 - 82 Expression and purification of E2/NS1 protein of hepatitis C virus and detection of anti-E2/NS1 antibodies in chronic liver disease patients; Pandya J et al.; Glycoproteins on the surface of viral particles present the main target of neutralizing antibodies . The structural proteins of most Flaviviruses are known to elicit neutralizing antibodies and, thus, to help in both the natural resolution of the infection and the protection from challenge with homologous hepatitis C virus (HCV) . Because such antigens are associated with the viral clearance in both humans and chimpanzees, we aimed to express the E2/NS1 protein of HCV and to study the role of anti-E2/NS1 antibodies in the natural resolution of HCV infection . The prevalence of anti-E2/NS1 antibodies to recombinant E2/NS1 protein was seen by Western blot in chronic liver disease patients (15 chronic hepatitis and 12 cirrhotic patients), who were positive for anti-HCV and negative for HBV infection . The study also included 2 negative controls (positive for HBV infection and negative for anti-HCV antibodies) and 2 healthy controls (negative for both HBV and HCV infection) . Anti-E2/NS1 was present in 20% of the chronic hepatitis and 16% of the cirrhosis patients . None of the controls were positive for anti-E2/NS1 antibodies . Serum samples positive for anti-E2/NS1 antibodies were also positive for HCV RNA by RT/PCR . Accordingly, the presence of anti-E2/NS1 may have very little or no role in the natural resolution of HCV infection . Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 611 - 4 Epub 2003 Feb 21. The organization of divalent cations in the active site of cadmium Escherichia coli fructose-1,6-bisphosphate aldolase; Hall DR et al.; Previously determined crystal structures of the zinc enzyme Escherichia coli class II fructose-1,6-bisphosphate aldolase display good agreement for the protein structure but a differing metal-ion organization in the active site . The structure of the enzyme with Cd(2+) in place of Zn(2+) has now been determined to 2.0 A resolution to facilitate cation identification . The protein structure was essentially identical to other structures and five Cd(2+) positions were identified . Two of the cations are at the active site; one corresponds to the catalytic ion and the other provides a structural contribution . These Cd(2+) sites are equivalent to two Zn(2+) ions observed when the enzyme is complexed with a transition-state mimic and confirm our assignment of the roles played by these ions. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 607 - 10 Epub 2003 Feb 21. Structure of a tetragonal crystal form of Escherichia coli 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; Kemp LE et al.; 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase is an essential enzyme in the mevalonate-independent pathway of isoprenoid biosynthesis . The structure of a tetragonal crystal form has been solved by molecular replacement and refined to 2.4 A resolution . Structure and sequence comparisons suggest that the enzyme is a suitable target for a structure-based approach to the development of novel broad-spectrum antibiotics . However, the absence of ligands in the enzyme active site together with the moderate resolution of the structure indicates that this tetragonal crystal form is inferior to that of a previously reported highly ordered monoclinic form {Richard et al . (2001), Nature Struct . Biol . 8, 641-647}. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 600 - 2 Epub 2003 Feb 21. Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase from Escherichia coli; Horsefield R et al.; The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then purified and crystallized . The initial crystals obtained were small and diffracted poorly . In order to facilitate structure determination, rational screening and sample-quality analysis using electron microscopy was implemented . The crystals of SQR from E . coli belong to the trigonal space group R32, with unit-cell parameters a = b = 138.7, c = 521.9 A, and diffract to 2.6 A resolution . The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 597 - 9 Epub 2003 Feb 21. The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral; Beddoe T et al.; Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight . These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools . An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized . Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 A resolution and belong to space group I4(1)22 . The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized . These crystals belong to space group P4(2)22 and diffract to 2.4 A resolution or better. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 587 - 90 Epub 2003 Feb 21. Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion protein; Zhu J et al.; Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers {(HR1-HR2)(3)} after the fusion between viral and cellular membranes . In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli . Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method . A complete data set was collected in-house to 1.9 A resolution from a single crystal . The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058 A . To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8 A using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 572 - 5 Epub 2003 Feb 21. Crystallization and preliminary analysis of native and N-terminal truncated isoforms of toluene-4-monooxygenase catalytic effector protein; Orville AM et al.; Single crystals have been obtained of the toluene 4-monooxygenase catalytic effector protein, the SeMet-enriched protein and a truncated isoform missing ten amino acids from the N-terminus . Complete X-ray diffraction data sets have been collected and analyzed to 2.0, 3.0 and 1.96 A resolution for the native, SeMet and truncated isoform crystals, respectively . The native and SeMet proteins crystallized in space group P6(1)22 (unit-cell parameters a = b = 86.41 +/- 0.15, c = 143.90 +/- 0.27 A), whereas the truncated isoform crystallized in space group P2(1)3 (a = b = c = 86.70 +/- 0.47 A) . Matthews coefficient calculations suggest either two or three molecules per asymmetric unit in the P6(1)22 space group and two molecules per asymmetric unit in the P2(1)3 space group . Experimental phases from MAD analysis of the SeMet isoform and molecular replacement of the truncated isoform confirm the presence of two molecules per asymmetric unit in each case . These crystallographic results are the first available for the evolutionarily related but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 569 - 71 Epub 2003 Feb 21. Crystallization and preliminary X-ray crystallographic studies of chorismate synthase from Helicobacter pylori; Ahn HJ et al.; Chorismate synthase (EC 4.6.1.4) catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate in the last step of the shikimate pathway . Chorismate synthase from Helicobacter pylori fused with an eight-residue C-terminal tag was overexpressed in soluble form in Escherichia coli . It was crystallized at 296 K using polyethylene glycol 400 as a precipitant . A set of X-ray diffraction data was collected to 2.5 A resolution using synchrotron radiation . The crystals belong to the tetragonal space group I4, with unit-cell parameters a = b = 145.79, c = 130.98 A . The asymmetric unit contains a tetramer, giving a crystal Volume per protein mass (V(M)) of 2.13 A(3) Da(-1) and a solvent content of 42.3%. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 563 - 5 Epub 2003 Feb 21. Crystallization and preliminary X-ray analysis of the Mj0684 gene product, a putative aspartate aminotransferase, from Methanococcus jannaschii; Yang JK et al.; A putative aspartate aminotransferase from the hyperthermophilic archaeon Methanococcus jannaschii encoded by the Mj0684 gene has been overexpressed in Escherichia coli and crystallized at 296 K using the sitting-drop vapour-diffusion method . The crystals belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 111.87, c = 60.86 A . They diffract to 2.2 A resolution using Cu Kalpha X-rays . The asymmetric unit contains a single subunit of the recombinant Mj0684 gene product, giving a corresponding V(M) of 2.25 A(3) Da(-1) and a solvent content of 45.3% by Volume . An X-ray diffraction data set has been collected to 2.2 A at 295 K. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 561 - 2 Epub 2003 Feb 21. Crystallization and preliminary X-ray crystallographic studies of phosphopantetheine adenylyltransferase from Helicobacter pylori; Eom SJ et al.; Phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) is an essential enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine to form 3'-dephospho-CoA . PPAT from Helicobacter pylori has been overexpressed in Escherichia coli and crystallized at 296 K using sodium chloride as a precipitant by the hanging-drop vapour-diffusion method . X-ray diffraction data have been collected to 2.00 A resolution at 100 K using synchrotron radiation . The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 80.50, c = 143.05 A, alpha = beta = 90, gamma = 120 degrees . Six monomers of PPAT are likely to be present in the asymmetric unit, giving a V(M) of 2.39 A(3) Da(-1) and a solvent content of 49%. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 558 - 60 Epub 2003 Feb 21. Expression, purification and preliminary crystallographic analysis of human sorbitol dehydrogenase; Darmanin C et al.; Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography . Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method . X-ray data were collected to 2.75 A resolution . The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees . This is the first crystallization report of human sorbitol dehydrogenase. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 544 - 6 Epub 2003 Feb 21. Purification, crystallization and preliminary X-ray analysis of Caenorhabditis elegans ubiquitin-conjugation enzyme M7.1; Gavira J JA et al.; M7.1 is a class IV ubiquitin-conjugation enzyme (UBC) that belongs to the ubiquitination cascade in Caenorhabditis elegans . The clone for this UBC has been overexpressed in Escherichia coli and the 16.7 kDa protein was purified from the soluble fraction . M7.1 was crystallized by sitting-drop vapor diffusion in 10% ethanol, 1.5 M NaCl at 277.5 K . Crystals diffracted to 1.75 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.3, b = 54.3, c = 60.2 A . The asymmetric unit contains a single monomer . A molecular-replacement model has been determined and refinement is in progress. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 532 - 4 Epub 2003 Feb 21. Expression, purification, crystallization and preliminary crystallographic analysis of Trypanosoma brucei phosphofructokinase; Keillor JW et al.; Phosphofructokinase from Trypanosoma brucei (TbPFK) was purified from a recombinant expression system in Escherichia coli by metal-affinity chromatography via its N-terminal His tag . The yield was 15-20 mg of pure enzyme per litre of culture . M(r) was shown to be 55 585 by mass spectrometry . Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with sodium formate as the precipitating agent . Monoclinic crystals of the apoenzyme grew within one week, as did orthorhombic crystals of PFK in the presence of enzymic reaction products or an active-site inhibitor . Initial attempts to solve the structure by molecular replacement with bacterial PFK structures as search models proved unrewarding, but a multiple-copy search with a polyalanine model was successful . In addition, heavy-atom soaking with platinum and mercury has yielded derivatives suitable for X-ray diffraction . A combination of the phase information from the molecular-replacement solution and the heavy-atom derivatives should allow structure solution of TbPFK . The availability of this first eukaryotic PFK structure will be of particular significance for structure-based drug design and will also provide important additional structural evidence for the allosteric control of PFK activity. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 522 - 5 Epub 2003 Feb 21. Crystallization and preliminary crystallographic studies of mung bean cytokinin-specific binding protein; Bujacz GD et al.; Cytokinins, or plant growth hormones, bind with very high affinity to cytokinin-specific binding proteins (CSBPs) . Recombinant mung bean CSBP has been overexpressed in Escherichia coli and crystallized in complex with zeatin, a natural plant growth hormone . The crystals belong to the hexagonal system, space group P6(2) or P6(4), with unit-cell parameters a = 113.62, c = 86.85 A, contain two to five copies of the protein in the asymmetric unit and diffract X-rays to 1.25 A resolution. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 512 - 4 Epub 2003 Feb 21. Crystallization and preliminary X-ray crystallographic analysis of SP1, a novel chaperone-like protein; Wang W et al.; SP1 (108 amino acids) is a boiling-stable stress-responsive protein . It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps) . SP1 activity is ATP-independent, similar to other small heat-shock proteins . Based on these features, it is expected that the structure-function relationship of SP1 will be unique . In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described . Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments . SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl . One to four single crystals appeared in each droplet within a few Days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks . Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 A . Efforts to crystallize the selenomethionine derivative of SP1 are in progress. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 509 - 11 Epub 2003 Feb 21. Crystallization and initial X-ray diffraction of BtuB, the integral membrane cobalamin transporter of Escherichia coli; Chimento DP et al.; BtuB, the cobalamin transporter from Escherichia coli, has been overexpressed, purified and crystallized . The purified protein was solubilized in n-octyl tetraoxyethylene (C(8)E(4)) and was crystallized using sitting-drop vapor diffusion with PEG 3350 and magnesium acetate as precipitants (pH 6.5) . Two crystal forms have been obtained . Crystal type I belongs to space group P3(1)21, with unit-cell parameters a = b = 81.6, c = 210.0 A, alpha = beta = 90, gamma = 120 degrees . Crystal type II belongs to space group P3(1)21, with unit-cell parameters a = b = 81.6, c = 226.0 A, alpha = beta = 90, gamma = 120 degrees . Each crystal form contains a monomer in the asymmetric unit . Diffraction for crystal type I extends to 2.0 A and diffraction for crystal type II extends to 2.7 A . Both crystal forms are suitable for structure determination. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 416 - 22 Epub 2003 Feb 21. Structural comparison of Escherichia coli L-asparaginase in two monoclinic space groups; Sanches M et al.; The functional L-asparaginase from Escherichia coli is a homotetramer with a molecular weight of about 142 kDa . The X-ray structure of the enzyme, crystallized in a new form (space group C2) and refined to 1.95 A resolution, is compared with that of the previously determined crystal form (space group P2(1)) . The asymmetric unit of the new crystal form contains an L-asparaginase dimer instead of the tetramer found in the previous crystal form . It is found that crystal contacts practically do not affect the conformation of the protein . It is shown that subunit C of the tetrameric form is in a conformation which is systematically different from that of all other subunits in both crystal forms . Major conformational differences are confined to the lid loop (residues 14-27) . In addition, the stability of this globular protein is analyzed in terms of the interactions between hydrophobic parts of the subunits. Nucleic Acids Res . 2003 Mar 1;31(5):e22. New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method; Kasarjian JK et al.; The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis . We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test . To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I) . Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)) . When multiple recognition sites were present, greater reductions in EOT values were observed . Once established in the cell, the plasmids were subjected to modification (EOT = 1.0) . We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells . Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively . Eco1158I, an isoschizomer of EcoBI, was also found in this study. Nucleic Acids Res, 2003 Mar 1, 31(5), 1554 - 64 Analysis of helicase activity and substrate specificity of Drosophila RECQ5; Ozsoy AZ et al.; RecQ5 is one of five RecQ helicase homologs identified in humans . Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms . To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity . However, little is known about other members of the RecQ family . Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases . Drosophila RECQ5 helicase is capable of unwinding 3' Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5' Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM. Nucleic Acids Res, 2003 Mar 1, 31(5), 1407 - 15 Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria; Yoon YG et al.; We have devised an efficient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques . In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modified genomes into E.coli . We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers . When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone . We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT-PCR . This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a first step towards true in vivo engineering of mammalian mitochondrial genomes. J Biol Chem, 2003 May 2, 278(18), 16381 - 8 Epub 2003 Feb 20. Magnesium ion-dependent activation of the RecA protein involves the C terminus; Lusetti SL et al.; Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP . We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange . In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur . The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure . The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels . Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro . We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations. J Biol Chem, 2003 Apr 18, 278(16), 13623 - 6 Epub 2003 Feb 20. F1F0-ATP synthase . Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit; Weber J et al.; The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque . In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1) . Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta . Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm) . Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding . When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1) . Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8 . Circular dichroism measurements showed that alpha N1-22 had significant helical content . Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface. Infect Immun, 2003 Mar, 71(3), 1527 - 37 Mutant Escherichia coli heat-labile toxin B subunit that separates toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions; Fraser SA et al.; The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function . Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane . Here we describe the properties of EtxB(H57S), a mutant B subunit with a His-->Ser substitution at position 57 . The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8(+)-T-cell apoptosis, (ii) activate nuclear translocation of NF-kappaB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant . However, its GM1 binding, cellular uptake, and delivery functions remained intact . This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line . These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB . Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking . Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits. Infect Immun, 2003 Mar, 71(3), 1497 - 504 Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells; Smith WE et al.; Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome . Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium . The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis . Stxs have one enzymatically active A subunit associated with five identical B subunits . The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA . We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity . We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8 . By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h . Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either . Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity . Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage . In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events. Infect Immun, 2003 Mar, 71(3), 1161 - 9 Rho GTPase is activated by cytotoxic necrotizing factor 1 in peripheral blood T lymphocytes: potential cytotoxicity for intestinal epithelial cells; Brest P et al.; Some strains of Escherichia coli related to acute cystitis or colitis produce a toxin named cytotoxic necrotizing factor 1 (CNF-1) . CNF-1 mediates its effects on epithelial cells or phagocytes via the permanent activation of small GTP-binding proteins, caused by the toxin-induced deamidation of Glu(63) of p21 Rho . The behavior of peripheral blood T lymphocytes during the acute phase of bacterial colitis has been poorly investigated . Our study was conducted to test whether (i) peripheral blood T lymphocytes can be activated by CNF-1 and (ii) CNF-1-activated T lymphocytes are cytotoxic against intestinal epithelial cells . Activation of T lymphocytes by CNF-1 was assessed by electrophoresis, flow cytometry, confocal microscopy, and electron microscopy studies . Assays for migration and adherence of CNF-1-treated T lymphocytes were performed in Transwell chambers with T84 intestinal epithelial cells grown on polycarbonate semipermeable filters . CNF-1 induced a decrease in the electrophoretic mobility of the GTP-binding protein Rho in treated T lymphocytes . CNF-1 provoked an increase in the content of actin stress fibers and pseudopodia in T lymphocytes . Several adherence molecules were clustered into cytoplasmic projections in CNF-1-treated T lymphocytes and adherence of such lymphocytes on the basolateral pole of T84 was increased, resulting in cytotoxicity toward epithelial cells . Such enhanced adherence in response to CNF-1 was dependent on p42-44(MAP) kinase activation of T lymphocytes . Taken together, these results suggest that CNF-1, by acting on T lymphocytes, may increase in an important fashion the virulence of certain strains of E . coli against the intestinal epithelia. J Mol Biol, 2003 Mar 7, 326(5), 1597 - 614 Quantitative analysis of aspartate receptor signaling complex reveals that the homogeneous two-state model is inadequate: development of a heterogeneous two-state model; Bornhorst JA et al.; The two-state model of receptor activation, in which a receptor population exists in equilibrium between a single on-state and a single off-state, has long been considered a viable model for the signaling behavior of bacterial chemoreceptors . Here, we show that this simple, homogeneous two-state model is adequate for a pure receptor population with just one adaptation state, but fails to account quantitatively for the observed linear relationship between the apparent attractant affinity (K(1/2)) and kinase activity (V(obs)(apo)) as the adaptation state is varied . Further analysis reveals that the available data are instead consistent with a heterogeneous two-state model in which covalent modification of receptor adaptation sites changes the microscopic properties of the on-state or off-state . In such a system, each receptor molecule retains a single on-state and off-state, but covalent adaptation generates a heterogeneous population of receptors exhibiting a range of different on-states or off-states with different microscopic parameters and conformations . It follows that covalent adaptation transforms the receptor from a simple, two-state toggle switch into a variable switch . In order to identify the microscopic parameters most sensitive to covalent adaptation, six modified, two-state models were examined in which covalent adaptation alters a different microscopic parameter . The analysis suggests that covalent adaptation primarily alters the ligand-binding affinity of the receptor off-state (K(D1)) . By contrast, models in which covalent adaptation alters the ligand-binding affinity of the receptor on-state, the maximal kinase stimulation of the on-state or off-state, cooperative interactions between receptors, or the assembly of the receptor-kinase signaling complex are inconsistent with the available evidence . Overall, the findings support a heterogeneous two-state model in which modification of the receptor adaptation sites generates a population of receptors with heterogeneous off-states differing in their attractant affinities.In the process of testing the effects of covalent adaptation on the assembly of the receptor-kinase signaling complex, a new method for estimating the stoichiometric ratio of receptor and CheA in the ternary signaling complex was devised . This method suggests that the ratio of receptor dimers to CheA dimers in the assembled complex is 6:1 or less. J Mol Biol, 2003 Mar 7, 326(5), 1539 - 47 Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue; Ohki S et al.; We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1 . Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase . We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge . There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation . The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle . Despite this conformational switch, there was little increase in the inhibitory potency with T38D . We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1. Biochim Biophys Acta, 2003 Mar 17, 1620(1-3), 32 - 8 Interaction of the parathyroid hormone receptor with the 14-3-3 protein; Tazawa H et al.; The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development . 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization . We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell . 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used . Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding . The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR . The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling. Exp Parasitol, 2002 Aug, 101(4), 210 - 4 Babesia gibsoni: molecular cloning and characterization of Rab6 and Rab11 homologues; Zhou J et al.; Members of the Rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes, individual Rab proteins have a remarkable degree of specific subcellular localization . As a first step towards developing a set of compartment specific probes for studying protein trafficking in Babesia-infected erythrocyte, here we describe the cloning and characterization of Rab6 and Rab11 gene homologues in Babesia gibsoni (BgRab6 and BgRab11) . The deduced amino acid sequence of both BgRab6 and BgRab11 contained the highly conserved GTP-binding consensus sequence and C-terminal cysteines . Northern blotting analysis of total RNA hybridized a 1.3 kb band on both BgRab6 and BgRab11 probed blots consistent with the expected size . Using a GTP-binding assay we demonstrated that Escherichia coli expressed recombinant BgRab6 and BgRab11 were able to bind GTP . BgRab6 and BgRab11 represent the first two molecular markers of B . gibsoni. Intensive Care Med, 2003 Feb, 29(2), 292 - 300 Epub 2003 Jan 14. Effects of epinephrine and norepinephrine on hemodynamics, oxidative metabolism, and organ energetics in endotoxemic rats; Levy B et al.; OBJECTIVE: To determine whether epinephrine increases lactate concentration in sepsis through hypoxia or through a particular thermogenic or metabolic pathway . DESIGN: Prospective, controlled experimental study in rats . SETTING: Experimental laboratory in a university teaching hospital . INTERVENTIONS: Three groups of anesthetized, mechanically ventilated male Wistar rats received an intravenous infusion of 15 mg/kg Escherichia coli O127:B8 endotoxin . Rats were treated after 90 min by epinephrine ( n=14), norepinephrine ( n=14), or hydroxyethyl starch ( n=14) . Three groups of six rats served as time-matched control groups and received saline, epinephrine, or norepinephrine from 90 to 180 degrees min . Mean arterial pressure, aortic, renal, mesenteric and femoral blood flow, arterial blood gases, lactate, pyruvate, and nitrate were measured at baseline and 90 and 180 min after endotoxin challenge . At the end of experiments biopsy samples were taken from the liver, heart, muscle, kidney, and small intestine for tissue adenine nucleotide and lactate/pyruvate measurements . MEASUREMENTS AND RESULTS: Endotoxin induced a decrease in mean arterial pressure and in aortic, mesenteric, and renal blood flow . Plasmatic and tissue lactate increased with a high lactate/pyruvate (L/P) ratio . ATP decreased in liver, kidney, and heart . The ATP/ADP ratio did not change, and phosphocreatinine decreased in all organs . Epinephrine and norepinephrine increased mean arterial pressure to baseline values . Epinephrine increased aortic blood flow while renal blood low decreased with both drugs . Plasmatic lactate increased with a stable L/P ratio with epinephrine and did not change with norepinephrine compared to endotoxin values . Nevertheless epinephrine and norepinephrine when compared to endotoxin values did not change tissue L/P ratios or ATP concentration in muscle, heart, gut, or liver . In kidney both drugs decreased ATP concentration . CONCLUSIONS: Our data demonstrate in a rat model of endotoxemia that epinephrine-induced hyperlactatemia is not related to cellular hypoxia. Ann N Y Acad Sci, 2002 Dec, 980, 13 - 22 Datamining protein structure databanks for crystallization patterns of proteins; Valafar H et al.; A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins . NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches . One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential . Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials . The most immediate result is an apparent correlation of crystallization potential with protein size . Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization . Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%. Am J Respir Cell Mol Biol, 2003 Mar, 28(3), 386 - 96 Shift toward an alternatively activated macrophage response in lungs of NO2-exposed rats; Garn H et al.; Inflammatory mechanisms are thought to play an important role in the pathogenesis of acute and chronic obstructive pulmonary diseases . In a rat inhalation model using continuous exposure to 10 ppm nitrogen dioxide for 1, 3, and 20 d, we investigated the inflammatory response with particular focus on the activation state of alveolar macrophages . Whereas the number of inflammatory cells and total protein concentration were increased in the bronchoalveolar lavage (BAL), the amount of the proinflammatory cytokine tumor necrosis factor-alpha was markedly reduced with increasing exposure time . In contrast, interleukin (IL)-10 and IL-6 were found at elevated levels and intracellular amounts of suppressor of cytokine signaling-3 protein increased in BAL cells . Upon in vitro lipopolysaccharide stimulation, BAL cells revealed reduced capability to produce the proinflammatory mediators tumor necrosis factor-alpha, IL-1 beta, and nitric oxide, but showed markedly increased transcription and protein release for IL-10 . In addition, elevated levels of IL-6, scavenger receptor B, and suppressor of cytokine signaling-3 mRNA were detected in BAL cells from exposed animals . Analyses of highly purified alveolar macrophages indicated that changes in the activation state of these cells were responsible for the observed effects . In conclusion, a priming toward development of the alternatively activated macrophage phenotype occurred in the lungs of rats following nitrogen dioxide inhalation. Am J Respir Cell Mol Biol, 2003 Mar, 28(3), 347 - 53 Surfactant protein A inhibits lipopolysaccharide-induced in vivo production of interleukin-10 by mononuclear phagocytes during lung inflammation; Chabot S et al.; We previously demonstrated that resident alveolar macrophages from naive mice do not synthesize interleukin (IL)-10, whereas mononuclear phagocytes (MP) recruited during the lung inflammatory process are transiently competent for IL-10 production when exposed to lipopolysaccharide (LPS) in vitro . As surfactant protein A (SP-A), a member of the collectin family, inhibits LPS-induced in vitro IL-10 formation by bone marrow-derived macrophages, we studied its effect on MP under in vivo inflammatory conditions . When mice with LPS-induced inflamed lungs were given a second intranasal LPS administration, IL-10 concentration recovered in the bronchoalveolar lavage fluids varied as a function of the time interval between the two LPS doses . Thus, IL-10 concentration increased with the number of MP up to Day 3, and then decreased to undetectable values within 24 h, despite a continued increase in the number of MP . Analysis of IL-10 mRNA from purified MP indicated that gene expression correlated with the IL-10 level in the bronchoalveolar lavage fluid . In contrast to IL-10 production, SP-A concentrations during LPS-induced inflammation decreased with a nadir at Day 3, and then increased significantly within 24 h . Furthermore, intranasal administration of exogenous SP-A to mice with LPS-induced inflamed lungs led to a repression of the IL-10 production . In summary, this study demonstrates for the first time an in vivo inhibitory role of SP-A on the anti-inflammatory activity of MP, through inhibition of IL-10 production. Avian Pathol, 2002 Dec, 31(6), 611 - 7 Improved detection of antibodies to Mycoplasma synoviae vaccine MS-H using an autologous recombinant MSPB enzyme-linked immunosorbent assay; Noormohammadi AH et al.; Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis . An indirect enzyme-linked immunosorbent assay (ELISA) has previously been devised in our laboratory using the major membrane antigen MSPB of M . synoviae strain WVU 1853 as antigen . However, sera from chickens inoculated with the M . synoviae vaccine strain MS-H showed lower optical densities in the assay than chickens infected with wild-type strains . In the present study, we investigate whether a low level of antibodies detected in MS-H-vaccinated birds is due to the limited ability of the vaccine to elicit antibodies, or to the reduced capacity of the antigen to specifically detect antibodies to this strain . Preliminary immunostaining experiments using native MSPBs from M . synoviae MS-H and WVU 1853 suggested that they were antigenically related but differed in at least some epitopes . Using a combination of polymerase chain reaction (PCR) and cloning, the gene encoding MSPB (vlhA) was cloned from strain MS-H, and its nucleotide sequence was partially determined . Analysis of the partial nucleotide sequence of the cloned vlhA gene revealed that it had a high identity (86%) with the previously published vlhA sequence from strain WVU 1853, but differed from it in several regions . Also, several nucleotide substitutions/deletions were detected in the conserved region (nucleotides 1 to 700) of the MS-H vlhA gene . A polypeptide, containing amino acids 27 to 299 of the MS-H MSPB, was expressed as a fusion protein in Escherichia coli and purified by affinity chromatography . An indirect ELISA was developed using the MS-H MSPB as coating antigen and compared with that of WVU 1853 MSPB and the commercial rapid serum agglutination test using a panel of sera from MS-vaccinated and/or challenged or unvaccinated specific pathogen free and commercial field chickens . Analysis of the absorbance values from specific pathogen free and field chicken sera showed that MS-H MSPB was species specific and more sensitive than the WVU-MSPB ELISA or the rapid serum agglutination test in detecting antibodies to the MS-H vaccine strain . These results emphasize the importance of using appropriate diagnostic antigens for sensitive detection of antibodies following vaccination or challenge with a M . synoviae strain. J Neuroimaging, 2003 Jan, 13(1), 75 - 8 MRI findings of hemolytic uremic syndrome with encephalopathy: widespread symmetrical distribution; Nakamura H et al.; The authors report the magnetic resonance imaging (MRI) findings in a 22-year-old woman with hemolytic uremic syndrome and encephalopathy secondary to verotoxin-producing Escherichia coli . Multiple lesions in the midbrain, cerebellum, occipital lobe, and basal ganglia showed high signal intensity on T2-weighted images with widespread symmetrical distribution . Most of these findings showed remarkable reduction on MRI images obtained 70 days after the onset . It is suggested that edema induced by local breakdown of blood-brain barrier might play an important role in the patient. DNA Seq, 2002 Oct, 13(5), 295 - 300 Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein; Mizrahi-Aviv E et al.; Salt stress results in a massive change in gene expression . An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis) . The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily . ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures . His6-tagged ScRAB protein was expressed in E . coli, and purified to homogeneity . The purified protein bound radiolabelled GTP . The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding. Protein Sci, 2003 Mar, 12(3), 577 - 85 Crystal structure of a defective folding protein; Saul FA et al.; Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system . MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment . We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding . Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells . MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein . Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes . The structure was determined by molecular replacement methods and refined to 1.85 A resolution . The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand . The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system. Protein Sci, 2003 Mar, 12(3), 551 - 9 Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding; Ferre H et al.; The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional . Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions . Their subsequent purification and refolding is complicated by the fact that (1) . correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2) . optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary . Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding . In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography . In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide . Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule. Protein Sci, 2003 Mar, 12(3), 480 - 90 In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation; Kosinski-Collins MS et al.; Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts . This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains . Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology . We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding . Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl . The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions . Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes . The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region . Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles . The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets . The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo. Protein Sci, 2003 Mar, 12(3), 458 - 67 A PDZ domain-based assay for measuring HIV protease activity: assay design considerations; Hamilton AC et al.; We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain*peptide ligand complexes . The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates . Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu(3+)chelate-labeled GST-PDZ ({Eu(3+)}GST-PDZ) . The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain*peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand . To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature . In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH . Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease . The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design. RNA, 2003 Mar, 9(3), 355 - 63 Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 A resolution; Correll CC et al.; GNRA tetraloops (N is A, C, G, or U; R is A or G) are basic building blocks of RNA structure that often interact with proteins or other RNA structural elements . Understanding sequence-dependent structural variation among different GNRA tetraloops is an important step toward elucidating the molecular basis of specific GNRA tetraloop recognition by proteins and RNAs . Details of the geometry and hydration of this motif have been based on high-resolution crystallographic structures of the GRRA subset of tetraloops; less is known about the GYRA subset (Y is C or U) . We report here the structure of a GUAA tetraloop determined to 1.4 A resolution to better define these details and any distinctive features of GYRA tetraloops . The tetraloop is part of a 27-nt structure that mimics the universal sarcin/ricin loop from Escherichia coli 23S ribosomal RNA in which a GUAA tetraloop replaces the conserved GAGA tetraloop . The adenosines of the GUAA tetraloop form an intermolecular contact that is a commonplace RNA tertiary interaction called an A-minor motif . This is the first structure to reveal in great detail the geometry and hydration of a GUAA tetraloop and an A-minor motif . Comparison of tetraloop structures shows a common backbone geometry for each of the eight possible tetraloop sequences and suggests a common hydration . After backbone atom superposition, equivalent bases from different tetraloops unexpectedly depart from coplanarity by as much as 48 degrees . This variation displaces the functional groups of tetraloops implicated in protein and RNA binding, providing a recognition feature. Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2296 - 9 Epub 2003 Feb 18. Molecular mechanism of recruitment of TFIIF- associating RNA polymerase C-terminal domain phosphatase (FCP1) by transcription factor IIF; Kamada K et al.; After mRNA transcription termination in eukaryotes, the hyperphosphorylated form of RNA polymerase II (pol II0) must be recycled by TFIIF-associating C-terminal domain phosphatase (FCP1), the phosphatase responsible for dephosphorylating the C-terminal domain of the largest polymerase subunit . Transcription factor (TF)-IIF stimulates the activity of FCP1, and the RNA polymerase II-associating protein 74 subunit of TFIIF forms a complex with FCP1 in both human and yeast . Here, we report a cocrystal structure of the winged-helix domain of human RNA polymerase II-associating protein 74 bound to the alpha-helical C terminus of human FCP1 (residues 944-961) . These results illustrate the molecular mechanism by which TFIIF efficiently recruits FCP1 to the pol II transcription machinery for recycling of the polymerase. J Bacteriol, 2003 Mar, 185(5), 1739 - 44 Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae; Zimmermann R et al.; Growth of Bartonella henselae is strongly heme dependent, and B . henselae is unable to synthesize heme itself . At least five outer membrane-associated proteins from B . henselae bind hemin, including the 31-kDa protein designated Pap31 . The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction . Complementation of the hemA-deficient mutant E . coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B . henselae. J Bacteriol, 2003 Mar, 185(5), 1726 - 9 tRNA2Thr complements temperature sensitivity caused by null mutations in the htrB gene in Escherichia coli; Mohri Y et al.; According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr . In order to investigate the reason that this nonessential tRNA nevertheless exists in Escherichia coli, we attempted to isolate tRNA2Thr-requiring mutants . Using strain JM101F(-), which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr . These mutants had a mutation in the htrB gene, whose product is an enzyme involved in lipid A biosynthesis . Although it is known that some null mutations in the htrB gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity . We discuss a possible mechanism for the requirement for tRNA2Thr. J Bacteriol, 2003 Mar, 185(5), 1701 - 4 The Escherichia coli methyl-directed mismatch repair system repairs base pairs containing oxidative lesions; Wyrzykowski J et al.; A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors . In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis . When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically . In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR . In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks . Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR . This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR . We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations . These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both. J Bacteriol, 2003 Mar, 185(5), 1616 - 23 The cyclic AMP-cyclic AMP receptor protein complex regulates activity of the traJ promoter of the Escherichia coli conjugative plasmid pRK100; Starcic M et al.; The TraJ protein is a central activator of F-like plasmid conjugal transfer . In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system . A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100 . The defect in the cya mutant was partially restored by addition of exogenous cAMP . Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription . Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region . DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter . Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site . Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100. J Bacteriol, 2003 Mar, 185(5), 1582 - 9 The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase; Jordan SW et al.; In an earlier study (S . W . Jordan and J . E . Cronan, Jr., J . Biol . Chem . 272:17903-17906, 1997) we reported a new enzyme, lipoyl-{acyl carrier protein}-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase . It was also shown that E . coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase . However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action . We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase . A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase . In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E . coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein . Prior genetic results (T . W . Morris, K . E . Reed, and J . E . Cronan, Jr., J . Bacteriol . 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP . We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase . A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions. J Bacteriol, 2003 Mar, 185(5), 1495 - 502 CheZ-mediated dephosphorylation of the Escherichia coli chemotaxis response regulator CheY: role for CheY glutamate 89; Silversmith RE et al.; The swimming behavior of Escherichia coli at any moment is dictated by the intracellular concentration of the phosphorylated form of the chemotaxis response regulator CheY, which binds to the base of the flagellar motor . CheY is phosphorylated on Asp57 by the sensor kinase CheA and dephosphorylated by CheZ . Previous work (Silversmith et al., J . Biol . Chem . 276:18478, 2001) demonstrated that replacement of CheY Asn59 with arginine resulted in extreme resistance to dephosphorylation by CheZ despite proficient binding to CheZ . Here we present the X-ray crystal structure of CheYN59R in a complex with Mn(2+) and the stable phosphoryl analogue BeF(3)(-) . The overall folding and active site architecture are nearly identical to those of the analogous complex containing wild-type CheY . The notable exception is the introduction of a salt bridge between Arg59 (on the beta3alpha3 loop) and Glu89 (on the beta4alpha4 loop) . Modeling this structure into the (CheY-BeF(3)(-)-Mg(2+))(2)CheZ(2) structure demonstrated that the conformation of Arg59 should not obstruct entry of the CheZ catalytic residue Gln147 into the active site of CheY, eliminating steric interference as a mechanism for CheZ resistance . However, both CheYE89A and CheYE89Q, like CheYN59R, conferred clockwise flagellar rotation phenotypes in strains which lacked wild-type CheY and displayed considerable (approximately 40-fold) resistance to dephosphorylation by CheZ . CheYE89A and CheYE89Q had autophosphorylation and autodephosphorylation properties similar to those of wild-type CheY and could bind to CheZ with wild-type affinity . Therefore, removal of Glu89 resulted specifically in CheZ resistance, suggesting that CheY Glu89 plays a role in CheZ-mediated dephosphorylation . The CheZ resistance of CheYN59R can thus be largely explained by the formation of the salt bridge between Arg59 and Glu89, which prevents Glu89 from executing its role in catalysis. Biochim Biophys Acta, 2003 Feb 20, 1625(3), 305 - 8 cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development; Zhu C et al.; cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library . The function of all cDNAs was analyzed by complementation in Escherichia coli . Transcript levels during different stages of flower development of G . lutea were determined and compared to the carotenoid composition . Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene . The transcript amount of the latter was strongly decreased . These results indicate that during flower development, carotenoid formation is enhanced . Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives. Phytochemistry, 2003 Apr, 62(7), 1081 - 6 A cDNA clone for 3-carene synthase from Salvia stenophylla; Hoelscher DJ et al.; The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component . Using an enriched cDNA library prepared from mRNA isolated from S . stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene synthase in sequence . Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene . This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S . stenophylla. Arch Biochem Biophys, 2003 Mar 1, 411(1), 56 - 62 Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii; Wang S et al.; Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli . The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa) . Its optimal pH and temperature were 7.6 and 90 degrees C, respectively . The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h) . The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor . K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate . The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation . Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent. J Helminthol, 2003 Mar, 77(1), 57 - 63 Molecular cloning and expression of the full-length tropomyosin gene from Trichinella spiralis; Nakada T et al.; A clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) of Trichinella spiralis through immunoscreening against infected sera . The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight) . A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene . The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position of T . spiralis . The fusion proteins encoded by the clone TsTM were produced in an Escherichia coli expression system and affinity purified, and the antibody was raised against the protein for the following studies . The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots . The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity. Biochemistry, 2003 Feb 25, 42(7), 2202 - 17 Thermal and urea-induced unfolding of the marginally stable lac repressor DNA-binding domain: a model system for analysis of solute effects on protein processes; Felitsky DJ et al.; Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding . Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH . Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0 </= C(3) </= 6 M . For unfolding the HTH, deltaG(o)(obs) decreases linearly with increasing C(3) at all temperatures examined, which directly confirms the validity of the linear extrapolation method (LEM) to obtain the intrinsic stability of this protein . At 25 degrees C (pH 7.3 and 50 mM K(+)), both linear extrapolation and extrapolation via the local-bulk domain model (LBDM) to C(3) = 0 yield deltaG(o)(obs) = 1.23 +/- 0.05 kcal mol(-)(1), in agreement with direct measurement (1.24 +/- 0.30 kcal mol(-)(1)) . Like deltaG(o)(obs), both deltaH(o)(obs) and deltaS(o)(obs) decrease linearly with increasing C(3); the derivatives with respect to C(3) of deltaG(o)(obs), deltaH(o)(obs) and TdeltaS(o)(obs) (in cal mol(-)(1) M(-)(1)) are -449 +/- 11, -661 +/- 90, and -203 +/- 91 at 25 degrees C, indicating that the effect of urea on deltaG(o)(obs) is primarily enthalpic . The deltaC(o)(P,obs) of unfolding (0.63 +/- 0.05 kcal mol(-)(1) K(-)(1)) is not detectibly dependent on C(3) or temperature . The urea m-value of the lacI HTH (-d deltaG(o)(obs),/dC(3) = 449 +/- 11 cal mol(-)(1) M(-)(1) at 25 degrees C) is independent of C(3) up to at least 6 M . Use of the LBDM to fit the C(3)-dependence of deltaG(o)(obs) yields the local-bulk partition coefficient for accumulation of urea at the protein surface exposed upon denaturation: K(P) = 1.103 +/- 0.002 at 25 degrees C . This partition coefficient is the same within uncertainty as those previously determined by LBDM analysis of osmometric data for solutions of urea and native (folded) bovine serum albumin, as well as LBDM analysis of the proportionality of m-values to changes in water accessible surface area upon protein unfolding . From the correspondence between values of K(P), we conclude that the average local urea concentration at both folded and unfolded protein surface exceeds the bulk by approximately 10% at 25 degrees C . The observed decrease in m-value for the lacI HTH with increasing temperature, together with the observed reductions in both deltaH(o)(obs) and deltaS(o)(obs) of unfolding with increasing urea concentration, demonstrate that K(P) for urea decreases with increasing temperature and that transfer of urea from the bulk solution to