Escherichia coli

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 Escherichia coli; Modrzewska M et al.; Escherichia coli null dnaJ and dnaKdnaJ mutants, when introduced to Hfr donor, impair its ability to DNA transfer during conjugation . The additive effect of both mutations was shown . Lack of DnaK and DnaJ chaperones also decrease the extent of proteolysis in mutant strains . This effect is seen only at 42 degrees C . The influence of double dnaKdnaJ deletion but not single dnaJ deletion on novobiocin susceptibility was also demonstrated.

Arch Insect Biochem Physiol, 2003 Mar, 52(3), 130 - 8
Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori; Uno T et al.; From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family . Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins . The other 4 proteins show low sequence similarity to any of the known Rab proteins . However, all of them contain the region conserved in rab protein . Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated . The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein . After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column . The protein bound {(3)H}-GDP with association constant of 1.02 x 10(11) M(-1) . Further, the protein was phosphorylated by protein kinase . This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation .

Science, 2003 Feb 14, 299(5609), 1067 - 70
Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands; Reissmann S et al.; NiFe-hydrogenases have an Ni-Fe site in which the iron has one CO and two CN groups as ligands . Synthesis of the CN ligands requires the activity of two hydrogenase maturation proteins: HypF and HypE . HypF is a carbamoyltransferase that transfers the carbamoyl moiety of carbamoyladenylate to the COOH-terminal cysteine of HypE and thus forms an enzyme-thiocarbamate . HypE dehydrates the S-carbamoyl moiety in an adenosine triphosphate-dependent process to yield the enzyme thiocyanate . Chemical model reactions corroborate the feasibility of this unprecedented biosynthetic route and show that thiocyanates can donate CN to iron . This finding underscores a striking parallel between biochemistry and organometallic chemistry in the formation of an iron-cyano complex.

Plant Physiol, 2003 Feb, 131(2), 725 - 35
A novel small heat shock protein gene, vis1, contributes to pectin depolymerization and juice viscosity in tomato fruit; Ramakrishna W et al.; We have characterized a novel small heat shock protein gene, viscosity 1 (vis1) from tomato (Lycopersicon esculentum) and provide evidence that it plays a role in pectin depolymerization and juice viscosity in ripening fruits . Expression of vis1 is negatively associated with juice viscosity in diverse tomato genotypes . vis1 exhibits DNA polymorphism among tomato genotypes, and the alleles vis1-hta (high-transcript accumulator; accession no . AY128101) and vis1-lta (low transcript accumulator; accession no . AY128102) are associated with thinner and thicker juice, respectively . Segregation of tomato lines heterogeneous for vis1 alleles indicates that vis1 influences pectin depolymerization and juice viscosity in ripening fruits . vis1 is regulated by fruit ripening and high temperature and exhibits a typical heat shock protein chaperone function when expressed in bacterial cells . We propose that VIS1 contributes to physiochemical properties of juice, including pectin depolymerization, by reducing thermal denaturation of depolymerizing enzymes during daytime elevated temperatures.

Plant Physiol, 2003 Feb, 131(2), 516 - 24
Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth; Jang IC et al.; Trehalose plays an important role in stress tolerance in plants . Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1) . The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P . The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1) . This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes . The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants . It has been reported that transgenic plants with E . coli TPS and/or TPP were severely stunted and root morphology was altered . Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose . Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses . Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Plant Physiol, 2003 Feb, 131(2), 463 - 71
Subcellular targeting of methylmercury lyase enhances its specific activity for organic mercury detoxification in plants; Bizily SP et al.; Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals . In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA . In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000) . To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall . Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme . MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells . These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification . In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 365 - 70
The regulatory elements of the Mycobacterium tuberculosis gene Rv3881c function efficiently in Escherichia coli; Satchidanandam V et al.; We report efficient expression of the Mycobacterium tuberculosis gene Rv3881c in Escherichia coli from its M . tuberculosis promoter, attributable to an E . coli consensus Pribnow box and ribosome binding site . The N-terminal sequence of the recombinant E . coli-generated protein was identical to the predicted open reading frame of Rv3881c and transcription of the Rv3881c gene initiated at the same nucleotide position in both bacteria . We demonstrate the utility of this promoter for rapid analysis of expression in E . coli of heterologous gene constructs, for subsequent expression from the genomes of slow-growing mycobacteria such as Mycobacterium bovis-BCG . M . tuberculosis Rv3881c homologues were present in other pathogenic mycobacteria such as M . bovis-BCG, Mycobacterium szulgai and Mycobacterium kansasii.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 351 - 7
Cyanobacterial leader peptides for protein secretion; Sergeyenko TV et al.; The leader peptide of the major secreted protein PilA1 of the cyanobacterium Synechocystis sp . strain PCC 6803 and several artificial leader peptides have been used to study secretion of the reporter protein lichenase to the culture medium . The strains of Synechocystis carrying lichenase with the leader sequences of PilA and with the leader sequence of Slr2016 efficiently secreted the reporter protein . The artificial leader sequence that was characterized by the overall positive charge (as PilA1 and Slr2016 leaders) also allowed secretion . The artificial leader with negative charge, however, did not allow secretion of the reporter protein . Moreover, no secreted proteins have been isolated from this strain using conventional techniques for preparation of secreted proteins . These data suggest that the general secretion pathway in cyanobacteria, at least for pilins, recognizes the overall charge of the leader sequences, and operates in a sequence-non-specific manner.

Biochim Biophys Acta, 2003 Feb 17, 1610(1), 46 - 50
Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli; Venturi M et al.; Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens . Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand . Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes . In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters . We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins . The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter .

FEBS Lett, 2003 Feb 11, 536(1-3), 167 - 72
A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages; Nakamura M et al.; Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically . BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E . coli by using the avidin-biotin-peroxidase complex method with electron microscopy . Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space . Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes . The periplasmic spaces in some infected cells were expanded and filled with reaction products . Some cells showed wavy lines of positive staining in the periplasmic space . BIO-phages were detected as thick filaments or clusters covered with reaction products . The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane . These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.

FEBS Lett, 2003 Feb 11, 536(1-3), 92 - 6
Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants; Shen W et al.; We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate . We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems . Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli . The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination . We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions . Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.

Chemistry, 2003 Feb 17, 9(4), 893 - 9
Fluorogenic stereochemical probes for transaldolases; Gonzalez-Garcia E et al.; Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate . As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose . The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E . coli transketolase for assembly of the final product . The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose . Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde . The latter primary product undergoes a beta-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone . A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3 . The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.

J Virol, 2003 Mar, 77(5), 3334 - 8
Ebola virus transcription activator VP30 is a zinc-binding protein; Modrof J et al.; Ebola virus VP30 is an essential activator of viral transcription . In viral particles, VP30 is closely associated with the nucleocapsid complex . A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys(3)-His motif comprising amino acids 68 to 95 . By using a colorimetric zinc-binding assay we found that the VP30-specific Cys(3)-His motif stoichiometrically binds zinc ions in a one-to-one relationship . Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding . Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.

Carcinogenesis, 2003 Feb, 24(2), 155 - 7
The discovery of a new family of mammalian enzymes for repair of oxidatively damaged DNA, and its physiological implications; Hazra TK et al.; Oxidatively damaged bases in the genome are likely to be responsible for mutations leading to sporadic carcinogenesis . Two structurally similar DNA glycosylases, NTH1 and OGG1, which are able to excise most of these damaged bases, were identified previously in mammalian cells . A distinct family, consisting of two human DNA glycosylases orthologous to enzymes in Escherichia coli, has recently been characterized; they have overlapping substrate ranges with NTH1 and OGG1 . The presence of multiple enzymes with potential back-up functions underscores the importance of removing both endogenously and exogenously generated oxidatively damaged bases from the genome, and may explain why no cancer or other disease phenotype has so far been linked to the deficiency of a single DNA glycosylase.

Plasmid, 2003 Jan, 49(1), 9 - 17
Construction of a novel set of transfer vectors to study vaccinia virus replication and foreign gene expression; Dvoracek B et al.; Vaccinia virus (VV) is a useful expression vector for many laboratory applications . To date, approximately 60% ( approximately 120) of the VV genes remain uncharacterized . The thought of smallpox being used as a biological weapon has gained attention . In light of this, it is imperative that we continue to study the basic replication of VV, a poxvirus that is closely related to smallpox . A set of plasmid vectors were constructed to generate gene deletions (pZIPPY-NEO/GUS) in VV or for foreign gene expression (pBR-EXPRESS) . The vectors contain the Escherichia coli neomycin resistance (neo) and beta-glucuronidase (gusA) genes as selectable markers to facilitate isolation of recombinant viruses . These are the first transfer vectors to use a neo/gusA selection system . We used these vectors to successfully generate a recombinant D9R deletion mutant of VV and to express E . coli lacZ gene . Results indicate that both vectors are highly suited for their designed purpose .

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 187 - 93
In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli; Kuper J et al.; Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes . We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli . The assay is based on (i) . the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii) . the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E . coli . We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate . This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding . As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 181 - 6
Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea; Alduina R et al.; Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes . Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed . Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb . The S . coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones . Hybridisation of the P . rosea library to selected probes indicates a good representation of the P . rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 107 - 13
Holin locus characterisation from lysogenic Xenorhabdus nematophila and its involvement in Escherichia coli SheA haemolytic phenotype; Brillard J et al.; Lysogeny has previously been described in the entomopathogenic bacteria of the genus Xenorhabdus . Screening of a X . nematophila prophage DNA library on blood agar resulted in the identification of a 5.7-kb locus that caused a haemolytic phenotype when cloned in Escherichia coli, but not in the E . coli sheA null mutant, lacking the SheA cryptic haemolysin . This locus exhibited similarity to lysis genes from lambdoid phages . In particular, it encoded a functional holin able to complement a lambda Sam7 mutant . It is the second time that a locus encoding a functional holin is shown to reveal the SheA haemolytic phenotype in E . coli . The possible role of the holin in extracellular release of SheA is discussed.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 93 - 9
A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity; Grant AW et al.; A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified . This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family . The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1 . Sequences encoding putative homologues of this enzyme exist in many other bacteria . The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal . The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM . Overexpression of the recombinant enzyme in E . coli leads to increased resistance to methylglyoxal . It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 47 - 50
Effect of amino acid starvation on glucose transport in two archaeal organisms; Scoarughi GL et al.; When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes . One of the events under SC is transport of metabolites . Thus, under amino acid starvation, E . coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog . Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified . In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius . The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly . Unlike E . coli, H . volcanii and S . acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation . Thus, in both Archaea glucose analog transport is not under SC, as it is in E . coli.

Intern Med, 2003 Jan, 42(1), 3 - 6
Myocarditogenic epitopes and autoimmune myocarditis; Izumi T et al.; Experimental autoimmune myocarditis is provoked by immunization with cardiac myosin . This animal model finally develops into dilated cardiomyopathy through repetitive myosin injections . To identify the myocardiogenic epitope, therefore, it is imperative not only to understand the mechanism of induction, but also to produce specific therapies, such as a blocking therapy to suppress the autoimmune process . Thus, we attempted to identify the myocarditogenic epitope using recombinant peptides . Beta-cardiac myosin heavy chain (CMHC) was amplified from rat mRNA by a reverse transcription polymerase chain reaction method . The PCR primers were designed to narrow the epitopic amino acid portion from each N-terminal to C-terminal site . These PCR products were cloned into an E . coli expression vector to produce fusion proteins consisting of a Histidine-tag and a myosin peptide . The segment of amplified CMHC including the epitopic amino acid sequence to provoke moderate myocarditis in vivo was reported previously . Each peptide solution was emulsified in an equal volume of complete Freund's adjuvant and given as an immunization to 7-week-old rats . On day 21 after immunization, the rats were sacrificed, and the fresh heart was observed pathologically . Through this immunization, we could restrict the myocardiogenic site . Lastly, this peptide was found to be located on residues from 1,124 to 1,153 . Using ELISA, the antibodies against myocarditogenic peptides were easily identified . Whether or not the antibody productivity is linked to myocarditogenecity is discussed.

Eur Biophys J, 2003 Feb, 31(8), 595 - 607 Epub 2002 Nov 01.
A water network within a protein: temperature-dependent water ligation in H64V-metmyoglobin and relaxation to deoxymyoglobin; Engler N et al.; The sperm whale myoglobin mutant H64V, where the distal histidine is mutated to valine, is known to be five coordinated in the ferric state at room temperature and physiological pH . A change of the ligation in this H64V-Mbmet has been observed by optical absorption spectroscopy as a function of temperature from 20 K to 300 K . Above the dynamical transition at about 180 K one observes the temperature-dependent equilibrium between five- and six-ligated heme . Below the dynamical transition the equilibrium is frozen-in at about 50% of six-coordinate molecules . The water ligation of the iron occurs at temperatures where protein-specific motions are present, as monitored by Mossbauer spectroscopy . The X-ray structures of H64V-Mbmet at 300 K and 110 K are reported with a resolution of 1.5 A and 1.3 A, respectively . The measurements at high resolutions are possible owing to crystallization in the space group P2(1), whereas all mutant myoglobins studies up to now have been carried out with crystals in the space group P6 . The overall structure at both temperatures is very close to the native myoglobin . The binding of water at the sixth coordination site at lower temperatures is possible owing to a stabilizing water network extending from the protein surface to the active centre . The reduction of the H64V-Mbmet by electrons obtained by X-ray irradiation of the water-glycerol solvent at 85 K produces an intermediate low-spin state of the water-ligated molecules where Fe(II) retains the six-fold coordination . Mossbauer spectroscopy shows that the relaxation of the metastable low-spin state to high-spin H64V-Mbdeoxy with dissociation of the Fe(II)-H(2)O bond starts at about 115 K and is completed at about 170 K . Differences in the dynamics properties of the native and mutant myoglobin and the connection to the dynamical transition around 180 K are discussed.

Theor Appl Genet, 2002 Mar, 104(4), 709 - 719
Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes; Breitler JC et al.; This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use influences the integration and expression of the co-transferred gene of interest . Two non-linked marker genes ( yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments . Three different DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins . Our results demonstrate that microprojectile bombardment-mediated transformation of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use of whole-plasmid DNA . Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization of molecules prior to integration was rarely observed in gene cassette transformants.

Theor Appl Genet, 2002 Apr, 104(5), 828 - 839 Epub 2002 Feb 6.
Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica; Wan Y et al.; Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter . Three Ae . cylindrica and two T . timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined . A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae . cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T . timopheevi HMW subunits as 1Gx, 1Ax and 1Ay . It was not clear whether or not a 1Gy HMW subunit was present in T . timopheevi . The PCR products from Ae . cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T . timopheevi were derived from 1Ax and 1Ay genes . The 1Cx, 1Gx and 1Gy sequences were not amplified successfully . The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat ( Triticum aestivum) . Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M(r) polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species . There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat . The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE . The truncated 1Dx gene from Ae . cylindrica failed to express in E . coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.

Theor Appl Genet, 2002 Jul, 105(1), 34 - 42 Epub 2002 May 18.
Molecular cloning, characterization, expression and chromosomal location of OsGAPDH, a submergence responsive gene in rice ( Oryza sativa L.); Arumugam Pillai M et al.; Differential clones from submergence stress and control treatment from rice seedlings were isolated by the differential screening method . One of the clones, OsGAPDH, represented a gene that was expressed at high level during 12-h submergence . A homology search of GenBank databases showed that OsGAPDH had significant sequence homology with maize non-reversible glyceraldehyde-3-Phosphate dehydrogenase . The OsGAPDH sequence consists of 1,772 bp with the longest open reading frame encoding 499 amino acids with a calculated relative mass of 54.2 kDa . Genomic Southern analysis indicated that one or two copies of the OsGAPDH gene occur in the Yukihikari genome . The chromosomal location of the OsGAPDH gene was identified by RFLP analysis indicating that OsGAPDH was located on chromosome 8 . Tissue-specific expression of OsGAPDH indicated that the high level of mRNA was detected in the panicle . Plants exposed to drought, submergence and ABA treatment showed an increased accumulation of OsGAPDH transcripts . The induction of Escherichia coli cells containing the pGST-OsGAPDH plasmid resulted in the accumulation of a large amount of the 83.2-kDa recombinant protein . The purified GAPDH enzyme showed an optimum activity at pH 8.5 and 50 degrees C, and was strongly inhibited by ATP and ADP.

Nucleic Acids Res, 2003 Feb 15, 31(4), 1351 - 63
Limited repair of 8-hydroxy-7,8-dihydroguanine residues in human testicular cells; Olsen AK et al.; Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects . These DNA lesions are predominantly removed by base excision repair . Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine . DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals . This excision was as low as with human mononuclear blood cell extracts . The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species . Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay . Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions . Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions . In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.

Nucleic Acids Res, 2003 Feb 15, 31(4), 1191 - 6
Identification of high excision capacity for 5-hydroxymethyluracil mispaired with guanine in DNA of Escherichia coli MutM, Nei and Nth DNA glycosylases; Hori M et al.; The oxidation and deamination of 5-methylcytosine (5mC) in DNA generates a base-pair between 5-hydroxymethyluracil (5hmU) and guanine . 5hmU normally forms a base-pair with adenine . Therefore, the conversion of 5mC to 5hmU is a potential pathway for the generation of 5mC to T transitions . Mammalian cells have high levels of activity of 5hmU-DNA glycosylase, which excises 5hmU from DNA . However, glycosylases that similarly excise 5hmU have not been observed in yeast or Escherichia coli . Recently, we found that E.coli MutM, Nei and Nth have DNA glycosylase activity for 5-formyluracil, which is another type of oxidation product of the thymine methyl group . In this study, we examined whether or not E.coli MutM, Nei and Nth have also DNA glycosylase activity that acts on 5hmU in vitro . When incubated with synthetic duplex oligonucleotides containing 5hmU:G or 5hmU:A, purified MutM, Nei and Nth cleaved the 5hmU:G oligonucleotide 58, 5 and 37 times, respectively, more efficiently than the 5hmU:A oligonucleotide . In E.coli, the 5hmU-DNA glycosylase activities of MutM, Nei and Nth may play critical roles in the repair of 5hmU:G mispairs to avoid 5mC to T transitions.

