Escherichia coli

J Mol Biol, 2004 Jul 23, 340(5), 1143 - 52
Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy; Kedrov A et al.; Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches . Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit . Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end . It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not . Additionally, some interactions appeared to occur between the secondary structural elements . After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane . After five seconds, the refolded polypeptide established all secondary structure elements of the native protein . One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular . Additionally, individual helices required characteristic periods of time to fold . Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.

J Mol Biol, 2004 Jul 23, 340(5), 1025 - 37
Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin; Sparla F et al.; Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB) . AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH) . Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP . This suggested a possible involvement of these residues in the regulatory mechanism . Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties . Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188 . NADH-dependent activity was unaffected . The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme . A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4 . We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH . A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.

J Mol Biol, 2004 Jul 23, 340(5), 965 - 79
Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E; Callaghan AJ et al.; The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli . The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA . The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome . However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity . The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins . The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components . The implications of these and other observations for the organization of the RNA degradosome are discussed.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Aug 25, 808(1), 105 - 9
Purification of human galectin-1 produced in high-cell density cultures of recombinant Escherichia coli: a comparison with classic shake flask cultivation; Lutomski D et al.; The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production . Gal1 is a beta-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis . These effects have attracted the attention of researchers in cell biology, biochemistry and immunology . However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments . To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Aug 25, 808(1), 91 - 7
Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli; Cabanne C et al.; Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli . Two pH of buffer were tested . Capture was done in an expanded mode whereas elution was done in a packed mode . The same conditions were chosen for evaluation of the three matrices . We observed a loss of EGFP (8-15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application . The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris . The two other matrices gave a good purification of the EGFP (7-15-fold) but the Q Hyper Z matrix appeared to give the best results . It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.

Biochemistry (Mosc), 2004 Jun, 69(6), 697 - 701
New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis; Volkov DA et al.; This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis . The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons . The corresponding cDNA fragment has been cloned and expressed in E . coli . The protein accumulated in inclusion bodies . The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose . Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain . It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases . We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range . The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

Biochemistry (Mosc), 2004 Jun, 69(6), 642 - 50
Cloning and expression of catalytic subunit of MLIII, the ribosome-inactivating protein from Viscum album; Tonevitsky AG et al.; We have cloned the gene encoding a precursor of mistletoe (Viscum album) toxin MLIII . Analyses of nucleotide and deduced amino acid sequences of this gene revealed significant differences between MLI and MLIII preprotoxin genes . Immunochemical properties of recombinant A-subunit expressed in Escherichia coli and renatured were investigated using a panel of monoclonal antibodies raised against three mistletoe toxins (MLI, MLII, and MLIII) . Ribosome-inactivating activity of recombinant MLIII A-subunit was detected in cell-free lysate of rabbit reticulocytes.

Biochemistry, 2004 Jul 13, 43(27), 8858 - 68
ER-60 domains responsible for interaction with calnexin and calreticulin; Urade R et al.; ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT) . In this study, we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule . ER-60 was shown to be composed of at least four domains, named a, b, b', and a', by limited proteolysis . Recombinant fragments of ER-60, a, b', and a'c, were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient . These fragments each gave the circular dichroism (CD) spectrum of the folded protein . On the other hand, fragment b, which did not undergo the cooperative unfolding transition along a urea gradient gel, did not show any sign of the folded structure on the CD measurement . However, subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb' . Both a and a'c, which have a catalytic center CGHC motif, showed activity almost equivalent to half of that of wild-type ER-60 . Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity, suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60 . The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.

Biochemistry, 2004 Jul 13, 43(27), 8766 - 77
Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein; Stewart RC et al.; In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA . Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)) . Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest . To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins . This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan {6-bromoacetyl-2-(dimethylamino)naphthalene} attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis . Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and an approximately 30% increase in signal intensity at 490 nm . The fluorescence of Badan-labeled CheY(M17C) also served as a sensitive reporter of CheY-CheA binding interactions, exhibiting an approximately 50% increase in emission intensity in the presence of saturating levels of CheA . Compared to wild-type CheY, Badan-labeled CheY exhibited reduced ability to autodephosphorylate and could not interact productively with the phosphatase CheZ . However, with respect to autophosphorylation and interactions with CheA, Badan-CheY performed identically to wild-type CheY, allowing us to explore CheA-CheY phosphotransfer kinetics and binding kinetics without interference from the fluorescence/absorbance properties of CheA and ATP . These results provide insights into CheY interactions with CheA, CheZ, and other components of the chemotaxis signaling pathway.