the local domain at the protein surface exposed on denaturation is enthalpically driven and entropically unfavorable. Biochemistry, 2003 Feb 25, 42(7), 1958 - 68 Entropic nature of the interaction between promoter bound CRP mutants and RNA polymerase; Krueger S et al.; The interaction between CRP, T127L, S128A, and CRP and RNA polymerase bound to a 104 bp synthetic promoter were determined by ITC at 298 K and ranges from a deltaG(b) degrees = 1.4 +/- 0.8 kJ mol(-)(1) (cAMP-ligated S128A) to 4.5 +/- 0.3 kJ mol(-)(1) (cAMP-ligated double mutant CRP) with endothermicities that range from 4 +/- 3 kJ mol(-)(1) (cAMP-ligated CRP) to 47 +/- 8 kJ mol(-)(1) (cGMP-ligated T127L) . The interaction is, thus, entropically driven, exhibits enthalpy-entropy compensation, and increases the binding affinity of the RNA polymerase to the promoter by factors ranging from 1.7 +/- 0.1 (cAMP-ligated S128A) to 6.1 +/- 0.1 (cAMP-ligated CRP) . Although the binding affinities to the promoter alone, except for cAMP-ligated S128A, are the same as to a shorter 40 bp duplex containing the same CRP consensus binding site sequence (conDNA), the binding enthalpies of CRP/mutant to the promoter are lower by factors of 2-3 x than the corresponding binding enthalpies to conDNA . Small angle neutron scattering measurements on the DNA-CRP/mutant complexes in D(2)O/H(2)O solutions exhibit an increase in the Rg of the CRP/mutant component from 22 to 27-31 A that can be attributed to a conformational change in the N-terminal domain of CRP . The Rg = 27 A for the bound conDNA can be attributed to a slight unwinding of the DNA in solution that would also enhance the activation of transcription . The Rg = 53 +/- 3 A for the bound promoter is attributed to bending of the promoter in solution that can be responsible for the lower CRP/mutant-promoter binding endothermicities. Biochemistry, 2003 Feb 25, 42(7), 1910 - 21 Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching; Flowers S et al.; DnaB helicase of E . coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis . We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB . Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI) . In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment . Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation . ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching . However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation . Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48 . However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone . The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis . There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity . The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis . This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action . We have correlated these results with partial structural models of alpha, beta, and gamma domains Rapid Commun Mass Spectrom, 2003, 17(5), 455 - 62 High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays; Koopmann JO et al.; A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated . Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry . Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule . Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E . coli . The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target . Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E . coli proteins were detected . Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins . Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated . Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array . The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins . Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry . J Biol Chem, 2003 May 2, 278(18), 16082 - 7 Epub 2003 Feb 16. An amino acid cluster around the essential Glu-14 is part of the substrate- and proton-binding domain of EmrE, a multidrug transporter from Escherichia coli; Gutman N et al.; EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds . Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved . This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons . The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the coupling between substrate and proton fluxes . Systematic cysteine-scanning mutagenesis of the residues in the transmembrane segment (TM1), where Glu-14 is located, reveals an amino acid cluster on the same face of TM1 as Glu-14 that is part of the substrate- and proton-binding domain . Substitutions at most of these positions yielded either inactive mutants or mutants with modified affinity to substrates . Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with modified affinity to protons and thereby impaired in the coupling of substrate and proton fluxes . Taken as a whole, the results strongly support the concept of a common binding site for substrate and protons and stress the importance of one face of TM1 in substrate recognition, binding, and H(+)-coupled transport. J Biol Chem, 2003 Apr 25, 278(17), 14820 - 6 Epub 2003 Feb 16. In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding; Nagamori S et al.; Insertion and folding of polytopic membrane proteins is an important unsolved biological problem . To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro . The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays . By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane . Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable . Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated . Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane . Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide . Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer . Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains. FEMS Immunol Med Microbiol, 2003 Jan 21, 35(1), 25 - 31 Antibody-inducing properties of a prototype bivalent herpes simplex virus/enterotoxigenic Escherichia coli DNA vaccine; Lasaro MO et al.; The antibody-inducing properties of a bacterial/viral bivalent DNA vaccine (pRECFA), expressing a peptide composed of N- and C-terminal amino acid sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) fused with an inner segment encoding the major structural subunit of enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae (CFA/I), was evaluated in BALB/c mice following intramuscular immunization . The bivalent pRECFA vaccine elicited serum antibody responses, belonging mainly to the IgG2a subclass, against both CFA/I and HSV gD proteins . pRECFA-elicited antibody responses cross-reacted with homologous and heterologous ETEC fimbrial antigens as well as with type 1 and type 2 HSV gD proteins, which could bind and inactivate intact HSV-2 particles . On the other hand, CFA/I-specific antibodies could bind but did not neutralize the adhesive functions of the bacterial CFA/I fimbriae . In spite of the functional restriction of the antibodies targeting the bacterial antigen, the present evidence suggests that fusion of heterologous peptides to the HSV gD protein represents an alternative for the design of bivalent DNA vaccines able to elicit serum antibody responses. Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 915 - 22 Transcription of ahpC, katG, and katE genes in Escherichia coli is regulated by polyamines: polyamine-deficient mutant sensitive to H2O2-induced oxidative damage; Jung IL et al.; Polyamines (putrescine and spermidine) are present in almost all living organisms and participate in numerous cellular processes . In this study, we report the protective roles of polyamines against hydrogen peroxide (H2O2)-induced oxidative stress . All of ahpC, katG, and katE genes, known to participate in the antioxidant defense mechanism against H2O2-induced stress in Escherichia coli, failed to induce in the absence of polyamines during normal aerobic growth . The induction of both oxyR and rpoS gene expression, whose products are essential to induce ahpC, katG, and katE genes, was also absolutely dependent on polyamines . Polyamine-deficient E . coli mutant has increased susceptibility to exogenous H2O2, and this cell cytotoxicity was relieved to a wild-type level by addition of putrescine or spermidine (1mM), which restored the transcriptional induction of ahpC, katG, and katE genes . H2O2-removing capacity was measured in the mutant, showing a significantly low H2O2-removing capacity compared to the wild type when polyamines were not present . We concluded that the increased susceptibility of the polyamine-deficient E . coli mutant to H2O2 treatment resulted from an intracellular low level of H2O2-removing capacity through the failure of their regulons, ahpC, katG, and katE induction, as well as the failure of oxyR and rpoS induction. Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 879 - 85 A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis; Tu H et al.; A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR . The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species . The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification . The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized . The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm . The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein. Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 819 - 24 Sequence analysis, expression, and paratope characterization of a single-chain Fv fragment for the eukaryote ribosomal P proteins; Lopez Bergami P et al.; The variable genes of monoclonal antibody (mAb) B10, specific for the C-terminal region of the eukaryotic ribosomal P protein, have been cloned as a single-chain Fv fragment (scFv) and expressed in Escherichia coli . The primary sequence of the variable regions of the B10 antibody, together with a detailed characterization of the reactive residues of the antigen, allowed the construction of a model of the paratope-epitope interaction, giving a first insight into the binding mechanisms of anti-P autoantibodies to their target peptides . The mAb and scFv could be useful for extensive P protein detection since both recognize the highly conserved motif DDxGF. Res Vet Sci, 2003 Apr, 74(2), 171 - 8 Efficacy of danofloxacin 18% injectable solution in the treatment of Escherichia coli diarrhoea in young calves in Europe; Sunderland SJ et al.; The efficacy of danofloxacin 18% against naturally occurring Escherichia coli diarrhoea was investigated in calves at seven European sites . Treatment commenced on day 0, with either a single subcutaneous injection of danofloxacin 18% (n=267) at 6 mg/kg repeated on day 2 if required, or reference product containing baquiloprim/sulphadimidine (n=37) or gentamicin (n=98) administered as recommended . E . coli was isolated from 90% to 100% of calves pre-treatment, and the prevalence of serotypes K99 and F41 was 8-46% and 46-92%, respectively . In both treatments, the majority of calves (93.2-93.9%) showed clinical improvement and completed the studies . There were significant reductions for both treatments, in severity of clinical signs on days 4 and 10 compared to day 0 (P<0.0001), and between days 4 and 10 (P<0.05), but no significant differences between treatments (P>0.05) . Danofloxacin 18% was clinically safe, and as effective as the reference products in the treatment of E . coli diarrhoea in calves. J Biol Inorg Chem, 2003 Feb, 8(3), 341 - 7 Epub 2002 Nov 16. Characterization of calcium binding properties of lithostathine; Lee BI et al.; The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones . Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity . Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation . To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy . Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry . EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT . Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT. Curr Genet, 2003 Jan, 42(4), 236 - 40 Epub 2002 Dec 17. Development of a transformation system for Crinipellis perniciosa, the causal agent of witches' broom in cocoa plants; Lima JO et al.; Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences . The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment . Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved . About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels . The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis . This is the first report of a C . perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen. Curr Genet, 2003 Feb, 42(5), 292 - 300 Epub 2003 Jan 18. Proteobacteria-like ferrochelatase in the malaria parasite; Sato S et al.; A gene encoding the heme biosynthetic enzyme ferrochelatase (FC) was found in the genomic DNA databases of Plasmodium spp . The predicted amino acid sequence of malarial FC is highly conserved and fairly well conserved by comparison with other orthologues . The FC genes of P . falciparum and P . yoelii are transcribed and the mRNAs are processed to encode polypeptides of the expected amino acid sequence . The cloned cDNA for the FC of P . falciparum successfully rescued a FC-null mutant of Escherichia coli, indicating that it encodes an active enzyme . Unlike eukaryotic FCs, the malarial enzyme lacks a characteristic extension at the C-terminus . In addition, the sequence of the malarial FC resembles proteobacterial orthologues rather than eukaryotic enzymes . Strikingly, the malarial FC lacks a bipartite presequence at its N-terminus, unlike delta-aminolevulinic acid dehydratase of the same organism . This suggests an unusual intracellular distribution of heme biosynthetic enzymes, involving multiple subcellular compartments. Curr Genet, 2003 Feb, 42(5), 260 - 7 Epub 2003 Jan 14. Cooperative action of the NIT2 and NIT4 transcription factors upon gene expression in Neurospora crassa; Mo X et al.; In Neurospora crassa, the nit-3 gene, which encodes nitrate reductase, an enzyme required for the utilization of inorganic nitrate, is subject to a high degree of genetic and metabolic regulation as a member of the nitrogen control circuit . The nit-3 gene promoter contains binding sites for a globally acting protein NIT2 and a pathway-specific protein NIT4 . Expression of the nit-3 gene absolutely requires both the NIT2 and NIT4 transcription factors and only occurs under conditions of nitrogen source derepression and nitrate induction . In the sulfur control circuit, the cys-14 gene encodes sulfate permease II, which facilitates the assimilation of sulfate . Expression of cys-14 is strongly regulated by only a single positive-acting factor, CYS3 . It was of interest to determine whether NIT2 or NIT4 alone was capable of turning on the expression of cys-14, since this structural gene is normally controlled by only one regulatory protein . NIT2- and/or NIT4-binding elements were introduced into the promoter of a wild-type cys-14 gene and these constructs were transformed into a cys-13(-) cys-14(-) mutant strain and into a nit-2(-) mutant host . We examined whether any of these cys-14 genes in these transformants could now be controlled as a nitrogen-regulated gene . Sulfate permease assays revealed that both NIT2 and NIT4 were required for cys-14 expression upon nitrate induction, while neither alone activated any detectable cys-14 expression . We thus conclude that neither NIT2 nor NIT4 is capable alone of activating gene expression in this context, but together they can cooperate to elicit strong activation. J Biol Chem, 2003 Apr 25, 278(17), 15341 - 8 Epub 2003 Feb 14. High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides; Katoh S et al.; EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell . We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy . The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops . It is stabilized by two tetrahedrally coordinated zinc ions . This structure is similar to that of other RING finger domains of proteins of known function . From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2) . The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix . We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b . This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5 . By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b . We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5. Vet Res, 2003 Jan-Feb, 34(1), 119 - 25 Proteic boost enhances humoral response induced by DNA vaccination with the dnaK gene of Chlamydophila abortus but fails to protect pregnant mice against a virulence challenge; Hechard C et al.; In order to enhance the quantity and the protective properties of the antibodies induced by DNA vaccination with the heat shock protein dnaK gene of Chlamydophila abortus AB7 as well as to elicit an efficient cellular immune response, we vaccinated mice with a DNA prime followed by a boost with the recombinant DnaK protein . In non-pregnant mice, this strategy induced the same predominance of the IgG2a isotype as DNA immunization alone with a substantial increased antibody level . The induced antibodies had no in vitro neutralizing properties on C . abortus infectivity . Moreover, the proteic boost probably failed to elicit an efficient cellular immune response since the pregnant or non-pregnant mice were not protected against the bacterial challenge. Chem Res Toxicol, 2003 Feb, 16(2), 129 - 36 Mutation of tyrosine 190 to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite; Lin HL et al.; We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine . Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225 . This peptide contains two tyrosine residues at positions 190 and 203 . In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1 . All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized . The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT . The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A . The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein . Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A . Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1 . Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149. Acta Microbiol Pol, 2002, 51(3), 217 - 24 Effect of null mutations in dnaK and dnaJ genes on conjugational DNA transfer, proteolysis and novobiocin susceptibility of Escherich