Nucleic Acids Res, 2003 Feb 15, 31(4), 1174 - 9
Expression of tetanus toxin Fragment C in tobacco chloroplasts; Tregoning JS et al.; Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus . Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA . To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts . We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts . Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes . Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies . Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.

J Biol Chem, 2003 May 2, 278(18), 16088 - 94 Epub 2003 Feb 11.
Crystal structure and biochemical analysis of the MutS.ADP.beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair; Alani E et al.; During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors . Despite intensive efforts, a MutS.ATP.DNA complex has eluded crystallographic analysis . Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP.beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases . Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2.MSH6 and MLH1.PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2.MSH6 from mismatch DNA . Crystallographic analysis of the Taq MutS.DNA.ABF complex indicated that although this complex was very similar to that of MutS.DNA.ADP, both ADP.Mg(2+) moieties in the MutS . DNA.ADP structure were replaced by ABF . Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered . Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF . We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.

J Biol Chem, 2003 May 23, 278(21), 19159 - 63 Epub 2003 Feb 11.
A receptor-binding region in Escherichia coli alpha-haemolysin; Cortajarena AL et al.; Escherichia coli alpha-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells . Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A . L., Goni, F . M., and Ostolaza, H . (2001) J . Biol . Chem . 276, 12513-12519) . The present study was aimed at identifying the glycophorin-binding region in the toxin . Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain . Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914-936 of HlyA . We therefore prepared a deletion mutant lacking these residues (HlyA Delta 914-936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type . This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column . The peptide Trp914-Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles . Moreover, the peptide Trp914-Arg936 protected red blood cells from hemolysis induced by wild type HlyA . It was concluded that amino acid residues 914-936 constitute a major receptor-binding region in alpha-hemolysin.

J Biol Chem, 2003 Apr 18, 278(16), 13919 - 27 Epub 2003 Feb 10.
A new class of N-hydroxycinnamoyltransferases . Purification, cloning, and expression of a barley agmatine coumaroyltransferase (EC 2.3.1.64); Burhenne K et al.; Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L.) seedlings . The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity for p-coumaroyl-CoA among various acyl donors with a specific activity of 29.7 nanokatal x mg(-1) protein . Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5.20 as determined by isoelectric focusing . The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues . The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT . Recombinant ACT produced and affinity-purified from Escherichia coli had a specific activity of 189 nanokatal x mg(-1) protein, thus confirming the identity of the purified native protein . A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT . Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily . The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis.

J Biol Chem, 2003 Apr 18, 278(16), 14406 - 13 Epub 2003 Feb 10.
Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine; Johnson A et al.; The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication . The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer . The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction . Neither delta nor delta' bind ATP . This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit . Thus, the delta' stator contributes to the motor function of the gamma trimer . Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled . This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed . Implications of these results to clamp loaders of other systems are discussed.

Curr Opin Struct Biol, 2003 Feb, 13(1), 64 - 70
S-domain assembly of the signal recognition particle; Sauer-Eriksson AE et al.; The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes . In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA . The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.

Curr Opin Struct Biol, 2003 Feb, 13(1), 23 - 30
Damage repair DNA polymerases Y; Yang W; The newly found Y-family DNA polymerases are characterized by low fidelity replication using an undamaged template and the ability to carry out translesion DNA synthesis . The crystal structures of three Y-family polymerases, alone or complexed with DNA and nucleotide substrate, reveal a conventional right-hand-like catalytic core consisting of finger, thumb and palm domains . The finger and thumb domains are unusually small resulting in an open and spacious active site, which can accommodate mismatched base pairs as well as various DNA lesions . Although devoid of a 3'-->5' exonuclease activity, the Y-family polymerases possess a unique "little finger" domain that facilitates DNA association, catalytic efficiency and interactions with auxiliary factors . Expression of Y-family polymerases is often induced by DNA damage, and their recruitment to the replication fork is mediated by beta-clamp, clamp loader, single-strand-DNA-binding protein and RecA in Escherichia coli, and by ubiquitin-modified proliferating cell nuclear antigen in yeast.

J Mol Biol, 2003 Feb 21, 326(3), 887 - 97
Localization of the trigger factor binding site on the ribosomal 50S subunit; Blaha G et al.; In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF) . TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding . We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation . Our data suggest that the TF binds in the form of a homodimer . On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit . The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone . Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.

J Mol Biol, 2003 Feb 21, 326(3), 761 - 7
Crystal structure of activated ModE reveals conformational changes involving both oxyanion and DNA-binding domains; Schuttelkopf AW et al.; ModE is a bacterial transcriptional regulator that orchestrates many aspects of molybdenum metabolism by binding to specific DNA sequences in a molybdate-dependent fashion . We present the crystal structure of Escherichia coli ModE in complex with molybdate, which was determined at 2.75A from a merohedrally twinned crystal (twin fraction approximately 0.30) with space group P4(3) . We now have structures of ModE in both its "switched on" (ligand-bound) and "switched off" (apo) states . Comparison with the apo structure shows that ligand binding leads to extensive conformational changes not only in the molybdate-binding domain, but also in the DNA-binding domain . The most obvious difference is the loss of the pronounced asymmetry between the two chains of the ModE dimer, which had been a characteristic property of the apo structure . Another major change concerns the relative orientation of the two DNA-interacting winged helix-turn-helix motifs . Manual docking of an idealized DNA structure suggests that this conformational change should improve DNA binding of the activated molybdate-bound ModE.

Mol Microbiol, 2003 Feb, 47(4), 1091 - 100
PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells; Jaktaji RP et al.; The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC . It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions . It is also a DNA helicase . We describe insertions near the 3' end of priA that interfere with PriA activity . These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage . The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops . In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.

Mol Microbiol, 2003 Feb, 47(4), 1045 - 60
Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster; Rahn A et al.; Escherichia coli group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic (cps) operons . Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart-RfaH antitermination mechanism, with the cps promoter being localized to a region immediately upstream of the JUMPStart sequence . A putative stem-loop structure located within the K30 cps cluster separates conserved genes with products that are required for surface expression of capsule from serotype-specific genes encoding enzymes for polymer repeat-unit synthesis and polymerization . This putative stem-loop structure significantly reduces transcription in a termination-probe vector and may contribute to differential expression of the cps genes . Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs (regulator of capsule synthesis) proteins . However, neither overexpression of the transcriptional activator RcsB nor an rcsB::aadA chromosomal insertion altered the level of transcription measured by cps::lacZ fusions . In the group 1 strains examined, an RcsAB box was found immediately upstream of galF, a gene involved in the production of sugar nucleotide precursors . Overexpression of RcsB was found to result in a threefold increase in transcription of a galF::lacZ chromosomal fusion . Moreover, overexpression of GalF gave rise to a two- to threefold increase in cell-free as well as cell-associated capsule, without affecting cps::lacZ activity . These results indicate that transcription of the E . coli group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive . However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing galF transcription and influencing the available pool of biosynthetic precursors.

Mol Microbiol, 2003 Feb, 47(4), 1015 - 27
Versatility of inner membrane protein biogenesis in Escherichia coli; Froderberg L et al.; To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far . The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains . We show that the assembly of the complete set of model IMPs is assisted (i.e . requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other . This indicates that IMP assembly is much more versatile than previously thought.

Mol Microbiol, 2003 Feb, 47(4), 975 - 91
AREA directly mediates nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi, but its activity is not affected by NMR; Mihlan M et al.; AREA (NIT2) is a general transcription factor involved in derepression of numerous genes responsible for nitrogen utilization in Gibberella fujikuroi and many other fungi . We have previously shown that the deletion of areA-GF resulted in mutants with significantly reduced gibberellin (GA) production . Here we demonstrate that the expression level of six of the seven GA biosynthesis genes is drastically reduced in mutants lacking areA . Furthermore, we show that, despite the fact that GAs are nitrogen-free diterpenoid compounds, which are not obviously involved in nitrogen metabolism, AREA binds directly to the promoters of the six N-regulated genes . The binding of AREA was analysed in more detail using the promoter of one of the GA-biosynthesis genes encoding the ent-kaurene oxidase (P450-4) . Deletion/mutation analysis of the P450-4 promoter fused to the Escherichia coli uidA gene, which encodes beta-glucuronidase, allowed the in vivo identification of functional GATA motifs . We have also analysed the nmr gene of G . fujikuroi (nmr-GF) which has high similarity to the Neurospora crassa nmr-1 and Aspergillus nidulans nmrA genes, both involved in nitrogen metabolite repression . In contrast to our expectation, deletion of nmr-GF did not result in significant derepression of the GA biosynthesis genes in the presence of ammonium, glutamine or glutamate . Overexpression of the nmr-GF gene fused to the strong promoter of the G . fujikuroi glutamine synthetase (gs) gene revealed only a very slight repression of the nitrate reductase (niaD) gene, resulting in weak resistance to chlorate . Surprisingly, this effect was only observed in the presence of high amounts of glutamate; cultivation on ammonium failed to induce any resistance to chlorate . Despite the limited effect of gene replacement and overexpression of nmr-GF on the nitrogen metabolism of G . fujikuroi itself, the gene fully restored nitrogen metabolite repression in A . nidulans and N . crassa nmr mutants . Therefore, we postulate that, in contrast to A . nidulans and N . crassa, NMR does not function independently as the main modulator of AREA in G . fujikuroi.

Mol Microbiol, 2003 Feb, 47(4), 961 - 74
The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression; Caillet J et al.; In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding . This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein . The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome . This is the first demonstration of a modular structure for a translational repressor and is reminiscent of that of transcriptional regulators . The mimicry between the operator and tRNA, suspected on the basis of previous experiments, is further supported by the fact that identical regions of the synthetase recognize both the operator and the tRNA anticodon arm . Based on these results, and recent structural data, we have constructed a computer-derived molecular model for the operator-threonyl-tRNA synthetase complex, which sheds light on several essential aspects of the regulatory mechanism.

Mol Microbiol, 2003 Feb, 47(4), 871 - 7
Differential analysis of DNA microarray gene expression data; Hatfield GW et al.; Here, we review briefly the sources of experimental and biological variance that affect the interpretation of high-dimensional DNA microarray experiments . We discuss methods using a regularized t-test based on a Bayesian statistical framework that allow the identification of differentially regulated genes with a higher level of confidence than a simple t-test when only a few experimental replicates are available . We also describe a computational method for calculating the global false-positive and false-negative levels inherent in a DNA microarray data set . This method provides a probability of differential expression for each gene based on experiment-wide false-positive and -negative levels driven by experimental error and biological variance.

Plant J, 2003 Feb, 33(3), 503 - 11
ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits; Frueauf JB et al.; Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme {Frueauf, J.B., Ballicora, M.A . and Preiss J . (2001) J . Biol . Chem., 276, 46319-46325} . The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases . In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits . It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified . To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu . Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants . By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits . These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.

Eur J Biochem, 2003 Feb, 270(4), 757 - 63
Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein; Chen S et al.; The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway . The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine . Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains . Residues 1-88 constituted a functional CM domain, which was also dimeric . Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains . The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state . Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.

Eur J Biochem, 2003 Feb, 270(4), 600 - 9
A simple protocol to study blue copper proteins by NMR; Gelis I et al.; In the case of oxidized plastocyanin from Synechocystis sp . PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids . A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues . The proposed protocol differs from previous approaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form . This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field . The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.

Genes Cells, 2003 Feb, 8(2), 179 - 87
Instability of sensory histidine kinase mRNAs in Escherichia coli; Aiso T et al.; BACKGROUND: Regulating mRNA stability is one of the essential mechanisms in gene expression . In order to identify genes from Escherichia coli whole genome whose expression is effectively modulated during the process of mRNA decay, we previously performed differential display-PCR as the first step . In the screening, it was suggested that two mRNAs from the histidine kinase genes, narX and yojN, in a two-component signal transduction system, were extremely unstable . In this study we analysed the stability of sensory kinase mRNAs, e.g . arcB, barA, rcsC, narQ, narX and evgS mRNA . RESULTS: The cellular level of the histidine kinase mRNAs was very low and the mRNAs were rapidly degraded in wild-type cells cultured at 37 degrees C in LB medium . Additional experiments using RNase E deficient cells indicated that the mRNAs existed abundantly and expressed a prolonged half-life in the cells . Monocistronic transcripts of the cognate response regulator genes, arcA, rcsB, narP and narL have a half-life of 1.5-3.4 min . CONCLUSIONS: mRNAs of the six histidine kinase genes in E . coli are synthesized efficiently, but rapidly degraded in wild-type cells.

J Chromatogr A, 2003 Jan 24, 985(1-2), 519 - 29
Hyperlayer hollow-fiber flow field-flow fractionation of cells; Reschiglian P et al.; Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing . Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design . The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage . which is particularly appealing for analytical bio-applications . Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae) . Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.

Acta Virol, 2002, 46(3), 147 - 51
Polyclonal antibodies to a recombinant coat protein of Potato virus A; Cerovska N et al.; Specific mouse antibodies against a recombinant coat protein (CP) of Potato virus A (PVA) were produced . The PVA CP gene was cloned in an expression vector pMPM4omega . After expression in Escherichia coli the presence of the expressed CP was proved by Western blot analysis using polyclonal and monoclonal antibodies (MAbs) . The expressed CP was purified by centrifugation in CsCl density gradient or on a sucrose cushion . The production of virus-like particles (VLPs) was proved by electron microscopy . The purified CP was used for preparation of a mouse antiserum which had a titer of 1:1024 in ELISA and reacted specifically in Western blot analysis and indirect plate-trapped antigen ELISA (PTA-ELISA).

Extremophiles, 2003 Feb, 7(1), 79 - 83 Epub 2002 Nov 13.
Multi-subunit assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular protection at high temperature; Laksanalamai P et al.; The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, alpha-crystallin-like protein in response to exposure to extreme temperatures, above 103 degrees C . The P . furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes . Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions . We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly . The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics . The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP . The amino terminal domain was not required for inter-species thermotolerance.

Extremophiles, 2003 Feb, 7(1), 63 - 70 Epub 2002 Oct 15.
A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample; Sunna A et al.; Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers . Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined . Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample . Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence . Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame . The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases . The purified recombinant enzyme, XynA expressed in E . coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C . The enzyme showed high specificity toward different xylans and xylooligosaccharides.

J Biol Chem, 2003 May 2, 278(18), 15608 - 14 Epub 2003 Feb 10.
Accumulation of glucose 6-phosphate or fructose 6-phosphate is responsible for destabilization of glucose transporter mRNA in Escherichia coli; Morita T et al.; Previously we found that a mutation in either pgi or pfkA, encoding phosphoglucose isomerase or phosphofructokinase A, respectively, facilitates degradation of the ptsG mRNA in an RNase E-dependent manner in Escherichia coli (1) . In this study, we examined the effects of a series of glycolytic genes on the degradation of ptsG mRNA and how the mutations destabilize the ptsG mRNA . The conditional lethal mutation ts8 in fda, encoding fructose-1,6-P(2) aldolase just downstream of pfkA in the glycolytic pathway, caused the destabilization of ptsG mRNA at the nonpermissive temperature . Mutations in any other gene did not destabilize the ptsG mRNA; rather, they reduced the ptsG transcription mainly by affecting the cAMP level . The rapid degradation of ptsG mRNA in mutant strains was completely dependent upon the presence of glucose or any one of its compounds, which enter the Embden-Meyerhof glycolytic pathway before the block points . A significant increase in the intracellular glucose-6-P level was observed in the presence of glucose in the pgi strain . An overexpression of glucose-6-phosphate dehydrogenase eliminated both the accumulation and the degradation of ptsG mRNA in the pgi strain . In addition, accumulation of fructose-6-P led to the rapid degradation of ptsG mRNA in a pgi pfkA mutant strain lacking glucose-6-P . We conclude that the RNase E-dependent destabilization of ptsG mRNA occurs in response to accumulation of glucose-6-P or fructose-6-P.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2000 Jun, 8(2), 97 - 100
{Synthesis, Cloning and Expression of a Multiple Epitope Antigen of BCR-ABL Fusion Gene}; Zheng W et al.; Chronic myeloid leukemia (CML) appears an ideal and exciting immunological target . Novel and rational immunotherapy may therefore play an important adjuvant role in the treatment of CML patients . Peptides derived from the BCR-ABL fusion region have been shown to be immunogenic and are able to stimulate the production of BCR-ABL-specific T cell lines and clones . In this study, A 280 bp multiple epitope region of BCR-ABL fusion antigen was designed and synthesized . This region contains three BCR-ABL antigen epitopes which can bind to HLA-A2, HLA-A3 and HLA-DR11 molecules, respectively, and epitopes of cholera toxin B (CTB) and tetanus toxoid (TT) which are able to elicit vigorous T cell responses . The fusion antigen gene has highly been expressed in E . coli and the purified fusion protein reserved satisfied activity and antigenicity . The results of this investigation provided a basis for further research on the developing specific T cell immunotherapy of CML.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2000 Dec, 8(4), 251 - 254
{Cloning and Expression of Various Deletants of Gsalpha Gene in Escherichia Coli}; Lou J et al.; Gsalpha gene mutation has been discovered in some human tumors . In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia . To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E . coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E . coli, respectively . As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies . The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro . The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.