Biotechnol Bioeng, 2004 Jul 20, 87(2), 170 - 7
Detection of alkanes, alcohols, and aldehydes using bioluminescence; Minak-Bernero V et al.; We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light . Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase . In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase . We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E . coli host cell line . Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader . We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo . This method is amenable to the high-throughput screening needs required for the identification of novel catalysts .

Biotechnol Bioeng, 2004 Jul 20, 87(2), 129 - 37
Identification and characterization of Cu(2)O- and ZnO-binding polypeptides by Escherichia coli cell surface display: toward an understanding of metal oxide binding; Thai CK et al.; We have used the FliTrx cell surface display system to identify disulfide-constrained dodecapeptides binding to the semiconducting metal oxides Cu(2)O and ZnO . Sequence analysis of the inserts revealed that the two populations exhibit similar, yet subtly different patterns of amino acid usage . Both sets of binders were enriched in arginine, tryptophan, and glycine with a higher degree of positional preference in the case of Cu(2)O binders . Tyrosine, proline, and serine were underrepresented in both populations . Peptides binding electrodeposited Cu(2)O or ZnO with high avidity could be subdivided into two classes based on pI and hydrophilicity . In the hydrophilic and positively charged Class I binders, the Arg-X-X-Arg tetrapeptide appears to be implicated in metal oxide binding, whereas Arg-Arg and Arg-Lys pairs allow for discrimination between Cu(2)O and ZnO . Molecular dynamics simulations of the disulfide-constrained peptides suggest that the aforementioned motifs are important to properly orient two basic residues that are likely to contact the metal oxides . The implications of our results in understanding the rules governing the interaction between peptides and inorganic compounds and in their use for the design of hybrid nanoarchitectures are discussed .

Gastroenterology, 2004 Jul, 127(1), 80 - 93
Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer; Martin HM et al.; BACKGROUND & AIMS: Altered mucosal glycosylation in inflammatory bowel disease and colon cancer could affect mucosal bacterial adherence . This study aimed to quantify and characterize mucosa-associated and intramucosal bacteria, particularly Escherichia coli, in these conditions . METHODS: Mucosa-associated bacteria were isolated, after dithiothreitol mucolysis, from biopsy samples obtained at colonoscopy (Crohn's disease, n = 14 patients; ulcerative colitis, n = 21; noninflamed controls, n = 24) and at surgical resection (colon cancer, n = 21) . Intramucosal bacteria were grown after gentamicin treatment followed by hypotonic lysis . RESULTS: Mucosa-associated and intramucosal bacteria were cultured more commonly in Crohn's disease (79%, P = 0.03; and 71%, P < 0.01, respectively), but not ulcerative colitis (38% and 48%), than in noninflamed controls (42% and 29%) and were commonly cultured from colon cancers (71% and 57%) . Mucosa-associated E . coli, which accounted for 53% of isolates, were more common in Crohn's disease (6/14; 43%) than in noninflamed controls (4/24, 17%), as also were intramucosal E . coli: Crohn's disease, 29%; controls, 9% . E . coli expressed hemagglutinins in 39% of Crohn's cases and 38% of cancers but only 4% of controls, and this correlated (P = 0.01) with adherence to the I407 and HT29 cell lines . Invasion was cell-line dependent . E . coli, including nonadherent isolates, induced interleukin-8 release from the cell lines . E . coli adhesins showed no blood group specificity, excepting 1 cancer isolate (HM44) with specificity for the Thomsen-Friedenreich antigen, but they could be blocked by soluble plantain fiber . CONCLUSIONS: These studies support a central role for mucosally adherent bacteria in the pathogenesis of Crohn's disease and colon cancer . Soluble plant fibers that inhibit their adherence have therapeutic potential.