Biotechnol Appl Biochem, 2003 Feb, 37(Pt 1), 51 - 61
Process characterization of the chromatographic steps in the purification process of a recombinant Escherichia coli-expressed protein; Rathore AS et al.; Process development and characterization studies were performed for the chromatographic steps in the purification process of a recombinant Escherichia coli -expressed protein product candidate . The objective of this work was to develop a robust and efficient purification process that would generate material of adequate purity and quantity . A resin screening procedure was developed to aid in picking out the optimal resin for each of the chromatographic columns . It was found that, as a result of resin screening, it was possible to come up with a process with only two column-chromatographic steps . The resulting process used a sulphopropyl (SP) and a quaternary amino (Q) column with intermittent ultrafiltration steps for purification . Effects of different process parameters such as the gradient slope, pH, flow velocity and protein loading on the column performance were evaluated . Buffer pH for the SP column, and buffer pH, gradient slope, protein loading and flow velocity for the Q column, were identified as parameters that could have a significant impact on the performance of the chromatographic step and would require further characterization to improve the robustness of the process . Further process characterization led to the findings that the gradient slope, load pH and buffer pH of the Q column have a significant impact on column performance (>15% change in step yield) . All other parameters under consideration did not have any significant impact on pool quality (>10% change in pool purity for the SP column and >5% for the Q column) . On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a large-scale robust process with step yields and product quality that were better than those at the small scale.

Biochemistry, 2003 Feb 18, 42(6), 1796 - 803
Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding, catalysis, and synthesis of allolactose, the natural lac operon inducer; Huber RE et al.; Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli) . Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made . Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much . This shows that Trp-999 is important for binding in the shallow mode . The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor) . Substitution greatly diminished the affinity for glucose . Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions . The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions . Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG . The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much . As a result, k(3) became small and rate determining for each substituted enzyme . The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols . This is probably because glucose can approach with less restriction in the absence of Trp-999 . However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small . Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.

Biochemistry, 2003 Feb 18, 42(6), 1711 - 7
Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel; Tomson FL et al.; Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel . These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants . Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined . Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel . The current work examines the properties of mutants of this conserved glutamate in the oxidase from R . sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II) . It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%) . Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a(3) prior to the reaction with O(2) . These are phenotypes expected of K-channel mutants . It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.

Biochemistry, 2003 Feb 18, 42(6), 1707 - 10
Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules; Shoemaker GK et al.; By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase . Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater . beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1) . This activity was found to be significantly different from that of the enzyme used to grow the crystals, which showed an activity distribution of 38,500 +/- 900 reactions min(-1) (enzyme molecule)(-1).

Biochemistry, 2003 Feb 18, 42(6), 1652 - 9
Metal dependency for transcription factor rho activation; Weber TP et al.; The Escherichia coli rho transcription termination factor terminates select transcripts and rho activity requires Mg(2+) . We investigated whether divalent metal ions other than Mg(2+) catalyze rho-dependent ATP hydrolysis to ADP and P(i) in vitro . The effects of 11 divalent metal ions (Be(2+), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), Sr(2+), VO(2+), Zn(2+)) on ATPase activity were determined in the absence and presence of MgCl(2) . Without MgCl(2), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), VO(2+), and Zn(2+) activated ATP hydrolysis with either hyberbolic (Ca(2+), Co(2+), Cu(2+), Hg(2+), VO(2+)), peak velocity (Cd(2+), Mn(2+), Zn(2+)), or sigmoidal (Ni(2+)) rate acceleration curves . Sr(2+) was found to be a nonactivator and Be(2+) an inhibitor of rho-dependent ATPase activity . The metals' effects were compared with Mg(2+) and gave different rank orders when either the velocity (V(max), V(peak)) or the efficiency (V(max)/K(M), V(peak)/K(M)) of ATP hydrolysis was used as the determinant (V: Mg(2+) approximately Mn(2+) > Zn(2+) > Co(2+) > Ni(2+) approximately Cd(2+) > Ca(2+) > Cu(2+) > Hg(2+) approximately VO(2+); V/K(M): Mg(2+) > Mn(2+) > Ca(2+) > Co(2+) > Zn(2+) > Cu(2+) > Ni(2+) > Hg(2+) > Cd(2+)) . Mg(2+) proved to be the most effective divalent metal . We observed that the metal-dependent rates were affected by metal ion interactions with rho, RNA, and the buffer constituents . Significantly, replacement of the octahedral Mg(2+) ion by metals that typically prefer coordination spheres less than six (Cd(2+), Co(2+), Ni(2+), VO(2+), Zn(2+)) led to ATPase activity, suggesting that the putative Mg x ATP(2-) coordination sphere in rho does not need to remain fully intact for ATP hydrolysis.

Biochemistry, 2003 Feb 18, 42(6), 1581 - 8
Catalytic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: site-directed mutagenesis and biochemical studies; Li Y et al.; The roles of a pair of conserved positively charged residues R82 and R92 at a catalytic loop of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) have been investigated by site-directed mutagenesis and biochemical analysis . In the structure of HPPK in complex with ATP and a 6-hydroxymethyl-7,8-dihydropterin (HP) analogue, the guanidinium group of R82 forms two hydrogen bonds with the alpha-phosphate and that of R92 two hydrogen bonds with the beta-phosphate . In the structure of HPPK in complex with alpha,beta-methyleneadenosine triphosphate (AMPCPP, an ATP analogue) and HP, the guanidinium group of R82 has no direct interaction with AMPCPP and that of R92 forms two hydrogen bonds with the alpha-phosphate . Substitution of R82 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 380, but there were no significant changes in the binding energy or binding kinetics of either substrate . Substitution of R92 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 3.5 x 10(4) . The mutation caused no significant changes in the binding energy or binding kinetics of MgATP . It did not cause a significant change in the binding energy of HP either but caused a decrease in the association rate constant for the binding of HP by a factor of approximately 4.5 and a decrease in the dissociation rate constant by a factor of approximately 10 . The overall structures of the ternary complexes of both mutants were very similar to the corresponding structure of wild-type HPPK as described in the companion paper . The results suggest that R82 does not contribute to the binding of either substrate, and R92 is dispensable for the binding of MgATP but plays a role in facilitating the binding of HP . Both R82 and R92 are important for catalysis, and R92 plays a critical role in the transition state stabilization.

Biochemistry, 2003 Feb 18, 42(6), 1573 - 80
Dynamic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: crystallographic studies; Blaszczyk J et al.; 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway . Arginine residues 82 and 92, strictly conserved in 35 HPPK sequences, play dynamic roles in the catalytic cycle of the enzyme . At 0.89-A resolution, two distinct conformations are observed for each of the two residues in the crystal structure of the wild-type HPPK in complex with two HP variants, two Mg(2+) ions, and an ATP analogue . Structural information suggests that R92 first binds to the alpha-phosphate group of ATP and then shifts to interact with the beta-phosphate as R82, which initially does not bind to ATP, moves in and binds to alpha-phosphate when the pyrophosphoryl transfer is about to occur . The dynamic roles of R82 and R92 are further elucidated by five more crystal structures of two mutant proteins, R82A and R92A, with and without bound ligands . Two oxidized forms of HP are observed with an occupancy ratio of 0.50:0.50 in the 0.89-A structure . The oxidation of HP has significant impact on its binding to the protein as well as the conformation of nearby residue W89.

Biochemistry, 2003 Feb 18, 42(6), 1499 - 507
Protein-protein interactions between cytochrome b and the Fe-S protein subunits during QH2 oxidation and large-scale domain movement in the bc1 complex; Darrouzet E et al.; The ubihydroquinone:cytochrome (cyt) c oxidoreductase, or bc(1) complex, and its homologue the b(6)f complex are key components of respiratory and photosynthetic electron transport chains as they contribute to the generation of an electrochemical gradient used by the ATP synthase to produce ATP . The bc(1) complex has two catalytic domains, ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) sites, that are located on each side of the membrane . The key to the energetic efficiency of this enzyme relies upon the occurrence of a unique electron bifurcation reaction at its Q(o) site . Recently, several lines of evidence have converged to establish that in the bc(1) complex the extrinsic domain of the Fe-S subunit that contains a {2Fe2S} metal cluster moves during catalysis to shuttle electrons between the Q(o) site and c(1) heme . While this step is required for electron bifurcation, available data also suggest that the movement might be controlled to ensure maximal energetic efficiency {Darrouzet et al . (2000) Proc . Natl . Acad . Sci . U.S.A . 97, 4567-4572} . To gain insight into the plausible control mechanism, we used a biochemical genetic approach to define the different regions of the bc(1) complex that might interact with each other . Previously, we found that a mutation located at position L286 of the ef loop of Rhodobacter capsulatus cyt b could alleviate movement impairment resulting from a mutation in the hinge region, linking the {2Fe2S} cluster domain to the membrane anchor of the Fe-S subunit . Here we report that various substitutions at position 288 on the opposite side of the ef loop also impair Q(o) site catalysis . In particular, we note that while most of the substitutions affect only QH(2) oxidation, yet others like T288S also hinder the rate of the movement of the Fe-S subunit . Thus, position 288 of cyt b appears to be important for both the QH(2) oxidation and the movement of the Fe-S subunit . Moreover, we found that, upon substitution of T288 by other amino acids, additional compensatory mutations located at the {2Fe2S} cluster or the hinge domains of the Fe-S subunit, or on the cd loop of cyt b, arise readily to alleviate these defects . These studies indicate that intimate protein-protein interactions occur between cyt b and the Fe-S subunits to sustain fast movement and efficient QH(2) oxidation and highlight the critical dual role the ef loop of cyt b to fine-tune the docking and movement of the Fe-S subunit during Q(o) site catalysis.

Biochemistry, 2003 Feb 18, 42(6), 1391 - 400
Substrate-induced conformational changes of the periplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling; Fanucci GE et al.; The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling . Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12) . BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine . In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes . The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration) . This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system . The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate . Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change . EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters . When substrate binds to BtuB, this first beta-strand remains folded . The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions . The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.

Biochemistry, 2003 Feb 18, 42(6), 1377 - 82
Aromatic stacking in the sugar binding site of the lactose permease; Guan L et al.; Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269) . We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring . Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type . In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max) . Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively . The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized . The results are discussed in the context of a current model for the binding site.

Shock, 2003 Feb, 19(2), 187 - 90
Induction of procalcitonin and proinflammatory cytokines in an anhepatic baboon endotoxin shock model; Meisner M et al.; Our objective was to evaluate the role of the liver for procalcitonin (PCT) and cytokine induction in a baboon endotoxin shock model . Complete liver resection with portocaval anastomosis was established in a baboon prior to the induction of endotoxin shock by intravenous administration of endotoxin (100 microg/kg LPS Escherichia coli) . Two baboons without surgical intervention were used as controls . Plasma concentrations of PCT, tumor necrosis factor (TNF)-alpha, interleukin (IL) 6, IL-8, endotoxin, and hemodynamic and metabolic parameters were measured pre- and postoperatively and until 6 h after endotoxin administration . PCT concentrations increased to 1.2 and 4.6 ng/mL in control animals at 6 h, but remained below 0.3 ng/mL in the anhepatic baboon . IL-6 and IL-8 increased only for few hours in controls, but remained elevated in the hepatectomized animal near their maximum (IL-6, 2-6 ng/mL) or several-fold higher (IL-8, 30-35 ng/mL), whereas TNF-alpha response was only a small fraction (0.3 ng/mL) of the controls . Endotoxin was much higher and longer persisting in the hepatectomized animal compared with controls . The near absence of PCT production in the anhepatic baboon suggests a primary role for the liver as a source of PCT production during endotoxin shock . Furthermore, the liver also seems to be an important source of TNF-alpha, but not IL-6 or IL-8.

Biol Neonate, 2003, 83(2), 107 - 12
Increased respiratory burst and increased expression of complement receptor-3 (CD11b/CD18) and of IL-8 receptor-A in neutrophil granulocytes from newborns after vaginal delivery; Gessler P et al.; To study neutrophil activation in cord blood as a function of the mode of delivery, we performed analysis of the function of neutrophil granulocytes by assessing their ability to produce reactive oxygen products (ROP) as well as neutrophil cell surface expression of CD11b/CD18 and interleukin (IL)-8 receptors quantified with flow cytometry . Plasma levels of granulocyte colony-stimulating factor (G-CSF) were measured using an immunoassay . Neutrophil granulocytes were derived from cord blood of term newborns delivered vaginally (n = 20) and by cesarean section (n = 10), and, for comparison, from adult peripheral blood (n = 15) . Blood neutrophil counts and the capacity of neutrophil granulocytes to produce ROP in response to stimulation with Escherichia coli was increased in newborns after vaginal delivery as compared to newborns delivered by cesarean section . The level of expression of the adhesion molecule/complement receptor CD11b/CD18 and the chemokine receptor IL-8 RA was also higher after vaginal delivery . Plasma concentrations of G-CSF in cord blood of newborns were higher than those of adults with no difference detectable between vaginal delivery and cesarean section . The data demonstrate a higher functional responsiveness and a higher expression level of functionally important receptors in neutrophils after vaginal delivery possibly due to activation of neutrophil granulocytes during labor .

Microbiology, 2003 Jan, 149(Pt 1), 205 - 15
Transcriptional analysis of the 5' terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans; Haase EM et al.; Fresh isolates of the oral bacterial pathogen Actinobacillus actinomycetemcomitans exhibit a fimbriated, rough colony phenotype . Evidence suggests that the fimbrial subunit gene flp is part of a cluster of 14 genes (flp to tadG) thought to encode proteins involved in the synthesis, assembly and export of these fimbriae . To determine the transcriptional organization of the 5' terminus of this gene cluster, total RNA from rough and smooth phenotype variants of A . actinomycetemcomitans strain 283 were analysed by RT-PCR . Primers designed to amplify regions spanning gene junctions or multiple genes yielded amplicons at each individual gene junction from flp to tadD for both the rough and smooth variants . Semi-quantitative RT-PCR of the rcpA to tadZ amplicon revealed that significantly more mRNA was transcribed from the rough than the smooth variant . Longer amplicons encompassing flp to tadZ (3.9 kb) and tadA to tadD (2.1 kb) were also detected, but only from the rough variant . Rapid amplification of cDNA ends (RACE) was used to identify the 5' end of the mRNA containing flp . Antisense primers located within rcpC, orfB and flp-2 enabled amplification of a RACE product that was subsequently isolated and subcloned into pGEM-T . DNA sequencing indicated that the 5' end of the mRNA was located at a G or T nucleotide -102 to -101 nt upstream of flp . Corresponding sigma(70) consensus sequences were located at -10 (TATAAT) and -35 (TTGCAT) relative to the transcription start site . These data confirm that the flp gene cluster is an operon transcribed as a polycistronic message commencing from a G or T nucleotide located in the intergenic region upstream of flp . Promoter function of the flp upstream region was confirmed using a lacZ reporter gene construct transformed into Escherichia coli . RT-PCR analysis further suggests that although transcription does occur in both the rough and smooth variants, full-length transcripts are rapidly degraded or are significantly downregulated in the smooth variant.

Anal Biochem, 2003 Feb 1, 313(1), 160 - 6
Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Humeny A et al.; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand . The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications . Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately . Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes . We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme . Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction . We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings .

Anal Biochem, 2003 Feb 1, 313(1), 145 - 54
Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli; Maharjan RP et al.; The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition . In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction . In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways . In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol . Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with {14C}glucose . The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds . For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots . In contrast, 80 or less spots were obtained with other extraction methods . Extraction also influenced quantitative analysis of particular compounds . Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform . The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis .

Anal Biochem, 2003 Feb 1, 313(1), 68 - 75
Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system; Gorokhovatsky AY et al.; Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP) . The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells . Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity . It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin . These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin .

Structure (Camb), 2003 Feb, 11(2), 187 - 96
ATP-mediated conformational changes in the RecA filament; VanLoock MS et al.; The crystal structure of the E . coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination . Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments . The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity . A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits . This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.

Structure (Camb), 2003 Feb, 11(2), 139 - 45
Dehydration converts DsbG crystal diffraction from low to high resolution; Heras B et al.; Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process . Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement . Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 A resolution . After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 A resolution . This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

Br J Nutr, 2003 Feb, 89(2), 159 - 66
Fate of genetically modified maize DNA in the oral cavity and rumen of sheep; Duggan PS et al.; The polymerase chain reaction (PCR) technique was used to investigate the fate of a transgene in the rumen of sheep fed silage and maize grains from an insect-resistant maize line . A 1914-bp DNA fragment containing the entire coding region of the synthetic cryIA(b) gene was still amplifiable from rumen fluid sampled 5 h after feeding maize grains . The same target sequence, however, could not be amplified from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line . PCR amplification of a shorter (211-bp), yet still highly specific, target sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively . These findings indicate that intact transgenes from silage are unlikely to survive significantly in the rumen since a DNA sequence 211-bp long is very unlikely to transmit genetic information . By contrast, DNA in maize grains persists for a significant time and may, therefore, provide a source of transforming DNA in the rumen . In addition, we have examined the biological activity of plasmid DNA that had previously been exposed to the ovine oral cavity . Plasmid extracted from saliva sampled after incubation for 8 min was still capable of transforming competent Escherichia coli to kanamycin resistance, implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria.

Zhong Yao Cai, 2000 Dec, 23(12), 762 - 4
{Preliminary screen of anti-endotoxin activity of Chinese materia medica}; Liu Y et al.; OBJECTIVE: To screen Chinese materia medica against E . coli . O111B4 endotoxin . METHOD: 70% alcohol extracts from 134 kinds of heat-clearing Chinese materia medica were demonstrated by using limulus test in vitro . The extracts which had anti-endotoxin effects were diluted to demonstrate their minimal anti-endotoxin concertnations . RESULTS: 75 kinds of Chinese materia medica possessed anti-endotoxin activity in various degrees in vitro . CONCLUSION: Most of heat-clearing Chinese materia medica possess anti-endotoxin effects in vitro.

Hua Xi Yi Ke Da Xue Xue Bao, 2002 Apr, 33(2), 172 - 4, 195
{Molecular cloning and expression of the immunodominant protein Ag85A from Mycobacterium tuberculosis H37Rv strain}; Xie Y et al.; OBJECTIVE: To provide the target gene and target antigen for the development of new vaccine against tuberculosis . METHODS: According to the gene sequence encoding protein Ag85A from Mycobacterium tuberculosis H37Rv strain, we designed a pair of oligonucleotide primers, obtained the gene by using polymerase chain reaction, and inserted the gene into the BamH I and EcoR I site of plasmid pBK-CMV to construct recombinant plasmid, and after that, the recombinant plasmid was transferred into E . coli XL1-Blue MRF' and induced with IPTG . The expression product of the gene was analyzed by using SDS-PAGE and western-blotting . RESULTS: The gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain was successfully amplified by using PCR . A recombinant shuttle plasmid was constructed . The recombinant plasmid stably expressed recombinant Ag85A protein relative molecular mass 32 x 10(3) in E . coli XL1-Blue MRF' . CONCLUSION: A recombinant plasmid which contains the gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain has been successfully constructed . The recombinant plasmid can stably express recombinant protein relative molecular mass 32 x 10(3) in E . coli XL1-Blue MRF' . These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of new vaccine against tuberculosis.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2231 - 6 Epub 2003 Feb 06.
Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes; Zhu Y et al.; The structures of membrane receptors mediating rapid, nongenomic actions of steroids have not been identified . We describe the cloning of a cDNA from spotted seatrout ovaries encoding a protein that satisfies the following seven criteria for its designation as a steroid membrane receptor: plausible structure, tissue specificity, cellular distribution, steroid binding, signal transduction, hormonal regulation, and biological relevance . For plausible structure, computer modeling predicts that the protein has seven transmembrane domains, typical of G protein-coupled receptors . The mRNA (4.0 kb) is only detected in the brain and reproductive tissues on Northern blots . Antisera only detect the protein (40 kDa) in plasma membranes of reproductive tissues . The recombinant protein produced in an Escherichia coli expression system has a high affinity (K(d) = 30 nM), saturable, displaceable, single binding site specific for progestins . Progestins alter signal transduction pathways, activating mitogen-activated protein kinase and inhibiting adenylyl cyclase, in a transfected mammalian cell line . Inhibition of adenylyl cyclase is pertussis toxin sensitive, suggesting the receptor may be coupled to an inhibitory G protein . Progestins and gonadotropin up-regulate both mRNA and protein levels in seatrout ovaries . Changes in receptor abundance in response to hormones and at various stages of oocyte development, its probable coupling to an inhibitory G protein and inhibition of progestin induction of oocyte maturation upon microinjection of antisense oligonucleotides are consistent with the identity of the receptor as an intermediary in oocyte maturation . These characteristics suggest the fish protein is a membrane progestin receptor mediating a "nonclassical" action of progestins to induce oocyte maturation in fish.

J Immunol, 2003 Feb 15, 170(4), 2214 - 20
Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis; Lee SH et al.; Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes . Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication . Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3) . To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI) . MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites . Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue . Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium . To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine . Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS . When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6) . Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6 . Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA . We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

J Immunol, 2003 Feb 15, 170(4), 2074 - 82
Suppression of NF-kappa B activation and proinflammatory cytokine expression by Shiga toxin-producing Escherichia coli; Hauf N et al.; The NF-kappaB family of transcription factors forms one of the first lines of defense against infectious disease by inducing the expression of genes involved in inflammatory and immune responses . In this study, we analyzed the impact of Shiga toxin-producing Escherichia coli (STEC) on the NF-kappaB DNA-binding activity in HeLa cells . After a period of weak initial activation, DNA binding of NF-kappaB was actively suppressed by viable, E . coli secreted protein B (EspB)-secreting STEC . Sustained NF-kappaB activity was observed either using an isogenic mutant lacking EspB or after gentamicin-based killing of STEC after allowing bacterial attachment . These observations indicate that the ability of STEC to cause NF-kappaB activation is suppressed by a translocated bacterial effector protein, which is either EspB itself or requires EspB for delivery into the host cell . We found that STEC, enterohemorrhagic E . coli, and enteropathogenic E . coli all interfere with NF-kappaB activation initiated by TNF-alpha, indicating that suppression of signal-induced NF-kappaB activity is a property common to several attaching and effacing bacteria . As a consequence of NF-kappaB suppression, wild-type STEC induces significantly lower mRNA levels of IL-8, IL-6, and IL-1alpha upon prolonged infection periods compared with bacteria lacking EspB . For IL-8 and IL-6, the suppressive effect was also reflected at the level of cytokine secretion . Suppression of both basal and signal-induced NF-kappaB DNA binding by attaching and effacing-inducing bacteria appears to be an active strategy to counteract host defense responses, thus favoring intestinal colonization by these pathogens.

J Immunol, 2003 Feb 15, 170(4), 1625 - 9
Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells; McCurdy JD et al.; Mast cells play a critical role in host defense against bacterial infection . Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms . The expression of TLRs by human mast cells and responses to known TLR activators was examined . Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4 . Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1beta and also induced substantial short-term cysteinyl leukotriene generation . In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production . The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1beta leukotriene, or degranulation response . These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.

J Biol Chem, 2003 Apr 18, 278(16), 13819 - 28 Epub 2003 Feb 06.
PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit . Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation; Kim YM et al.; PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus . In the present study, we characterized the biochemical properties of PNUTS . A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells . A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity . The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe . Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1 . The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells . In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA . PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs . Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads . Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.

Hypertension, 2003 Feb, 41(2), 302 - 7
Adrenomedullin overexpression to inhibit cuff-induced arterial intimal formation; Yamasaki M et al.; Adrenomedullin (AM) inhibits vascular smooth muscle cell proliferation stimulated by fetal calf serum and platelet-derived growth factor in vitro . In this study, an adenovirus expressing AM (AxCAAM) was created to examine the in vivo action of AM . Femoral arteries of Wistar rats were wrapped with a silicone cuff and treated with adenovirus expressing Escherichia coli beta-galactosidase (AxCALacZ) or AxCAAM . Immunoreactivity for endothelial nitric oxide synthase (eNOS) was reduced in the endothelium of cuff-injured arteries and was associated with increased local DNA synthesis . Consequently, the intimal formation measured by the intimal-to-medial ratio was significantly increased at 14 and 28 days after the cuff placement . AxCAAM-infected arteries increased the expression of eNOS in the endothelium and inducible NOS in the media and the adventitia . AxCAAM significantly decreased the intimal-to-medial ratio by 40% at 14 days and 51% at 28 days, whereas AxCALacZ showed no changes compared with cuff-injured control arteries . AM overexpression effectively limits intimal hyperplasia by reducing cell proliferation through a nitric oxide-dependent pathway of eNOS . Our findings suggest the possibility of a therapeutic use of the AM gene for the prevention of vascular proliferative disorders.

Arch Biochem Biophys, 2003 Feb 15, 410(2), 307 - 16
Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme; Kopcho LM et al.; Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD) . Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD . The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane . Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE . The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands . We find no significant differences in ligand binding properties among these various constructs . These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment . Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.

Arch Biochem Biophys, 2003 Feb 15, 410(2), 261 - 8
Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product; Hirakawa K et al.; DNA damage mediated by photosensitizers participates in solar carcinogenesis . Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA) . Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment . The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA . Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself . In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer . Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light.

Arch Biochem Biophys, 2003 Feb 15, 410(2), 238 - 45
The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne); Pudney PD et al.; We have characterized a cold-induced, boiling stable antifreeze protein . This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects . Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure . Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone . These results are discussed in light of the currently proposed mechanisms of AFP action.

Biochim Biophys Acta, 2003 Feb 21, 1645(2), 228 - 40
Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP(C)-like) or amyloid (PrP(Sc)-like) conformation; Alvarez-Martinez MT et al.; Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity . We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)) . A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid . The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation . Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography . An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein . About 10 mg of PrP protein per liter of bacterial culture was obtained . The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy . This protein had the structural characteristics of PrP(C) and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP(Sc)) . The sensitivity of these two forms to high pressure was investigated . We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.

Biochim Biophys Acta, 2003 Feb 21, 1645(2), 212 - 7
Chloroplast fructose 1,6-bisphosphatase with changed redox modulation: comparison of the Galdieria enzyme with cysteine mutants from spinach; Reichert A et al.; Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E . coli . The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria . In spinach chloroplast FBPase, Cys(155) and Cys(174) are engaged in the formation of the disulfide bridge . The corresponding mutants are active when expressed in E . coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme . The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg(2+) as is the case for the wild-type enzyme . In the sequence of Galdieria FBPase, the Cys that corresponds to Cys(179) in the spinach enzyme is lacking . However, the Galdieria FBPase, in contrast to the spinach Cys(179) mutant, does not show any indication for a comparable redox modulation of its activity . Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties . Upon reduction, the lost activity can be recovered.

Biochim Biophys Acta, 2003 Feb 21, 1645(2), 183 - 92
Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase; Boulatnikov I et al.; Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general . In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4) . These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated . Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell . The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography . The purified kinase was stable and was not subjected to reassortment of the subunits . The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation . These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity.

Biochim Biophys Acta, 2003 Feb 21, 1645(2), 139 - 45
Construction and characterization of a chimeric myoglobin; Musto R et al.; In order to investigate the functional and structural role of modular structure in globins, we have engineered a chimeric myoglobin (ChimMb) in which the first and third exon come from the gene coding for the sperm whale Mb and the second exon from the gene coding for Aplysia limacina Mb . This ChimMb, fused to the Maltose Binding Protein (MBP) and expressed in Escherichia coli as an apoprotein, binds protoheme in a 1:1 stoichiometric ratio . Based on some functional and spectroscopic properties, we conclude that the central core of the ChimMb (which derives from A . limacina) is native-like . On the other hand, the ChimMb deprived (by proteolytic digestion) of the fused MBP displays a considerably reduced stability . These results suggest that the sperm whale A-G-H nucleus does not contribute significantly to the overall stability of the ChimMb.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 152 - 7
Quantitative monitoring for secreted production of human interleukin-2 in stable insect Drosophila S2 cells using a green fluorescent protein fusion partner; Shin HS et al.; Human interleukin-2 (hIL-2) production in Escherichia coli and insect cell/baculovirus expression systems can be inefficient . Here we investigated secreted production of hIL-2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogaster S2 cells . This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins . We report that, following stable transfection and induction, S2 cells secreted hIL-2 as a fusion protein (approximately 2.3 microg/mL yield), with a secretion efficiency of approximately 90% . Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL-2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system . The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL-2 expression.

Aviakosm Ekolog Med, 2002, 36(5), 42 - 5
{The bactericide effect of laser infrared radiation}; Voskanian KSh; The investigation had the purpose to study the lethal effect of laser infrared radiation (1220-1320 nm) on various genotypes of E . coli K-12 cells . Laser was produced by a parametric LiNbO3 light generator pumped up by the NdYAG second harmonic radiation . Results were compared with effects of X-ray radiation at 200 kV . The irradiated bacteria were cultivated in a solid full nutrient medium over 24 hrs at 37 degrees C . Bacteria spread in monolayer on the surface of "starvation" agar (4% agar-agar) were exposed to each type of radiation at room temperature . Survivability of the cells was determined by counting macrocolonies after 2 days of growth at 37 degrees C . Laser radiation at a wave-length of 1270 nm was found to have the highest lethal effect . This corresponds to one of the peaks of molecular oxygen consumption . Based on comparative analysis of the lethal effectiveness, the bacterial strains revealed equal relative sensitivity to the laser and X-ray radiation . Laser effectiveness was power dependent . Hence, proof has been exhibited that laser radiation at a wave-length of 1270 nm can be used as a bactericide means . The apparent advantage of the means over the photodynamic therapy is that no dyes are required.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2001, 19(1), 15 - 8
Cloning and expressing of the three repeat fragments of Plasmodium falciparum 11.1 gene; Hu W et al.; OBJECTIVE: To clone and express the 3R, 6R and 9R repeat fragments of Plasmodium falciparum(Pf11.1) gene . METHODS: Three repeat fragments from the genomic DNA of Plasmodium falciparum 3D7 strain cultivated were amplified by using the designed primers . The PCR products were cloned into the pT7 vector for bi-direction sequencing . The sequencing results were analysised by GENETYX-MAC . And then the amplified fragments were subcloned into pET32a(+) or pET32b(+) in order to express the recombinant proteins under the induction of IPTG in E . coli BL21 . RESULTS: 3R,6R and 9R fragments with sizez of 552 bp, 630 bp and 444 bp respectively were successfully amplified by PCR . The sequence analysis showed that there were 4 more 3AA units and one more 6AA unit in Pf11.1 gene of 3D7 strain as compared with Palo Alto strain . The homologies of the nucleotide sequence between the 3R fragment and the 6R fragment of the two strains were 92.8% and 95.1%, respectively . The amplified 9R fragment contains 139AA repeat units . The three recombinant proteins were expressed in BL21 strain with molecular weights of 45, 60 and 42 kDa . CONCLUSION: We got the 3R, 6R and 9R fragments separately by PCR and expressed them in E . coli successfully . The Pf11.1 gene of 3D7 strain is highly homologous to that of the Palo Alto strain.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2001, 19(2), 80 - 3
{Expression and immunocompetence characterization of Plasmodium falciparum lactate dehydrogenase}; Wu YS et al.; OBJECTIVE: To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E . coli TG1 and analyse its immunocompetence . METHODS: The LDH gene of the P . falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX-4T-1 vector for expression of fusion protein with glutathione S-transferase(GST) . The recombinant plasmid was transformed into the E . coli TG1 . Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected . The immunocompetence of recombinant protein was analysed by ELISA and Western-blot . RESULTS: The LDH gene of P . falciparum was successfully expressed in the E . coli TG1 . The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P . falciparum . The specific humoral responses were induced in mice and the titer of the specific antibody was 1:16 by two-dimensional diffusion assay . CONCLUSION: The LDH gene of P . falciparum has been successfully expressed in the E . coli TG1 and the expressed protein has high antigencity.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2001, 19(4), 209 - 12
{Aedes albopictus: cloning and identification of the acetylcholinesterase gene fragment from the mosquito}; Wu MW et al.; OBJECTIVE: To isolate, clone and identify the acetylcholinesterase (AChE) fragment from the mosquito, Aedes albopictus, in relation to exploring mechanism of insectide resistance . METHODS: The genome DNA extracted from the mosquito was used for degenerate polymerase chain reaction (PCR) and the two pairs of oligonucleotides encoding the highly conserved protein sequences were used as primers . The reaction products were cloned to T-vector and transfected into E . coli JM 109 . The replicative form DNA of recombinant vector extracted from E . coli JM 109 through alkalilysis was identified by the methods of digestion with EcoR I and Sal I and PCR . RESULTS: The products of degenerate primers polymerase chain reaction were obtained and the identified clone belongs to the AChE fragment of the mosquito . CONCLUSION: The clone was identified as the AChE fragment of Aedes albopictus.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2001, 19(4), 201 - 4
{Study on molecular phylogeny of Schistosoma sinensium based on nuclear ribosomal DNA}; Zhang GJ et al.; OBJECTIVE: To determine the phylogenetic relationships between Schistosoma sinensium and other Schistosomatid species using DNA sequence data . Two segments of the nuclear rDNA repeat, the second internal spacer (ITS2) and large subunit (LSU/12S) were selected for sequencing . METHODS: Adult worms stored in 100% methanol were washed 3 times with 0.1 x TE (pH8.0) and the genomic DNA was extracted by the GNT-K method . The target regions were amplified by PCR using specific primers . The PCR products were purified before ligation into the plasmid pT-adv (Clontech) . Recombinant plasmids were amplified in E . coli (strain TOP10), extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long-read auto-sequencer . Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE . Gene trees were constructed in PHYLIP (Version 3.6 alpha July, 2,000) and MEGA (version 2.0 beta build 3) using both Maximum Parsimony and Neighbor-Joining methods . For parsimony analysis, all characters were treated as unordered and with equal weights . At least 3,000 cycles of bootstrapping were carried out . For analysis in MEGA, all gap columns were deleted . Schistosomatium douthitti and Trichobilharzia were used as outgroups . RESULTS: The ITS2 and LSU sequences of Schistosoma sinensium were obtained . The ITS2 sequence of Trichobilharzia sp . was reported here for the first time . CONCLUSION: The phylogenetic trees from these data of nuclear rDNA suggested that S . sinensium belongs to the Asian schistosome group . And this species might be an ancient member in the Asian clade.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2001, 19(4), 198 - 200
{Induced expression of the variable region of AMA-1 from Plasmodium falciparum}; Nie BY et al.; OBJECTIVE: To express the variable region of AMA-1 gene from Plasmodium falciparum in Escherichia coli . METHODS: Genomic DNA of FCC1/HN was used as template and the variable region of AMA-1 gene was amplified by polymerase chain reaction(PCR) . The PCR products were digested by endonuclease BamH I and Hind III, cloned into pBlu2KSP . The nucleotide sequences of the variable region of AMA-1 gene were determined by sequencing . The AMA-1 gene fragment was subcloned into plasmid pQE, expressed in E . coli and induced by IPTG . The fusion product as identified by SDS-PAGE gel electrophoresis and Western blotting were proceeded with anti-AMA-1 sera from rabbit . RESULTS: The size of the variable region of AMA-1 gene from FCC1/HN was 506 bp and encoded 168 amino acids . On SDS-PAGE gel dyed with Coomassie brilliant blue R250, no specific protein band can be discerned, but Western blotting proceeded with anti-AMA-1 sera from rabbit demonstrated that the specific protein band was about 23.0 kDa . CONCLUSION: The variable region of AMA-1 gene from FCC1/HN was able to be expressed in E . coli and analysis of Western blotting demonstrated that the AMA-1 fussion protein contained specific antigenic epitopes.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1586 - 91 Epub 2003 Feb 05.
The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein; Rohdich F et al.; Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate . A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain . The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E . coli mutant . This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents . On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1) . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1) . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1) . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

J Biol Chem, 2003 Apr 18, 278(16), 14523 - 32 Epub 2003 Feb 05.
Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis; Wuebbens MM et al.; Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans . In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin . Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins . The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus . Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined . A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed . Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction . An intermediate of the synthase reaction was identified and characterized . This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE . Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.