Extremophiles, 2004 Dec, 8(6), 455 - 462 Epub 2004 Jul 2.
Extrinsic factors potassium chloride and glycerol induce thermostability in recombinant anthranilate synthase from Archaeoglobus fulgidus; Byrnes WM et al.; Thermostable anthranilate synthase from the marine sulfate-reducing hyperthermophile Archaeoglobus fulgidus has been expressed in Escherichia coli, purified, and characterized . The functional enzyme is an alpha(2)beta(2) heterotetrameric complex of molecular mass 150+/-15 kDa . It is composed of two TrpE (50 kDa) and two TrpG (18 kDa) subunits . The extrinsic factors glycerol (25%) and potassium chloride (2 M) stabilized the recombinant enzyme against thermal inactivation . In the presence of these extrinsic factors, the enzyme was highly thermostable, exhibiting a half-life of thermal inactivation of about 1 h at 85 degrees C . The kinetic constants for the enzyme under these conditions were: K(m) (chorismate) 84 muM, K(m) (glutamine) 7.0 mM, k(cat) 0.25 s(-1), and pH optimum 8.0 . The enzyme was competitively, though non-cooperatively, inhibited by tryptophan.

Nat Struct Mol Biol, 2004 Aug, 11(8), 697 - 705 Epub 2004 Jul 04.
Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment; Ramachandran R et al.; Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores . Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques . Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden beta-strand that forms the monomer-monomer interface and is required for oligomer assembly . The beta-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane beta-hairpins of adjacent monomers in the proper register for insertion . Collectively, these data reveal a novel membrane binding-dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.

J Nucl Med, 2004 Jul, 45(7), 1217 - 23
Relationship between neutrophil-binding affinity and suitability for infection imaging: comparison of (99m)Tc-labeled NAP-2 (CXCL-7) and 3 C-terminally truncated isoforms; Rennen HJ et al.; The CXC chemokines are a family of closely related chemoattractant cytokines that bind to, attract, and activate neutrophils to variable degrees . In this study, the relationship between neutrophil-binding affinity and suitability for infection imaging was investigated in a selected group of CXC chemokines . Neutrophil-activating peptide-2 (NAP-2, 70 residues; also called CXCL7) binds with high affinity to the CXCR2 receptor on neutrophils . Recently, C-terminally truncated NAP-2-variants have been described that have enhanced neutrophil-binding affinity and neutrophil-stimulating capacity . Here, NAP-2 and its C-terminal shortened variants NAP-2(1-68), NAP-2(1-66), and NAP-2(1-63) were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator and their potential for imaging of infection was investigated in a rabbit model of infection . The CXC chemokine interleukin-8 (IL-8) was used for comparison . In addition, a series of (99m)Tc-labeled CXC chemokines were screened for their potential to image infection, including CTAP-III, GCP-2, ENA-78, PF-4, and IP-10 . METHODS: The receptor-binding affinity of HYNIC-conjugated NAP-2 and its analogs was compared in competitive binding assays on Jurkat cells transfected with the CXCR2 receptor gene . Biodistribution of labeled NAP-2 (analogs) and other CXC chemokines in rabbits with intramuscular Escherichia coli infections was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection . RESULTS: The CXCR2-binding affinity of the HYNIC-conjugated NAP-2 analogs relative to NAP-2 was as follows: NAP-2(1-68), 2.5-fold; NAP-2(1-66), 10-fold; and NAP-2(1-63), 3-fold . In the rabbit model, uptake in the abscess (in percentage injected dose per gram +/- SEM) was 0.084 +/- 0.015 for NAP-2, 0.098 +/- 0.010 for NAP-2(1-68), 0.189 +/- 0.044 for NAP-2(1-66), and 0.114 +/- 0.017 for NAP-2(1-63) at 6 h after injection . In comparison, higher uptake in the abscess was found for labeled IL-8, a modest uptake was found for GCP-2 and ENA-78, and a low uptake was found for CTAP-III, PF-4, and IP-10 . CONCLUSION: This study showed a clear relationship between affinity to receptors on neutrophils and suitability for infection imaging . Of the NAP-2 variants, NAP-2(1-66) combined highest affinity to CXCR2 with the best characteristics for imaging . IL-8 binds to both CXCR1 and CXCR2 with high affinity and showed a superior imaging quality . The other CXC chemokines tested bind to neutrophils with lower affinity and were shown to be less suitable for infection imaging in this study.