Appl Environ Microbiol, 2003 Feb, 69(2), 1295 - 8
Enhanced production of recombinant proteins in Escherichia coli by filamentation suppression; Jeong KJ et al.; During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation . Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein . Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation . By the coexpression of the E . coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved . Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.

Appl Environ Microbiol, 2003 Feb, 69(2), 725 - 33
Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobacter blasticus; Inui M et al.; A 3.4-kb cryptic plasmid was obtained from a new isolate of Rhodobacter blasticus . This plasmid, designated pMG160, was mobilizable by the conjugative strain Escherichia coli S17.1 into Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris . It replicated in the latter strains but not in Rhodospirillum rubrum, Rhodocyclus gelatinosus, or Bradyrhizobium species . Plasmid pMG160 was stably maintained in R . sphaeroides for more than 100 generations in the absence of selection but showed segregational instability in R . palustris . Instability in R . palustris correlated with a decrease in plasmid copy number compared to the copy number in R . sphaeroides . The complete nucleotide sequence of plasmid pMG160 contained three open reading frames (ORFs) . The deduced amino acid sequences encoded by ORF1 and ORF2 showed high degrees of homology to the MobS and MobL proteins that are involved in plasmid mobilization of certain plasmids . Based on homology with the Rep protein of several other plasmids, ORF3 encodes a putative rep gene initiator of plasmid replication . The functions of these sequences were demonstrated by deletion mapping, frameshift analysis, and analysis of point mutations . Two 6.1-kb pMG160-based E . coli-R . sphaeroides shuttle cloning vectors were constructed and designated pMG170 and pMG171 . These two novel shuttle vectors were segregationally stable in R . sphaeroides growing under nonselective conditions.

Curr Protein Pept Sci, 2003 Feb, 4(1), 73 - 80
Rapid translation system (RTS): a promising alternative for recombinant protein production; Betton JM; Rapid Translation System (RTS) is a cell-free protein production system employing an enhanced Escherichia coli lysate to perform coupled in vitro transcription-translation reactions . A continuous supply of energy substrates, nucleotides and amino acids combined with the removal of by-products guarantees a high yield of protein production . The gene to express is either cloned into a plasmid vector or introduced as a PCR product amenable to automation . The main property of this alternative system to cellular expression systems is its open design allowing direct manipulation of the reaction conditions and applications that are impossible or difficult in cell-based systems . RTS offers new promising possibilities in the postgenomic era.

J Med Chem, 2003 Feb 13, 46(4), 591 - 600
Design, synthesis, and biological activities of classical N-{4-{2-(2-amino-4-ethylpyrrolo{2,3-d}pyrimidin-5-yl)ethyl}benzoyl}-l-glutamic acid and its 6-methyl derivative as potential dual inhibitors of thymidylate synthase and dihydrofolate reductase and as potential antitumor agents; Gangjee A et al.; Two novel analogues, N-{2-amino-4-ethyl{(pyrrolo{2,3-d}pyrimidin-5-yl)ethyl}benzoyl}-l-glutamic acid (2) and N-{2-amino-4-ethyl-6-methyl{(pyrrolo{2,3-d}pyrimidin-5-yl)ethyl}benzoyl}-l-glutamic acid (4), were designed and synthesized as potent dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents . Compound 2 had inhibitory potency against human DHFR similar to N-{4-{2-(amino-3,4-dihydro-4-oxo-7H-pyrrolo{2,3-d}pyrimidin-5-yl)ethyl}benzoyl}-L-glutamic acid (LY231514) and 1, whereas 4 was inactive against human DHFR . Both 2 and 4 were more potent than LY231514 against E . coliTS . Against human TS, 2 was 7-fold less potent than LY231514 and 4 showed similar inhibitory activity as LY231514 . In contrast to 2, which was an efficient substrate of human folypolyglutamate synthetase (FPGS), 4 was a poor substrate of FPGS . Compound 2 showed GI50 values in the nanomolar range against more than 18 human tumor cell lines in the standard NCI preclinical in vitro screen.

J Small Anim Pract, 2003 Jan, 44(1), 13 - 6
Renal abscess in a dog with transient diabetes mellitus; Hess RS et al.; A nine-year-old, intact female dalmatian with diabetes mellitus and a renal abscess is described . The renal abscess was treated surgically by nephrectomy, and the diabetes mellitus resolved with ovariohysterectomy . Abdominal ultrasound and ultrasound-guided aspiration of the abscess were helpful in establishing a diagnosis . To the authors' knowledge, this is the first report of a renal abscess in a dog with diabetes mellitus.

Fundam Clin Pharmacol, 2002 Aug, 16(4), 303 - 9
Nimesulide and diclofenac inhibit lipopolysaccharide-induced hypothermia and tumour necrosis factor-alpha elevation in rats; Dogan MD et al.; The effects of nimesulide and diclofenac on lipopolysaccharide (LPS)-induced rectal temperature changes and serum tumour necrosis factor (TNF)-alpha elevation were investigated in rats . LPS (Escherichia coli O111:B4; 50 microg/kg, intraperitoneally) produces a dual body temperature response, in which initial hypothermia precedes fever . Serum TNF-alpha levels rise during the initial phase of the induced hypothermia . Nimesulide, a preferential inhibitor of cyclooxygenase-2 (0.05, 0.5 or 1 mg/kg, subcutaneously) completely abolished the hypothermia, resulting in an acceleration of the fever phase . However, the peak and plateau phases of fever were not changed by nimesulide treatment . Nimesulide (0.5 mg/kg) partially prevented serum TNF-alpha elevation . The non-selective cyclooxygenase inhibitor diclofenac inhibited hypothermia at all doses tested (0.03, 0.3 or 3 mg/kg, subcutaneously) although fever was completely abolished at the 3 mg/kg dose only . Diclofenac also partially abolished the elevation in serum TNF-alpha levels, but at the highest dose only (3 mg/kg) . These data suggest that nimesulide and diclofenac can preferentially inhibit LPS-induced hypothermia at doses that do not abolish fever in rats . Both these drugs also reduced elevated TNF-alpha levels, a fact which may, at least partly, explain the antihypothermic effect of nimesulide.

Clin Exp Allergy, 2002 Nov, 32(11), 1620 - 7
Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils; Iacovacci P et al.; BACKGROUND: Cypress pollinosis is an important cause of respiratory allergies . Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed . The native counterpart of this allergen has been purified and characterized by our group . It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract . OBJECTIVE: To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart . To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one . METHODS: Recombinant Cup a1 was expressed in E . coli . IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils . RESULTS: Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen . Moreover, only native molecule was capable of inducing histamine release by this group of sera . Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation . CONCLUSION: A large number of sera reactive with the major allergen recognize carbohydrate epitopes only . IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999 Jun 30, 13(2), 117 - 20
{Expression of non-structural region 3 gene of the Chinese HGY and analysis of the antigenicity of the recombinant proteins}; Chen G et al.; OBJECTIVE: This study is to analyze the antigenicity of the NS3 proteins of Chinese HGV and their potential use in the serological diagnosis . METHODS: All three gene fragments of NS3 region of Chinese HGV were cloned into the pRSET vectors to construct recombinant plasmids . In E . coli BL21, all three recombinant plasmids achieved a high expression level with induction of IPTG . The expressed products were analyzed with Western blot and ELISA . RESULTS: The recombinant protein PA, P3 and P4 have a molecular weight of 42,000, 30,000 and 24,000, respectively . They all could react with HGV positive sera in Western blot and ELISA . Among them, the protein that covers the N terminal of NS3 region of HGV had a stronger reaction with HGV positive sera than the other two proteinsdid . CONCLUSION: The N terminal in the NS3 region of Chinese HGV includes an dominant antigenic determinant, and its gene product has relatively strong antigenicity.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999 Jun 30, 13(2), 113 - 6
{Expression of recombinant HIV-1 envelope glycoprotein gp41}; Teng Z et al.; OBJECTIVE: To construct and express HIV-1 env gp41 gene for develoing a simple and rapid test for HIV-1 infection . METHODS: HIV-1 env gp41 gene of BH10 strain (nt6,977-7,497) was constructed into expressing vector pBV221 and expressed in E . Coli HB101 . The expressed proteins were purified on 15% SDS-PAGE, the specific protein gel was cut down, transferred onto nitrocellulose membrane and stained with ponceau for 10 minutes . The membrane was detected with positive and negative serum respectively . The membrane was blocked with blocking buffer and cut into 2 mm of each strip and fixed into the well of thin plastic plate . RESULTS: We obtained a strand plasmid expressing HIV-1 env gene and the protein . CONCLUSION: The results showed that: (1) HIV-1 env gp41 protein can be used to detect HIV-1 antibody in serum of individual; (2) The expressed protein is a nonfusion protein and has high specificity and sensitivity to HIV-1.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1999 Apr, 21(2), 81 - 7
{The characterization of a novel testis-specific nucleoporin gene}; Gao Y et al.; OBJECTIVE: To investigate the structure and function of testis-specific gene and spermatogenesis in human . METHODS: Screening cDNA expression library, 5' rapid amplification of cDNA ends, Northern blot and fluorescent in situ hybridization(FISH) were used . Gene expressing, purified of expressed protein by affinity chromatography and SDS-PAGE as well as phosphorylation of expressed protein in vitro by PKC and p34cdc2 were observed . RESULTS: A cDNA designated as BS-63 was isolated and found to consist of 2,209 bp with an open reading frame of 2,100 bp and assigned the accession number U64675 by GenBank . The deduced polypeptide consisted of 700 amino acid residues containing XFXFG or FG motifs that were characteristic of nuclear pore complex (NPC) protein and acted as potential binding sites for Ran . The N-terminal region had high homology with Ran BP2/Nup 358, a nucleoporin component, showing that BS-63 was a member of the NPC family . Northern blot analysis of mRNA prepared from various human tissues showed that BS-63 gene was transcribed in two forms: 6.0 and 8.5 kb . The 8.5 kb transcript was present in low amounts in several somatic tissues; whereas the 6.0 kb transcript was expressed only in testis . Analysis by FISH method mapped the BS-63 gene in 2q11.2-12 . A protein band with an estimated Mr of 80,000 was detected with E . coli BL21 (DE3) transfected with recombinant plasmid pET30a (+)-BS-63 . In vitro phosphorylation test indicated the BS-63 recombinant protein could be phosphorylated by PKC and p34cdc2 . CONCLUSIONS: The study was the first demonstration that the BS-63 gene encoding a nucleoporin-related protein with Ran binding sites was expressed in germ cells of human testis.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1999 Feb, 21(1), 62 - 8
{Gene cloning and expression of Mycobacterium leprae alpha 2 antigen}; Yin Y et al.; OBJECTIVES: The recombinant alpha 2 antigen of M . leprae was prepared using the molecular biologic tools and the recombinant DNA expression technology . METHODS: Screening of the M . leprae expression library was performed by the plaque hybridization technique . Nucleotide sequences were determined by dideoxy termination method . RESULTS: The gene coding for alpha 2 antigen of M . leprae was cloned and characterized, and the complete nucleotide sequence data has been assigned in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databank . The over expression system of alpha 2 antigen gene in E . coli was constructed, and the recombinant alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95% . More than 10 mg of recombinant alpha 2 antigen has been obtained from 200 ml of liquid culture . CONCLUSION: The recombinant alpha 2 antigen of M . leprae could be used as one of the specific antigens for the sero-diagnosis of leprosy.

Planta, 2003 Feb, 216(4), 692 - 8 Epub 2002 Oct 18.
Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells; Sobajima H et al.; In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production . Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level . Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme . The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1) . The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA . Thus the rice enzyme was termed OsOPR1 . The physiological roles of OsOPR1 are discussed . This is the first report of the cloning of an OPR gene from a monocot plant.

J Biotechnol, 2003 Mar 6, 101(2), 189 - 98
Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100; Lee MH et al.; We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100 . The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli . The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts . Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium . After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column . When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab . The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations . The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45% . Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E . coli cells .

J Biotechnol, 2003 Mar 6, 101(2), 101 - 17
Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method; Zhao J et al.; A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids . By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified . This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source . The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system . Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand . The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization .

J Am Chem Soc, 2003 Feb 12, 125(6), 1501 - 7
Designing protein dimerizers: the importance of ligand conformational equilibria; Carlson JC et al.; In an effort to elucidate the role of ligand conformation in induced protein dimerization, we synthesized a flexible methotrexate (MTX) dimer, demonstrated its ability to selectively dimerize Escherichia coli dihydrofolate reductase (DHFR), and evaluated the factors regulating its ability to induce cooperative dimerization . Despite known entropic barriers, bis-MTX proved to possess substantial conformational stability in aqueous solution (-3.8 kcal/mol >/= DeltaG(fold) >/= -4.9 kcal/mol), exerting a dominant influence on the thermodynamics of dimerization . To dimerize DHFR, bis-MTX must shift from a folded to an extended conformation . From this conclusion, the strength of favorable protein-protein interactions in bis-MTX-E . coli DHFR dimers (-3.1 kcal/mol >/= DeltaG(c) >/= -4.2 kcal/mol), and the selectivity of dimerization for E . coli DHFR relative to mouse DHFR (>10(7)) could be determined . The crystal structure of bis-MTX in complex with E . coli DHFR confirms the feasibility of a close-packed dimerization interface and suggests a possible solution conformation for the induced protein dimers . Consequently, the secondary structure of this minimal foldamer regulates its ability to dimerize dihydrofolate reductase in solution, providing insight into the complex energy landscape of induced dimerization.

J Am Chem Soc, 2003 Feb 12, 125(6), 1472 - 3
Enzymes do what is expected (chalcone isomerase versus chorismate mutase); Hur S et al.; Madicago sativa chalcone isomerase (CI) catalyzes the isomerization of chalcone to flavanone, whereas E . coli chorismate mutase (CM) catalyzes the pericyclic rearrangement of chorismate to prephenate . Covalent intermediates are not formed in either of the enzyme-catalyzed reactions, K(M) and k(cat) are virtually the same for both enzymes, and the rate constants (k(o)) for the noncatalyzed reactions in water are also the same . This kinetic identity of both the enzymatic and the nonenzymatic reactions is not shared by a similarity in driving forces . The efficiency (DeltaG(o)() - DeltaG(cat)()) for the CI mechanism involves transition-state stabilization through general-acid catalysis and freeing of three water molecules trapped in the E.S species . The contribution to lowering DeltaG(cat)() by an increase in near attack conformer (NAC) formation in E.S as compared to S in water is not so important . In the CM reaction, the standard free energy for NAC formation in water is 8.4 kcal/mol as compared to 0.6 kcal/mol in E.S . Because the value of (DeltaG(o)() - DeltaG(cat)()) is 9 kcal/mol, the greater percentage of NACs accounts for approximately 90% of the kinetic advantage of the CM reaction . There is no discernible transition-state stabilization in the CM reaction . These results are discussed . In anthropomorphic terms, each enzyme has had to do what it must to have a biologically relevant rate of reaction.

J Am Chem Soc, 2003 Feb 12, 125(6), 1450 - 1
Converting the sacrificial DNA repair protein N-ada into a catalytic methyl phosphotriester repair enzyme; He C et al.; Mutation of the active-site residue Cys38 of N-Ada converts it from a sacrificial DNA repair protein to an enzyme that uses methanethiol as an external sacrificial reagent to repair DNA methyl phosphotriesters catalytically.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2002, 20(3), 158 - 60
{Expression and characterization of 21.7 kDa membrane protein of Schistosoma japonicum}; Liu JF; OBJECTIVE: To express, purify and characterize the 21.7 kDa membrane protein of Chinese strain S . japonicum (SjC21.7) . METHODS: The gene of SjC21.7 was subcloned into the expression vector pGEX-4T-3 to form recombinant plasmid . The recombinant plasmids were transformed into E . coli BL21 and the GST-SjC21.7 fusion protein was expressed by IPTG induction . The recombinant SjC21.7 molecule was prepared by affinity chromatography and digested by thrombin . The Kunming strain mice were immunized with the recombinant SjC21.7 molecule to produce anti-SjC21.7 antibody . The purified SjC21.7 was recognized by the immunized mouse serum and the sera of rabbits infected by S . japonicum . RESULTS: The SjC21.7 gene was subcloned into expression vector pGEX-4T-3, then transformed into E . coli BL21 to express the GST-SjC21.7 fusion protein . The recombinant SjC21.7 molecule obtained from the fusion protein could stimulate the mice to produce a high titer of specific antibody and could be recognized by sera of both the immunized and infected rabbits . The sera of immunized mice could also recognize the 21.7 kDa protein molecule of the adult worm antigen (AWA) . CONCLUSION: The recombinant and purified SjC21.7 was prepared and showed similar immunological characteristics to the natural SjC21.7 molecule, providing a basis for further investigation of the immunological protection of the recombinant SjC21.7.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2002, 20(3), 152 - 4
{Construction and expression of single chain Fv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum}; Song XT et al.; OBJECTIVE: To construct single chain Fv (scFv) gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum . METHODS: The heavy and light chain variable region genes of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum were inserted into two corresponding sites of expression vector pTHA90, and a scFv gene was constructed with a short peptide (Gly4Ser)3 linker gene . The recombinants were determined by digesting with XhoI/SpeI, XbaI/EcoRI and XhoI/EcoRI, and then were introduced into E . coli Top10 . The antigen binding activity of expressed product was detected with ELISA . RESULTS: The recombinants were determined by digesting with endonucleases and expected bands were identified . The value of expressed scFv was 3 times higher than negative control by ELISA(OD492 = 1.06) . CONCLUSION: The scFv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was successfully cloned, and the expressed scFv fragment could interact specifically with antigen NP48.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2002, 20(3), 141 - 4
{Purification and immunogenicity identification of the recombinant protein related with specific IgE against Schistosoma japonicum}; Wang Y et al.; OBJECTIVE: To purify the specific IgE antibody-related recombinant protein of Schistosoma japonicum and to identify its immunogenicity . METHODS: The recombinant plasmid Sj43B/pGEX-6p-1 was expressed in E . coli BL 21 . The inclusion body of the fusion protein was washed by TNMFX buffer and separated by FPLC . After renaturation, the fusion protein was used to vaccinate the mice . The specific IgG and IgE antibodies were detected by dot-ELISA and Western blotting analysis, respectively . RESULTS: Most of the proteins mixed with the inclusion body of the recombinant protein could be eliminated by washing with TNMFX buffer . The purified recombinant fusion protein could be obtained by FPLC separation . The experiment on mice immunized with the fusion protein showed that the specific IgE antibody was generated against the target part of the fusion protein, but not the specific IgG antibody . CONCLUSION: The fusion protein expressed by the recombinant plasmid Sj43B/pGEX-6p-1 could induce specific IgE response of the immunized mice.

Yan Ke Xue Bao, 2001 Sep, 17(3), 154 - 7
Construction of the enhanced yellow fluorescent protein expression vector carrying IFN-gamma gene; Lan Y et al.; PURPOSE: To construct the enhanced yellow fluorescent protein (EYFP) vector carrying interferon-gamma gene (ifn-gamma) in order to provide an ideal reporter in the expression of ifn-gamma and location of protein in vitro and in vivo . METHOD: According to the nucleotide sequence of ifn-gamma gene, a pair of oligonucleotides was designed as primer whose two end contained nucleotide sequence of EcoR V and Not I restriction endonuclease respectively . The gene encoding for inf-gamma was amplified using PCR technqiue . After the PCR product was retrieved and purified, it was digested with EcoR V and Not I restriction endonuclease, and then cloned into the plasmid pIRES-EYFP . The recombinant plasmid pIRES-EYFPIFN-gamma was identified by restriction endonuclease enzyme analysis and DNA sequence analysis . RESULTS: The ifn-gamma was successfully amplified and verified by partial DNA sequence analysis . The recombinant plasmid was correctly screened . CONCLUSION: The EYFP expression vector carrying ifn-gamma gene was successfully established . This research work has formed a base for monitoring the ifn-gamma gene expression and protein position in living cells.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2002, 20(2), 86 - 9
{Sequence analysis of rDNA-LSU gene of Orientobilharzia turkestanicum from mainland of China}; Zhang GJ et al.; OBJECTIVE: To classify the taxonomic status of O . cheni in relation to O . turkestanicum var . tuberculata from the mainland of China by comparing their nucleotide sequences of nuclear ribosomal partial large subunit gene (LSU) . METHODS: The genomic DNA of adult worms were extracted by the GNT-K method . The target gene was amplified by PCR using specific primers . The PCR products were purified before ligation into the plasmid PCR-blunt (Invitrogen) . Recombinant plasmids were amplified in E . coli, extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long-read auto-sequencer . Sequences of O . turkestanicum was retrieved from GenBank and aligned with our data in BioEdit . RESULTS: The nucleotide sequences of LSU between O . turkestanicum var . tuberculata and O . cheni was 100% identical, and 99.99% identical between O . turkestanicum var . tuberculata and O . turkestanicum . CONCLUSION: This study demonstrated high similarity in LSU nucleotide sequences, and the results do not support O . cheni as an independent species . O . cheni may be a synonym of O . turkestanicum var . tuberculata, and O . turkestanicum var . tuberculata is probably also a synonym of O . turkestanicum.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2002, 20(1), 10 - 3
{Study on molecular phylogeny of Schistosoma sinensium based on mitochondrial genes}; Zhang GJ et al.; OBJECTIVE: To determine the phylogenetic position of Schistosoma sinensium in the genus Schistosoma using mitochondrial cytochrome C oxidase 1 (CO1) and NADH dehydrogenase 1(ND1) as molecular markers . METHODS: The genomic DNA of adult worms were extracted by the GNT-K method . The target regions were amplified by PCR using specific primers . The PCR products were purified before ligation into the plasmid Zero-Blunt . Recombinant plasmids were amplified in E . coli, extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long-read auto-sequencer . Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE . Gene trees were constructed in PHYLIP and MEGA using both maximum parsimony and neighbor-joining methods . For parsimony analysis, all characters were treated as unordered and with equal weights . At least 3,000 cycles of bootstrapping were carried out . For analysis in MEGA, all gap columns were deleted . The third position of codon was included . RESULTS: The nucleotide and amino acid sequences of CO1 and ND1 of S . sinensium were obtained . CONCLUSION: The phylogenetic trees from these molecular data suggested that S . sinensium belongs to the Asian schistosome group, and the results coincided with the previous rDNA (ITS2 & LSU) analysis results.

J Biomol NMR, 2003 Jan, 25(1), 11 - 23
Data requirements for reliable chemical shift assignments in deuterated proteins; Hitchens TK et al.; The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens et al., 2002) . In particular, the consequences of missing amide resonances on the reliability of assignments derived from C alpha and CO or from C alpha and C beta chemical shifts was investigated . Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments . More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra . However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment . It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using C alpha and CO chemical shifts and C alpha inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process.

Biol Neonate, 2003, 83(1), 42 - 8
Endogenous production of nitric oxide in endotoxemic piglets; Kaftan HA et al.; We sought to assess the relation between endotoxin-induced pulmonary hypertension and the production of nitric oxide (NO) in neonatal animals . Adult animals respond to endotoxin by increasing exhaled NO and plasma NO metabolites . The response of neonatal animals has not previously been reported . We administered 20 microg/kg of Escherichia coli lipopolysaccharide (LPS) to 12- to 18-day-old and to 5- to 7-week-old piglets . Pulmonary vascular resistance increased significantly in both age groups . Exhaled NO in the 12- to 18-day-old animals and in the 5- to 7-week-old piglets did not increase significantly . A similarly treated group of adult rats did show a significant increase in exhaled NO (2.6 +/- 1.0 to 109.5 +/- 54.3 ppb; p = 0.028) . Plasma NO metabolite measurements followed the same pattern of no increase in both porcine groups, and a large increase in the rat group . However, immunostaining of lungs from 12- to 18-day-old piglets did reveal an increase in inducible NO synthase . These results suggest that piglets demonstrate a limited ability to modulate LPS-induced pulmonary hypertension by elevations in exhaled NO . They also demonstrate the differential response to LPS between species .

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1700 - 5 Epub 2003 Feb 03.
Designed to be stable: crystal structure of a consensus ankyrin repeat protein; Kohl A et al.; Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla . This finding suggested the use of AR proteins as designed binding molecules . Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues . We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat . Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability . We determined the crystal structure of one of those library members to 2.0-A resolution, providing insight into the consensus AR fold . Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the beta-turn and loop regions are noteworthy . Furthermore, all residues found in the turn region of the Ramachandran plot are glycines . Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion . We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues . This scaffold represents an excellent basis for the design of novel binding molecules.

J Biol Chem, 2003 Apr 18, 278(16), 14203 - 10 Epub 2003 Feb 03.
Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline; Fujita T et al.; The first step of proline biosynthesis is catalyzed by gamma-glutamyl kinase (GK) . To better understand the feedback inhibition properties of GK, we randomly mutagenized a plasmid carrying tomato tomPRO1 cDNA, which encodes proline-sensitive GK . A pool of mutagenized plasmids was transformed into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-dl-proline . Thirty-two mutations that conferred 3,4-dehydro-dl-proline resistance were obtained . Thirteen different single amino acid substitutions were identified at nine different residues . The residues were distributed throughout the N-terminal two-thirds of the polypeptide, but 9 mutations affecting 6 residues were in a cluster of 16 residues . GK assays revealed that these amino acid substitutions caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline accumulation in vivo . GK belongs to a family of amino acid kinases, and a predicted three-dimensional model of this enzyme was constructed on the basis of the crystal structures of three related kinases . In the model, residues that were identified as important for allosteric control were located close to each other, suggesting that they may contribute to the structure of a proline binding site . The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.

J Exp Med, 2003 Feb 3, 197(3), 375 - 85
Second class minors: molecular identification of the autosomal H46 histocompatibility locus as a peptide presented by major histocompatibility complex class II molecules; Sahara H et al.; CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown . We developed a new method to identify CD4 T cell-stimulating antigens . LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes . The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4-induced IL4i1 gene . The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by A(b) major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells . Both allelic peptides bind A(b) and a single alanine to methionine substitution at p2 defines nonself . These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity.

Genome Res, 2003 Feb, 13(2), 216 - 23
Global RNA half-life analysis in Escherichia coli reveals positional patterns of transcript degradation; Selinger DW et al.; Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-type Escherichia coli MG1655 . RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons . The half-life of total mRNA was 6.8 min under the conditions tested . We also observed significant relationships between gene functional assignments and transcript stability . Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition . This supports a novel mechanism of transcription for the tdc operon, whose promoter lacks any recognizable sigma binding sites . Probe by probe analysis of all known and predicted operons showed that the 5' ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3' direction, supporting and further generalizing the current model of a net 5' to 3' directionality of degradation . Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive . We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants . The 16 ORF dcw gene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation . We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.

Free Radic Biol Med, 2003 Feb 15, 34(4), 429 - 33
Superoxide-dependence of the short chain sugars-induced mutagenesis; Benov L et al.; Short chain sugars such as glycolaldehyde are produced at the initial stages of nonenzymatic glycosylation . Because their carbonyl groups cannot be blocked by cyclization, such compounds tautomerize to enediols, which are prone to autoxidation . Superoxide radical serves as an initiator and a propagator of this autoxidation . The biological importance of the involvement of superoxide in sugar autoxidation in vivo was examined using superoxide dismutase (SOD)-deficient and SOD-replete strains of Escherichia coli . Glycolaldehyde, glyceraldehyde, and dihydroxyacetone greatly enhanced the mutation rates in SOD-deficient E . coli . The effect was oxygen-dependent and was suppressed by SOD or by a SOD mimetic . The mutagenic effect of glycolaldehyde coincided with intracellular accumulation of glyoxal, a product of glycolaldehyde autoxidation.

Bioorg Med Chem Lett, 2003 Feb 10, 13(3), 339 - 42
Stereochemical studies on phosphopantothenoylcysteine decarboxylase from Escherichia coli; Strauss E et al.; Phosphopantothenoylcysteine decarboxylase catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine (2) to form 4'-phosphopanthetheine (3), an intermediate in the biosynthesis of Coenzyme A . In this study we investigated the stereochemistry of this reaction . Our results show that the decarboxylation proceeds with retention of stereochemistry, and that the pro-R proton at C(beta) of the cysteine moiety of 2 is removed during a reversible oxidation of the thiol to a thioaldehyde intermediate.

Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 430 - 6
Characterization of the Escherichia coli YedU protein as a molecular chaperone; Malki A et al.; We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein . The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution . Like other chaperones, YedU interacts with unfolded and denatured proteins . It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions . YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin . In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity . We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.

Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 350 - 7
Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp . strain P2; Pinyakong O et al.; Five sets of large and small subunits of terminal oxygenase (ahdA1{a-e} and ahdA2{a-e}) and a single gene set encoding ferredoxin (ahdA3) and ferredoxin reductase (ahdA4) were found to be scattered through 15.8- and 14-kb DNA fragments of phenanthrene-degrading Sphingobium sp . strain P2 . RT-PCR analysis indicated the inducible and specific expression of ahdA3, ahdA4, and three sets of genes for terminal oxygenase (ahdA1{c-e} and ahdA2{c-e}) in this strain grown on phenanthrene . The biotransformation experiments with resting cells of Escherichia coli JM109 harboring recombinant ahd genes revealed that AhdA2cA1c, AhdA1dA2d, and AhdA1eA2e can all function as a salicylate 1-hydroxylase which converts salicylate, a metabolic intermediate of phenanthrene, to catechol in cooperation with the electron transport proteins AhdA3A4 . The first two oxygenases exhibited a broad range of substrate specificities such that they also catalyzed the hydroxylation of methyl- and chloro-substituted salicylates to produce their corresponding substituted catechols.

Toxicon, 2003 Mar 1, 41(3), 261 - 7
Protection against dermonecrotic and lethal activities of Loxosceles intermedia spider venom by immunization with a fused recombinant protein; Araujo SC et al.; We report the use of a recombinant Loxosceles intermedia spider protein in the form of a fusion protein as an antigen for immunization in rabbits and mice . The aim is to produce model protective antisera in these animals against dermonecrotic and lethal activities of the venom from the Brazilian spider responsible for 3000 cases, reported annually, of spider bites in South Brazil . A protein homologous to the dermonecrotic toxin was cloned from a cDNA expression library made with L . intermedia venom glands, expressed in E . coli cells as a fusion protein with beta-galactosidase and the recombinant protein (Li-rec protein) was purified by molecular filtration and affinity chromatography {Kalapothakis et al., Toxicon (2002) in press} . The Li-rec protein was characterized and used as an antigen to generate antibodies in rabbits and mice . These specifically raised antibodies recognized the native venom . In vitro neutralization assay of lethal effects indicated that 1 ml of rabbit serum raised against Li-rec protein was able to neutralize 25 LD(50) of the whole venom . In vivo protection experiments, the fusion proteins induced a long-term protection in rabbits against the dermonecrotic activity of the native venom . Immunized mice were challenged with various doses of the Loxosceles venom . Mice were fully protected against 2.5 LD(50) of venom . This result provides basic data for the use of such recombinant spider proteins as immunogens in the development of anti-venoms for clinical use or can be used as a vaccine providing efficient immune protection against L . intermedia venom.

Thromb Res, 2002 Sep 15, 107(6), 357 - 63
Effect of some new thioglycosides on endotoxin-induced disseminated intravascular coagulation in rabbits; Szabo G et al.; Disseminated intravascular coagulation (DIC) is a systemic thrombohemorrhagic disorder seen in association with many clinical situations, e.g . sepsis, malignancy, obstetrical complications and intravascular hemolysis . In our model, disseminated intravascular coagulation was induced in rabbits by two consecutive intravenous bolus injections of endotoxin from Escherichia coli, 80 and 40 microg/kg . The control group was treated with 0.9% saline . The activity of thioglycosides was compared to unfractionated heparin (UFH) and efegatran with and without administration of endotoxin . Drugs were administered in the following doses: heparin 50 and 100 IU/kg/h i.v . infusion; efegatran 0.25 and 0.5 mg/kg/h i.v . infusion; GYKI 39521 (RGH-1875) as well as GYKI 39541 (RGH-1962) 12.5 and 25 mg/kg per os . Thioglycosides did not modify coagulation parameters in this model {prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT)} as compared with endotoxin/vehicle group . The changes in TFPI level after administration of thioglycosides and heparin were similar in the mentioned model to those without endotoxin . Endotoxin-induced changes of leukocyte count were not affected by GYKI 39521 and GYKI 39541 treatment in our model . Diminution of fibrinogen level and platelet count was prevented by GYKI 39521 and GYKI 39541 . Fibrin degradation products and fibrinolysis were significantly decreased by GYKI 39521 and GYKI 39541 . The thioglycosides may have a lower risk of bleeding in the treatment of disseminated intravascular coagulation than heparin .