Physiol Behav, 2004 Aug, 82(1), 63 - 8
Nociceptin/orphanin FQ acts as a functional antagonist of corticotropin-releasing factor to inhibit its anorectic effect; Ciccocioppo R et al.; Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP opioid receptor (previously referred to as ORL1 or OP4 receptor), exerts a variety of behavioral effects . N/OFQ as well as the synthetic NOP receptor agonist Ro 64-6198 have been reported to possess antistress properties and to elicit a pronounced hyperphagic effect in freely feeding rats . These findings have raised our interest to investigate possible interactions in the control of ingestive behavior between N/OFQ and corticotropin-releasing factor (CRF), which is well known to be a major mediator of stress and to possess anorectic properties . These studies have shown that intracerebroventricular injections of N/OFQ or of Ro 64-6198 reverse the anorectic action evoked by intracerebroventricular administration of CRF . The anti-anorectic effect of N/OFQ or Ro 64-6198 is antagonized by the selective NOP receptor antagonist {Nphe1}N/OFQ1-13NH2, providing evidence that it is mediated by this receptor . The effect occurs at doses that are not hyperphagic per se and is clearly selective versus the anorectic action of CRF since N/OFQ or Ro 64-6198 do not influence the anorectic effect of Escherichia coli lipopolysaccharide (LPS) . Neither N/OFQ nor Ro 64-6198 shows affinity for CRF receptors, suggesting that NOP receptor agonists might act as functional antagonists of CRF with regard to its anorectic action . Microinjection studies have revealed that the bed nucleus of the stria terminalis (BNST) is highly sensitive to the anorectic action of CRF, as well as to the anti-anorectic action of N/OFQ; pretreatment with 0.025-0.25 microg/site of N/OFQ into the BNST blocked the anorectic action of 0.1 microg/site of CRF given in the same area . On the other hand, intra-BNST microinjection of 0.025-0.25 microg/site of N/OFQ did not modify basal food intake . Thus, the BNST may be the site where the functional antagonism between N/OFQ and CRF takes place . These findings raise interest for the N/OFQ-NOP receptor system as a pharmacological target to block the anorectic effect of CRF . In comparison to CRF receptor antagonists, NOP receptor agonists may have the advantage of not inhibiting the hypothalamic-pituitary-adrenal (HPA) axis.

J Microbiol Methods, 2004 Aug, 58(2), 189 - 95
Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C . parvum oocysts; Kniel KE et al.; The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite . Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein . Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C . parvum oocysts and appeared to localize to the apical end of the parasite . Anti-rCPV40 serum was capable of detecting as few as 1 C . parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C . parvum oocyst protein or specific for the 41 kDa oocyst surface antigen . Water samples were seeded with C . parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity . Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence . While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C . By 3 months at 20 degrees C, the C . parvum oocysts were found to be non-infectious, but retained a high CPV signal . This study indicates that CPV is an excellent target for sensitive detection of C . parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.

Arch Biochem Biophys, 2004 Aug 1, 428(1), 99 - 108
Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications; Mast N et al.; Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study . All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta lacked the C-terminal proline-rich region . The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer . They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold . The two forms, Delta 46A1 Delta and H Delta 46A1 Delta, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

Arch Biochem Biophys, 2004 Aug 1, 428(1), 64 - 72
cDNA cloning, functional expression, and characterization of chicken sulfotransferases belonging to the SULT1B and SULT1C families; Wilson LA et al.; A search of the chicken expressed sequence tag (EST) database identified 2 cDNA clones that appeared to represent members of the SULT1B and SULT1C enzyme families . These cDNAs were fully sequenced and found to contain full-length inserts . Phylogenetic analysis of the derived amino acid sequences clearly placed them as the first members of the chicken SULT1B and SULT1C families, respectively, to be identified, and we propose they be named SULT1B1 and SULT1C1 . (CHICK)SULT1B1 shares approximately 60% amino acid sequence identity with mammalian SULT1B enzymes, whereas the closest neighbor to (CHICK)SULT1C1 was the ortholog (RAT)SULT1C1, with 68% identity . We cloned these cDNAs into the bacterial expression vectors from the pET series . Transformed Escherichia coli cells strongly expressed the recombinant proteins . Purification of the recombinant enzymes from E . coli was accomplished by a three-step procedure involving ammonium sulfate precipitation, anion exchange chromatography, and affinity chromatography . The purified enzymes displayed subunit molecular weights of approximately 35,000Da on SDS-PAGE, as predicted, and were both able to sulfate a wide range of compounds, including xenobiotics and endogenous substrates such as iodothyronines . Detailed kinetic analysis showed SULT1C1 was more prolific in that it was able to sulfate dopamine, tyramine, and apomorphine, which SULT1B1 was not . 2-Bromophenol was the best substrate for both enzymes . We also raised antibodies against these proteins, which were able to detect the SULTs by ELISA, and which were able to strongly inhibit the recombinant enzymes . This is the first detailed characterization of sulfotransferases from the chicken, and it demonstrates that the avian and mammalian SULT1 enzymes are closely related in both structure and function.

Neurochem Int, 2004 Oct, 45(5), 753 - 8
Characterization of novel Pur alpha-binding proteins in mouse brain; Zeng LH et al.; Pur alpha is an abundant protein in the brain and binds to a (GGN)n sequence, PUR element . It has been shown that Pur alpha not only interacts with single stranded DNA and RNA, but also with various proteins . In the present study, we tried to search for Pur alpha-binding proteins (PurBPs) in mouse brain by the overlay assay with GST-Pur alpha as a ligand . Three PurBPs of 35, 38 and 40 kDa were found mostly in the nuclear extract (N.Ext.) and they were not detected by the pretreatment of N.Ext . with trypsin, but not with RNase or DNase . The three PurBPs disappeared by the addition of ssCRE (single stranded cAMP response element) containing a PUR element, but not by DeltaGGN ssCRE (deletion of the PUR element from the ssCRE) . The PurBPs were abundantly expressed in the brain as Pur alpha . We also determined a region in Pur alpha which is required for the association with the PurBPs by using deletion mutants of Pur alpha . These biochemical properties of the PurBPs are different from the reported nuclear Pur alpha-binding proteins such as Sp1 and pRb.

Environ Pollut, 2004 Sep, 131(2), 255 - 62
Biosensors for detection of mercury in contaminated soils; Bontidean I et al.; Biosensors based on whole bacterial cells and on bacterial heavy metal binding protein were used to determine the mercury concentration in soil . The soil samples were collected in a vegetable garden accidentally contaminated with elemental mercury 25 years earlier . Bioavailable mercury was measured using different sensors: a protein-based biosensor, a whole bacterial cell based biosensor, and a plant sensor, i.e . morphological and biochemical responses in primary leaves and roots of bean seedlings grown in the mercury-contaminated soil . For comparison the total mercury concentration of the soil samples was determined by AAS . Whole bacterial cell and protein-based biosensors gave accurate responses proportional to the total amount of mercury in the soil samples . On the contrary, plant sensors were found to be less useful indicators of soil mercury contamination, as determined by plant biomass, mercury content of primary leaves and enzyme activities.

Eur J Biochem, 2004 Jul, 271(14), 3064 - 7
Cd(2+)-induced aggregation of Escherichia coli pyrophosphatase; Zimenkov YV et al.; We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd(2+) . This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd(2+) concentration . Aggregation was enhanced by Mg(2+), the natural cofactor of pyrophosphatase, and Mn(2+) . Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules, suppressed aggregation . These findings indicate that aggregation is affected by Cd(2+) binding to the peripheral metal-binding site, probably by strengthening intermolecular Trp149-Trp149' stacking interactions.