Biochemistry, 2003 Feb 11, 42(5), 1301 - 8
Basal and hydrogen peroxide stimulated sites of phosphorylation in heterogeneous nuclear ribonucleoprotein C1/C2; Stone JR et al.; Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration . The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood . Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells . Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein . Under resting conditions, the protein is phosphorylated at S247 and S286 . In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228 . Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain . These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1999, 17(5), 285 - 7
{Sequencing and analysis of a cDNA clone CO111 of Plasmodium falciparum}; Wang Y et al.; AIM: To sequence and analyse a cDNA clone CO111, reacting with immune sera obtained from rabbits immunized with Plasmodium falciparum and one McAb against P . falciparum . METHODS: cDNA clone was amplified by PCR . The PCR product was purified and polished with Klenow enzyme and ligated into the M13mp18 vector (digested by SamI) and then transformed into E . coli JM109 . The positive recombinant was screened out by PCR and sequenced by the PRISM Dye Primer Sequencing Kit(ABI) . The sequence was analyzed by the program from Geneva University and was compared by GenBank of EMBL . RESULTS: The nucleotide sequence of this cDNA clone contains an open-reading frame of 233 bp, which encodes a predicted polypeptide of 77 amino acid residues . The ratio between A + T and G + C is 3.16 . The polypeptide is highly hydrophilic and flexible . Comparison among cDNA of P . falciparum from GenBank of EMBL showed that no sequence identical to this cDNA was found . CONCLUSION: A novel cDNA clone reacted with the antibodies against P . falciparum was isolated.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1999, 17(2), 97 - 100
{Association expression of genes encoding gst of Schistosoma japonicum and enterotoxigenic Escherichia coli}; Zhang W et al.; AIM: To study the association expression of two genes encoding GST of Schistosoma japonicum and K99 of enterotoxigenic Escherichia coli . METHODS: PCR technique was used to gain the K99 gene . After digestion with BamH I and EcoR I, the gene was cloned into the plasmid vector pUC18 by using recombinant DNA techniques . The other target gene of GST in pSj5 plasmid was obtained by EcoR I digestion, and was then ligated into the recombinant plasmid pUC18-K99 . The expressed product was assayed by SDS-PAGE, Western blotting, reversal IHA, ELISA and transmission electron microscopy . RESULTS: Co-expression product of around 50 kDa was obtained . The protein was recognized by anti-GST and anti-K99 antibodies (ELISA and IHA), and acted as pilus on the surface of transformed DH5 alpha E . coli bacteria . CONCLUSION: Co-expression of the genes encoding GST and K99 was successfully achieved.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1999, 17(4), 215 - 7
{Fusion expression of porcine IFN-gamma and Cysticercus cellulosae antigen cC1 in Escherichia coli cells}; Guo Y et al.; AIM: To construct an expression vector, including a chimeric cDNA of porcine IFN-gamma and Cysticercus cellulosae antigen cC1 . METHODS: DNA fragments of porcine IFN-gamma and cC1 including linkers were generated by polymerase chain reaction (PCR) . The recombinant vector pJLA-PRcC1 was constructed by inserting a chimeric cDNA of porcine IFN-gamma and Cysticercus cellulosae antigen cC1, and transformed to E . coli XL-Blue . RESULTS: The recombinant vector was identified by restriction analysis . An inserted fragment about 1.5 kb could be found . After induction, a 52 kDa new protein band appeared in SDS-PAGE . CONCLUSION: The fusion expression of porcine IFN-gamma and cC1 antigen in Escherichia coli cells is successful.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1999, 17(4), 205 - 8
{Expression and identification of recombinant 22.6 kDa fusion protein of Schistosoma japonicum}; Su C et al.; AIM: To obtain a large amount of purified 22.6 kDa antigen of Schistosoma japonicum (Sj22.6) in large quantity . METHODS: The sequence of the gene fragment encoding Sj22.6 was reformed by PCR and subcloned into plasmid vector pGEX-1 lambda T that coded for the 26 kDa GST antigen of Schistosoma japonicum (Sj26 GST) . The recombinant plasmid was transformed into E . coli TG2 and then the positive recombinant clone was expressed by induction with IPTG . RESULTS: The recombinant Sj22.6/Sj26 GST fusion protein was expressed in 5.1% of total bacterial protein and was easy to be purified with glutathione sepharose 4B . Moreover, the purified recombinant Sj22.6 antigen could be cut off easily from the fusion protein with thrombin and had high immunogenicity . CONCLUSION: The purified recombinant Sj22.6 protein and Sj22.6/Sj26 GST fusion protein had the same immunological activity as the native Sj22.6 kDa protein.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1814 - 9 Epub 2003 Jan 31.
Kep1 interacts genetically with dredd/caspase-8, and kep1 mutants alter the balance of dredd isoforms; Di Fruscio M et al.; The Drosophila kep1 gene encodes an RNA binding protein related to the murine QUAKING apoptotic inducer . We have previously shown that kep1 can induce apoptosis when transfected into different cell lines . To better define the role of Kep1 in apoptosis, we generated kep1 null flies . These flies were viable, but females displayed reduced fertility, with approximately half of the eggs laid from kep1- homozygotes failing to hatch . In addition, loss of kep1 suppressed GMR-rpr-mediated apoptosis in the Drosophila eye, and kep1 mutant flies had increased susceptibility to Escherichia coli infection . We found that Kep1 bound dredd RNA in vitro, and that extracts prepared from kep1 mutant ovaries had markedly reduced proteolytic cleavage activity toward the caspase-8 target substrate IETD-7-amino-4-trifluoromethyl coumarin . We observed increased levels of the beta isoform of dredd mRNA in kep1 mutants as compared with wild-type . Taken together, our results suggest that Kep1 regulates apoptosis by influencing the processing of dredd RNA.

J Bacteriol, 2003 Feb, 185(4), 1299 - 315
Genetic and biochemical analysis of phosphatase activity of Escherichia coli NRII (NtrB) and its regulation by the PII signal transduction protein; Pioszak AA et al.; Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII . Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity . The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents . Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII . Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations . One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII . The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain . The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII . The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII . Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII . This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

J Bacteriol, 2003 Feb, 185(4), 1174 - 80
Interactions between phage-shock proteins in Escherichia coli; Adams H et al.; Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export . Expression of the operon requires the alternative sigma factor sigma54 and the transcriptional activator PspF . In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one . In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated . Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo . Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein . Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD . Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC . These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

J Bacteriol, 2003 Feb, 185(4), 1167 - 73
In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase; Ekberg M et al.; Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells . In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells . The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR . This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays . E . coli RNR is composed of two homodimeric proteins, R1 and R2 . The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site . The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues . Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity . In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

J Bacteriol, 2003 Feb, 185(4), 1161 - 6
YfiK from Escherichia coli promotes export of O-acetylserine and cysteine; Franke I et al.; yfiK was discovered as a gene augmenting cysteine production when it was overexpressed in an industrial Escherichia coli production strain . The gene product is an integral membrane protein with about six predicted transmembrane helices; it belongs to the RhtB family of export proteins . YfiK overproduction from a plasmid leads to drastic and parallel secretion of O-acetylserine and cysteine into the medium but only when the organism possesses a serine transacetylase that is feedback insensitive to cysteine . Externally provided O-acetylserine obviated this requirement for cysteine secretion both in the yfiK-carrying transformant and in the wild type . A DeltayfiK mutant did not show any phenotype, and it exported O-acetylserine and cysteine when transformed with a plasmid carrying ydeD, a previously characterized, alternate O-acetylserine/cysteine exporter . Since a ydeD-yfiK double mutant showed the same pattern, it appears that YfiK and YdeD act independently . The necessity for the cell to regulate the size of the internal pool of O-acetylserine via synthesis of exporter proteins could be connected to the fact that this compound (when supplied externally) inhibits growth . Overexpression of either ydeD or yfiK leads to alleviation of this inhibition paralled by increased resistance to azaserine, which is an analog of O-acetylserine.

J Biol Chem, 2003 Apr 11, 278(15), 13382 - 9 Epub 2003 Jan 31.
Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin-1 repeats and a unique GON domain; Llamazares M et al.; We have cloned a mouse brain cDNA encoding a new protein of the ADAMTS family (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats), which has been called ADAMTS-20 . This protein shows a domain organization similar to that described for other ADAMTSs including signal sequence, propeptide, metalloproteinase domain, disintegrin domain, central TS-1 motif, cysteine-rich region, and C-terminal TS module . However, this last module is more complex than that of other ADAMTSs, being composed of a total of 14 repeats . The structural complexity of ADAMTS-20 is further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module . This domain has been tentatively called GON domain and can also be recognized in some ADAMTSs such as gon-1 from Caenorhabditis elegans and human and mouse ADAMTS-9 . The presence of this domain is a hallmark of a novel subfamily of structurally and evolutionarily related ADAMTSs, called GON-ADAMTSs . Expression analysis demonstrated that ADAMTS-20 transcripts can be detected at low levels in several human and mouse tissues, especially in testis . This gene is also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas . Western blot analysis using polyclonal antibodies raised against recombinant ADAMTS-20 produced in Escherichia coli showed the presence of a 70-kDa band in mouse brain and testis extracts . This recombinant ADAMTS-20 hydrolyzed a synthetic peptide used for assaying matrix metalloproteinases . These data suggest that this novel enzyme may play a role in the tissue remodeling process occurring in both normal and pathological conditions.

J Biol Chem, 2003 Apr 11, 278(15), 13192 - 5 Epub 2003 Jan 31.
Rotational on-off switching of a hybrid membrane sensor kinase Tar-ArcB in Escherichia coli; Kwon O et al.; Signal transduction in biological systems typically involves receptor proteins that possess an extracytosolic sensory domain connected to a cytosolic catalytic domain . Relatively little is known about the mechanism by which the signal is transmitted from the sensory site to the catalytic site . At least in the case of Tar (methyl-accepting chemotaxis protein for sensing aspartate) of Escherichia coli, vertical piston-like displacements of one transmembrane segment relative to the other within the monomer induced by ligand binding has been shown to modulate the catalytic activity of the cytosolic domain . The ArcB sensor kinase of E . coli is a transmembrane protein without a significant periplasmic domain . Here, we explore how the signal is conveyed to the catalytic site by analyzing the property of various Tar-ArcB hybrids . Our results suggest that, in contrast to the piston-like displacement that operates in Tar, the catalytic activity of ArcB is set by altering the orientation of the cytosolic domain of one monomer relative to the other in the homodimer . Thus, ArcB represents a distinct family of membrane receptor proteins whose catalytic activity is determined by rotational movements of the cytosolic domain.

J Biol Chem, 2003 Apr 11, 278(15), 12864 - 72 Epub 2003 Jan 31.
Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP); Lin AP et al.; Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated octameric enzyme composed of two types of homologous subunits designated IDH1 and IDH2 . Based on sequence comparisons and structural models, both subunits are predicted to have adenine nucleotide binding sites . This was tested by alanine replacement of residues in putative sites in each subunit . Targets included adjacent aspartate/isoleucine residues implicated as important for determining cofactor specificity in related dehydrogenases and a residue in each IDH subunit in a position occupied by histidine in other cofactor binding sites . The primary kinetic effects of D286A/I287A and of H281A replacements in IDH2 were found to be a dramatic reduction in apparent affinity of the holoenzyme for NAD(+) and a concomitant reduction in V(max) . Ligand binding assays also showed that the H281A mutant enzyme fails to bind NAD(+) under conditions that are saturating for the wild-type enzyme . In contrast, the primary effect of corresponding D279A/D280A and of R274A replacements in IDH1 is a reduction in holoenzyme binding of AMP, with concomitant alterations in kinetic and isocitrate binding properties normally associated with activation by this allosteric effector . These results suggest that the nucleotide cofactor binding site is primarily contributed by the IDH2 subunit, whereas the homologous nucleotide binding site in IDH1 has evolved for regulatory binding of AMP . These results are consistent with previous studies demonstrating that the catalytic isocitrate binding sites are comprised of residues primarily contributed by IDH2, whereas sites for regulatory binding of isocitrate are contributed by analogous residues of IDH1 . In this study, we also demonstrate that a prerequisite for holoenzyme binding of NAD(+) is binding of isocitrate/Mg(2+) at the IDH2 catalytic site . This is comparable to the dependence of AMP binding upon binding of isocitrate at the IDH1 regulatory site.

Clin Exp Immunol, 2003 Feb, 131(2), 225 - 33
Enhanced chemokine response in experimental acute Escherichia coli pyelonephritis in IL-1beta-deficient mice; Hertting O et al.; The aim of the present study was to investigate the effects of IL-1beta and Escherichia coli on the expression and secretion of MIP-2, the mouse equivalent to human IL-8, MCP-1 and RANTES in the kidneys of mice with acute pyelonephritis . Female Bki NMRI, as well as IL-1beta deficient mice and their wild-type littermates, were transurethrally infected with either E . coli CFT 073 or injected with NaCl 0.9% (w/v) and thereafter obstructed for 6 h . The Bki NMRI mice were killed at 0, 24, 48 h and 6 days and the IL-1beta-deficient mice at 48 h . Chemokine mRNA and protein levels peaked at 24 h for the tested chemokines with the mRNA expression localized in the tubular epithelial cells and for MIP-2 also in neutrophils . Obstruction per se, also induced a chemokine expression similar to E . coli infection although at a lower level . Interestingly, MIP-2 levels were higher in the IL-1beta deficient mice as compared with the wild-type littermates . Likewise, the inflammatory changes were more frequent and, when present, more widespread in the IL-1beta-deficient mice than in the wild-type mice . Stimulation of a human renal tubular epithelial cell line (HREC), A498 and of primary human mesangial cells (HMC) with the same bacterial antigen depicted gene expression of the same chemokines . A rapid release of IL-8 and MCP-1 was observed from both cell types . RANTES response was delayed both in the HREC and the HMC . We conclude that acute E . coli pyelonephritis induces a MIP-2/IL-8, MCP-1 and RANTES expression and secretion localized primarily to the epithelial cells and that this production is confirmed after in vitro stimulation with the same bacterial antigen of human epithelial and mesangial cells . Blockade of induction of chemokine response may thus be an attractive target for possible therapeutic intervention.

J Egypt Soc Parasitol, 1999, 29(3), 859 - 72
Effect of dual infections of Escherichia coli and pure caecal Eimeria sp . in broiler chickens; Hegazy SH et al.; The interactive effect of Escherichia coli and pure caecal Eimeria spp . in broiler chickens was studied . Single and dual orally inoculations were assessed in six groups of 20 chicks each . Neither mortalities nor clinical signs were recorded on inoculated groups or control . There was a significant decrease in body weights in groups infected, either with E . coli or caecal Eimeria spp . or with both pathogens . The total oocyst output and caecal lesion score were high and severe in chicks infected with caecal Eimeria sp . alone or with E . coli prior to caecal Eimeria infection . Low oocyst output and moderate caecal lesion score were noticed in chicks inoculated with E . coli prior to or together with caecal Eimeria infection . The results suggested a synergistic action between E . coli and caecal Eimeria sp . in producing gross lesions . The electrophoretic distribution of serum proteins decreased in all chicks compared with control . The changes in serum protein fractions differed in inoculated groups and all protein components decreased quantitatively compared with controls . It was also shown that E . coli did not always invade the blood stream.

Wei Sheng Yan Jiu, 2002 Feb, 31(1), 49 - 52
{Expression of fused protein A-green fluorescent protein (PA-GFP)}; Zhao Z et al.; The green fluorescent protein (GFP) of jellyfish victoria is an unusual protein with strong visible fluorescence both in vivo and in vitro . In this study, about 750 bp fragment of gfp gene was amplified by PCR and was inserted into pRIT2T vector containing protein A gene . The recombinant plasmid was transferred into E . coli JM101 . GFP and protein A were fused and expressed in E . coli Top10 . The bright green fluorescence on the plates could be visible under UV at 365 nm . The bacteria cells were disrupted by sonic oscillation . The fused protein PA-GFP could be obtained from the supernatants.

Sheng Wu Gong Cheng Xue Bao, 2002 Sep, 18(5), 644 - 7
{The PTD domain of Tat protein enhance GFP protein delivering into myeloma cell SP2/0}; Li ZQ et al.; In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector . The TAT-GFP fusion protein was expressed in the E . coli ER2566 . Most of the fusion protein was presented in the inclusion body . The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea . The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0 . It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.

Sheng Wu Gong Cheng Xue Bao, 2002 Sep, 18(5), 556 - 60
{Screening of TNF-alpha antagonist peptides from a random peptide library displayed with Escherichia coli flagellar}; Li C et al.; Tumor necrosis factor(TNF-alpha) plays an improtant role in the process of anti-infection and anti-cancer . It can both protect and make damage to the host . In order to find new way of inhibiting its host-damaging activity, An E . coli flagella displayed random peptide library was constructed and TNF-alpha antagonist peptides were screened using the peptide library . After 5 rounds of panning and DNA sequencing, six peptide sequences were obtained . Two of them(TBP2, TBP3) have the same sequence frame of V--N-WG . After primary comparation of TNF-alpha binding ability, four peptides were synthesised and purified with RP-HPLC . The activity of inhibiting TNF-alpha was detected with L929 cell and MTT method . The data show that TBP2 and TBP3 can inhibit 90% of TNF-alpha activity when TNF-alpha gives about 30% cell toxicity on L929 . The two sequences have not been reported.

Science, 2003 Jan 31, 299(5607), 700 - 4
Architecture of succinate dehydrogenase and reactive oxygen species generation; Yankovskaya V et al.; The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone . It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide . This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS . Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.

Nucleic Acids Res, 2003 Feb 1, 31(3), 1108 - 17
Small worlds in RNA structures; Wuchty S; I consider conformational spaces of tRNA(phe) defined by sets of suboptimal structures from the perspective of small-world networks . Herein, the influence of modifications on typical small-world network properties and the shape of energy landscapes is discussed . Results indicate that natural modifications influence the degree of local clustering and mean path lengths far more than random or no modifications . High frequencies in the thermodynamic ensemble coincide with high numbers of neighboring structures that one conformation can adopt by one elementary move . Conformation spaces indicate the existence of modular substructures . It can be shown that modifications leave the nature of small-world topology untouched albeit natural modifications have a reasonable enhancing and streamlining effect on the degree of clustering and therefore on the substructures of the conformational space.

Nucleic Acids Res, 2003 Feb 1, 31(3), 1006 - 12
Stimulation of D-loop formation by polypurine/polypyrimidine sequences; Biet E et al.; Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair . The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy . One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ . We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein . We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides . This result was reproduced with various PPT sequences and oligonucleotides . The stimulatory effect was observed at loci as far as 4000 bp from the PPT . The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein . The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA . We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches . We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity . Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.

Nucleic Acids Res, 2003 Feb 1, 31(3), 899 - 910
DNA pairing is an important step in the process of targeted nucleotide exchange; Drury MD et al.; Modified single-stranded DNA oligonucleotides can direct the repair of genetic mutations in yeast, plant and mammalian cells . The mechanism by which these molecules exert their effect is being elucidated, but the first phase is likely to involve the homologous alignment of the single strand with its complementary sequence in the target gene . In this study, we establish the importance of such DNA pairing in facilitating the gene repair event . Oligonucleotide-directed repair occurs at a low frequency in an Escherichia coli strain (DH10B) lacking the RECA DNA pairing function . Repair activity can be rescued by using purified RecA protein to catalyze the assimilation of oligonucleotide vectors into a plasmid containing a mutant kanamycin resistance gene in vitro . Electroporation of the preformed complex into DH10B cells results in high levels of gene repair activity, evidenced by the appearance of kanamycin-resistant colonies . Gene repair is dependent on the formation of a double-displacement loop (double-D-loop), a recombination intermediate containing two single-stranded oligonucleotides hybridized to opposite strands of the plasmid at the site of the point mutation . The heightened level of stability of the double-D-loop enables it to serve as an active template for the DNA repair events . The data establish DNA pairing and the formation of the double-D-loop as important first steps in the process of gene repair.

Nucleic Acids Res, 2003 Feb 1, 31(3), 819 - 25
Site-specific protein modification to identify the MutL interface of MutH; Toedt GH et al.; We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach . For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for MutH . All MutH variants displayed wild type activity both in vivo and in vitro . These variants were then site-specifically modified at their cysteine residues with thiol-specific reagents and then tested for their ability to be stimulated in their DNA cleavage activity by the activator protein MutL . Thereby we were able to identify a defined region in the MutH protein that is important for interaction with MutL, and most likely represents the MutL binding site of MutH.

FEBS Lett, 2003 Jan 30, 535(1-3), 200 - 4
Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis; Jeong JJ et al.; The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli . The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized . The inhibition constants for four competitive inhibitors were determined . The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer . The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA . Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat) . The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.

FEBS Lett, 2003 Jan 30, 535(1-3), 34 - 8
The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine; Griffin SD et al.; Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important . HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses {Harada, T . et al., (2000) J . Virol . 74, 9498-9506} . We show that p7 can be cross-linked in vivo as hexamers . Escherichia coli expressed p7 fusion proteins also form hexamers in vitro . These and HIS-tagged p7 function as calcium ion channels in black lipid membranes . This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza {Hay, A.J . et al . (1985) EMBO J . 4, 3021-3024; Duff, K.C . and Ashley, R.H . (1992) Virology 190, 485-489} and has recently been shown to be active in combination with current HCV therapies.

J Mol Biol, 2003 Feb 14, 326(2), 585 - 92
Interaction of trigger factor with the ribosome; Maier R et al.; The trigger factor of Escherichia coli is a prolyl isomerase and a chaperone . It interacts with the ribosome and affects the folding of newly formed protein chains . Therefore, the dynamics of the interactions of trigger factor with the ribosome and with unfolded protein chains should be tailored for this function . Previously, we had found that binding of unfolded proteins to trigger factor is fast and that the lifetime of the complex between these two components is only about 100 ms . Here, we have labeled the trigger factor in its amino-terminal, ribosome-binding domain with a fluorescent dye and investigated how it interacts with the ribosome . We found that this association, as well as the dissociation of the complex, are rather slow processes . The average lifetime of the complex is about 30 seconds (at 20 degrees C) . The strong differences in the dynamics of the interactions of trigger factor with the ribosome and with protein substrates might ensure that, on the one hand, trigger factor remains bound to the ribosome while a protein chain is being synthesized, and, on the other hand, allows it to scan the newly formed protein for prolyl bonds that need catalysis of isomerization.

J Mol Biol, 2003 Feb 14, 326(2), 569 - 83
The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase; Svensson AK et al.; The propensity for peptide bonds to adopt the trans configuration in native and unfolded proteins, and the relatively slow rates of cis-trans isomerization reactions, imply that the formation of cis peptide bonds in native conformations are likely to limit folding reactions . The role of the conserved cis Gly95-Gly96 peptide bond in dihydrofolate reductase (DHFR) from Escherichia coli was examined by replacing Gly95 with alanine . The introduction of a beta carbon at position 95 is expected to increase the propensity for the trans isomer and perturb the isomerization reaction required to reach the native conformation . Although G95A DHFR is 1.30 kcal mol(-1) less stable than the wild-type protein, it adopts a well-folded structure that can be chemically denatured in a cooperative fashion . The mutant protein also retains the complex refolding kinetic pattern attributed to a parallel-channel mechanism in wild-type DHFR . The spectroscopic response upon refolding monitored by Trp fluorescence and the absence of a Trp/Trp exciton coupling apparent in the far-UV CD spectrum of the wild-type protein, however, indicated that the tertiary structure of the folded state for G95A DHFR is altered . The addition of methotrexate (MTX), a tight-binding inhibitor, to folded G95A DHFR restored the exciton coupling and the fluorescence properties through five slow kinetic events whose relaxation times are independent of the ligand and the denaturant concentrations . The results were interpreted to mean that MTX-binding drives the formation of the cis isomer of the peptide bond between Ala95 and Gly96 in five compact and stable but not wild-type-like conformations that contain the trans isomer . Folding studies in the presence of MTX for both wild-type and G95A DHFR support the notion that the cis peptide bond between Gly95 and Gly96 in the wild-type protein forms during four parallel rate-limiting steps, which are primarily controlled by folding reactions, and lead directly to a set of native, or native-like, conformers . The isomerization of the cis peptide bond is not a source of the parallel channels that characterize the complex folding mechanism for DHFR.

J Mol Biol, 2003 Feb 14, 326(2), 543 - 51
Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli; Laursen BS et al.; The infB gene encodes translation initiation factor IF2 . We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490 . These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24 . The mutations causing the suppression phenotype are located within infB . The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490 . Both positions are located in the GTP-binding G-domain of IF2 . A model of the G-domain of E.coli IF2 is presented in . Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated . At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain . Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins . The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes . The temperature-induced conformational change of the wt IF2 is a two-state process . In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2 . The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.

J Mol Biol, 2003 Feb 14, 326(2), 503 - 16
Biosynthesis of pteridines . Reaction mechanism of GTP cyclohydrolase I; Rebelo J et al.; GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate . The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit . Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc . These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate . The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity . The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group . Several basic amino acid residues interact with the triphosphate moiety of the substrate . The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding . Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113 . Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112 . In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP . Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc . The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis . The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.

J Mol Biol, 2003 Feb 14, 326(2), 485 - 92
Mega-Dalton biomolecular motion captured from electron microscopy reconstructions; Chacon P et al.; The vibrational analysis of elastic models suggests that the essential motions of large biomolecular assemblies can be captured efficiently at an intermediate scale without requiring knowledge of the atomic structure . While prior work has established a theoretical foundation for this analysis, we demonstrate here on experimental electron microscopy maps that vibrational modes indeed describe functionally relevant movements of macromolecular machines . The clamp closure in bacterial RNA polymerase, the ratcheting of 30S and 50S subunits of the ribosome, and the dynamic flexibility of chaperonin CCT are extracted directly from single electron microscopy structures at 15-27 A resolution . The striking agreement of the presented results with experimentally observed motions suggests that the motion of the large scale machinery in the cell is surprisingly independent of detailed atomic interactions and can be quite reasonably described as a motion of elastic bodies.

J Mol Biol, 2003 Feb 14, 326(2), 397 - 412
Sequence of the loxP site determines the order of strand exchange by the Cre recombinase; Lee L et al.; Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate . The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence . We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein . We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre) . We found that the initial strand exchange occurs preferentially next to the scissile G residue . Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue . Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.

J Mol Biol, 2003 Feb 14, 326(2), 381 - 96
Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization; Tachedjian G et al.; The tryptophan repeat motif of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is comprised of a cluster of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs . To determine the contributions of each of these residues for HIV-1 RT dimerization, we introduced changes into cloned DNA and tested the mutant subunits for their capacity to mediate heterodimerization in the yeast two-hybrid system . Changes of residue 401 to either leucine or alanine (but not phenylalanine) and residue 414 to leucine resulted in major reductions in beta-galactosidase activity produced from the reporter gene as compared to yeast expressing wild-type p66 bait and p51 prey fusions . Subunit selective mutagenesis revealed that the effect of these mutations was mediated mainly through the p66 subunit . Introduction of tryptophan mutants into the bacterial expression vector pRT6H/NB-PROT showed that RTs containing W401A or W401L substitutions (but not W401F) and W414L were defective for dimerization in vitro . Consistent with their dimerization defect, the W401A, W401L and W414L mutants were devoid of RT activity . Using the yeast two-hybrid system, we identified several second-site suppressors in p66 that restored interaction of the p66W401A bait to the p51W401A prey . The suppressors (T409I, D110G, V372A and I393M) also restored heterodimerization of bacterially expressed W401A subunits . When introduced into the W401A mutant, T409I was able to restore RT activity to 50% of the wild-type level . Examination of the RT structures revealed that K331 in p51 makes multiple hydrogen bond contacts with residues in the p66 loop spanned by W401 and W414 . Consistent with this observation, the K331A RT mutant was dimerization-defective . We conclude that mutations at codons 401 and 414 in p66 impair dimerization by altering the proper positioning of structural elements in between these residues that make important contacts with p51.

Mol Genet Metab, 2003 Jan, 78(1), 44 - 50
The AGT gene in Africa: a distinctive minor allele haplotype, a polymorphism (V326I), and a novel PH1 mutation (A112D) in Black Africans; Coulter-Mackie MB et al.; We describe a novel missense mutation (A112D) and polymorphism (V326I) in the human AGT gene in two black African patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44) . V326I was found in DNA from normal control Blacks with an allele frequency of 3% . Expression studies confirmed that A112D reduced AGT enzyme activity by 95% while V326I had no effect . Both A112D and V326I were homozygous in both patients and lie on a variant of the minor allele of the AGT gene . This variant haplotype, Mi(A), includes an intron 1 duplication and intron 4 VNTR (38 repeat) but lacks the P11L and I340M normally associated with the minor allele in Caucasians . Among the South African Blacks tested, the Mi(A) haplotype had an allele frequency of 12% compared to 3 % for the Caucasian-type minor allele haplotype.

Gene, 2003 Jan 16, 303, 187 - 96
Sucrose-phosphatase gene families in plants; Lunn JE; Sucrose-phosphatase (SPP; EC 3.1.3.24) catalyzes the final step in the pathway of sucrose biosynthesis and higher plants contain multiple isoforms of the enzyme encoded by different genes . The genome of the dicotyledonous plant Arabidopsis thaliana (thale cress) contains four SPP-like genes on chromosomes 1 (AtSPP1), 2 (AtSPP2) and 3 (AtSPP3a and AtSPP3b), all of which are expressed . The genome of the monocotyledonous plant rice (Oryza sativa) also contains four SPP-like genes, which have very similar exon-intron structures to those from A . thaliana . Two cDNA clones that encode catalytically active SPP enzymes have been isolated from maize (Zea mays), showing that this species contains at least two functional SPP genes . Multiple SPP-like cDNA clones have also been identified from wheat (Triticum aestivum), barley (Hordeum vulgare) and tomato (Lycopersicon esculentum) . The genomes of two cyanobacteria, Synechocystis sp . PCC 6803 and Anabaena sp . PCC 7120, contain single spp genes . The cyanobacterial SPPs and the N-terminal region of the higher plant enzyme share significant similarity with members of the haloacid dehalogenase (HAD) superfamily of hydrolases/phosphatases . In addition to the HAD phosphatase domain, SPP from higher plants also contains a shorter, C-terminal domain of unknown function . An SPP-like sequence from the bryophyte (moss) Physcomitrella patens also contains this C-terminal domain, indicating that its acquisition was an early event in the evolution of higher plants.

Gene, 2003 Jan 16, 303, 111 - 9
Characterization of a novel ubiquitin-fusion gene Uba256 from Spodoptera litura nucleopolyhedrovirus; Li Z et al.; The complete nucleotide sequence of Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene, encoding ubiquitin fused to GP37 protein of 256 amino acids was determined . The first 76 amino acids of the SpltMNPV ubiquitin showed 78-88, 77 and 81-84% amino acid sequence identity to baculovirus, Melanoplus sanguinipes entomopoxvirus and eukaryotes ubiquitins, respectively . The deduced amino acid sequence of SpltMNPV GP37 protein was similar to other baculovirus GP37 proteins and to entomopoxvirus fusolin proteins . The GP37 protein also showed a distant similarity to Pseudaletia separata entomopoxvirus enhancing factor, bacterial chitinase B and chitin-binding protein 1, but the significance of this is unclear . The mRNA start site of Uba256 fusion gene was mapped within a consensus baculovirus late promoter sequence (ATAAG), commonly found for baculovirus late genes . Uba256 transcripts were present from 48 h p.i . and remained detectable until 72 h p.i . Western blot analysis of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed to free ubiquitin and GP37 protein . Whereas expression Uba256 gene in Escherichia coli did not result in processing of the fusion protein . Tunicamycin treatment of SpltMNPV-infected cells confirmed that SpltMNPV GP37 protein is N-glycosylated . These findings provide additional information on the evolution of ubi genes and insight into genomic variation in baculoviruses.

Gene, 2003 Jan 16, 303, 89 - 97
An Escherichia coli strain, BJ5183, that shows highly efficient conservative (two-progeny) DNA double-strand break repair of restriction breaks; Takahashi N et al.; We examined the mode of recombination in an Escherichia coli strain, BJ5183, which has been frequently used in recovery and cloning of eukaryotic DNA . One of the important criteria in characterizing a homologous recombination mechanism is whether it produces two recombinant DNA molecules or only one recombinant DNA molecule out of two parental DNA molecules . Our previous work transferring plasmid molecules with a restriction break into Escherichia coli cells distinguished two modes in recombination stimulated by a double-strand break . In a recBC sbcA mutant strain, where recET genes on the Rac prophage are responsible for recombination (RecE pathway), recombination is often conservative, in the sense that it generates two recombinants out of two parental DNAs . In a recBC sbcBC mutant strain, in which recA and recF genes are responsible (RecF pathway), recombination is non-conservative, in the sense that it generates only one recombinant out of two parental DNAs . Unexpectedly, BJ5183, described as recBC sbcBC, showed very efficient conservative (two-progeny) double-strand break repair . Moreover, this recombination was not eliminated by disruption of its recA gene, which is essential to the RecF pathway . Our polymerase chain reaction analysis detected a recET gene homologue in this strain . This region was easily replaced by a RECT::Tn10 through general transduction and the resulting recT-negative derivative was defective in the conservative double-strand break repair . These results led us to conclude that, in strain BJ5183, the action of recET homologue is responsible for the conservative double-strand break repair as in the RecE pathway . BJ5183 carries a mutation in the endA gene, which codes for Endonuclease I . An endA mutation conferred a higher double-strand break-repair activity to a recBC sbcA mutant strain.

Int J Biol Macromol, 2002 Dec 20, 31(1-3), 19 - 27
Optical characterization of armadillo acyl-CoA binding protein; Cavagnari BM et al.; Acyl-CoA binding protein (ACBP) and fatty acid binding protein (FABP) are intracellular transporters of activated and free fatty acids, respectively . Unlike other tissues with active lipid metabolism, armadillo Harderian gland contains much more ACBP than FABP . To characterize armadillo ACBP structure and binding properties, we produced it in Escherichia coli and carried out detailed fluorescence and circular dichroism spectroscopy studies . The K(D) for palmitoyl-CoA, measured directly by fluorescence and rotatory power, was 34+/-12 and 75+/-39 nM, respectively . The structure of armadillo ACBP appears to be very similar to that of bovine and rat liver ACBPs.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 186 - 92
{Analysis the influence of palindrome structure to gene expression by constructing combination system}; Zhang R et al.; Palindrome sequence is very common in DNA, it usually acts as cis-elements of many regulative factors . In this paper, we design two palindrome sequences which is different in length, construct Cre-loxp combination system, then co-transform two kinds of plasmids, one contains cre gene, the other contains loxp site to E . coli DH5a strain . We judge the expression degree of cre gene in E . coli by examining the changes of plasmid maps of co-transformed strains, then conclude that 20 bp or shorter than 20 bp palindrome sequence uninhibit the expression of downstream genes, however, 34 bp or longer than 34 bp palindrome sequence can inhibit that.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 181 - 5
{Construction of DNA transfer system of Streptomyces tenebrarius}; Xia H et al.; To establish a gene transfer ssystem in Streptomyces tenebrarius, several methods including PEG-mediated transformation of protoplasts, conjugal transfer were investigated . Many attempts were made to introduce plasmid pIJ702 into Sreptomyces tenebrarius . It was found that plasmid pIJ702 isolated from S . lividans TK24 failed to transform the protoplasts of Sreptomyces tenebrarius . No transformant was achieved even if the protoplast was inactivated by heat treatment or dsDNA was converted ssDNA before transformation . All the results suggested that Sreptomyces tenebrarius exists a strong restriction and modification system for the transformation of foreign DNA . A recombinant E . coli ET12567 (pUZ8002, pHZ132) was obtained by transforming E . coli ET12567(pUZ8002) with oriT-containing E . coli-Sreptomyces shuttle plasmid pHZ132 . In mating experiments, E . coli ET12567 (pUZ8002, pHZ132) was the donor, and the recipient was Sreptomyces tenebrarius 9904 spores after pregerminating by heat shock . Matings between dornor and recipient were conducted . Plasmid pHZ132 was introduced into Sreptomyces tenebrarius 9904 by conjugation from E . coli ET12567 . The transfer system of Sreptomyces tenebrariu was established by conjugation . S . tenebrarius 9904 protoplasts were transformed by plasmid DNA modified by the host itself, and the transformation frequency was about 10(3)/microgram DNA (pHZ132).

What Is Nitrification?, What Is Growth Medium?, What Is Staphylococcus Aureus?, What Is Listeria Monocytogenes?, What Is Botulism?, r, Microbiology, s, Bacteria, a, Microorganism, i, Bacterium, r, Microbes, c, Fermentations, a, Halophilic bacterium, r, Escherichia coli, i, Bacillus anthracis, n, Bacillus, c, S. cerevisiae, n, Microorganisms, e, Thermophile, c, Microorganism, c, Enterobacters, c, Halophilic bacterium, o, Escherichia coli, a, Rhizobacterium, r, Pseudomonas aeruginosa, n, Staphylococcus, e, Escherichia coli, i, Serratia, i, E coli O157, c, Neisseria, e, Sepsis, e, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005