Eur J Biochem, 2004 Jul, 271(14), 3036 - 42
The transmembrane domain of subunit b of the Escherichia coli F1F(O) ATP synthase is sufficient for H(+)-translocating activity together with subunits a and c; Greie JC et al.; Subunit b is indispensable for the formation of a functional H(+)-translocating F(O) complex both in vivo and in vitro . Whereas the very C-terminus of subunit b interacts with F(1) and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper reconstitution of F(O) into liposomes . Here, we show that a synthetic peptide, residues 1-34 of subunit b (b(1-34)) {Dmitriev, O., Jones, P.C., Jiang, W . & Fillingame, R.H . (1999) J . Biol . Chem.274, 15598-15604}, corresponding to the membrane domain of subunit b was sufficient in forming an active F(O) complex when coreconstituted with purified ac subcomplex . H(+) translocation was shown to be sensitive to the specific inhibitor N,N'-dicyclohexylcarbodiimide, and the resulting F(O) complexes were deficient in binding of isolated F(1) . This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H(+) translocation across the membrane, whereas the binding of F(1) to F(O) is mainly triggered by C-terminal residues beyond Glu34 in subunit b . Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself, but might organize subunits a and c in F(O) assembly . Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b(1-34).

Biochem J, 2004 Nov 1, 383(Pt . 3), 517 - 27
Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes; Nagegowda DA et al.; 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA . In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli . Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa . It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes . It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1) . Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory . His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA) . Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity . Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA . Substitution S359A resulted in a 10-fold increased specific activity . Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1 . Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

J Biol Chem, 2004 Sep 3, 279(36), 37860 - 9 Epub 2004 Jul 01.
Rat brain cortex mitochondria release group II secretory phospholipase A(2) under reduced membrane potential; Macchioni L et al.; Activation of brain mitochondrial phospholipase(s) A(2) (PLA(2)) might contribute to cell damage and be involved in neurodegeneration . Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown . Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca(2+)-dependent PLA(2) having biochemical properties characteristic to secreted PLA(2) (sPLA(2)) and immunoreacting with the antibody raised against recombinant type IIA sPLA(2) (sPLA(2)-IIA) . Under identical conditions, no release of fumarase in the extramitochondrial medium was observed . The release of sPLA(2) from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC(6)(3) as a probe . The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA(2) release . The increase of sPLA(2) specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts . The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA(2) has mainly nuclear localization . In addition to sPLA(2), mitochondria contain another phospholipase A(2) that is Ca(2+)-independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane . We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA(2)-IIA that might contribute to cell damage.

J Bacteriol, 2004 Jul, 186(14), 4818 - 23
Loop deletions indicate regions important for FhuA transport and receptor functions in Escherichia coli; Endriss F et al.; Precise deletions of cell surface-exposed loops of FhuA resulted in mutants of Escherichia coli with distinct phenotypes . Deletion of loop 3 or 11 inactivated ferrichrome transport activity . Deletion of loop 8 inactivated receptor activity for colicin M and the phages T1, T5, and phi80 . The loop 7 deletion mutant was colicin M resistant but fully phage sensitive . The loop 4 deletion mutant was resistant to the TonB-dependent phages T1 and phi80 but fully sensitive to the TonB-independent phage T5 . The phenotypes of the deletion mutants revealed important sites for the multiple FhuA transport and receptor activities . The ligand binding sites are nonidentical and are distributed among the entire exposed surface . Presumably, FhuA evolved as a ferrichrome transporter and was subsequently used as a receptor by the phages and colicin M, which selected the same as well as distinct loops as receptor sites .

J Bacteriol, 2004 Jul, 186(14), 4802 - 7
Role of Escherichia coli DNA polymerase IV in in vivo replication fidelity; Kuban W et al.; We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli . We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome . Virtually no contribution of Pol IV to the chromosomal mutation rate was observed . On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F'(pro-lac) episome .

J Bacteriol, 2004 Jul, 186(14), 4556 - 67
Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica; Schuhle K et al.; The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate . The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied . This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins . Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation . Phenylphosphate carboxylation was restored when the 18-kDa subunit was added . The following reaction model is proposed . The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate . Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate . The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis . They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique . The 18-kDa subunit belongs to a hydratase/phosphatase protein family . Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen .

J Bacteriol, 2004 Jul, 186(14), 4520 - 7
Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells; Penfold CN et al.; The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood . It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation . We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain . After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9 . On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9 . The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry . The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins . The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB .

J Bacteriol, 2004 Jul, 186(14), 4510 - 9
Synthesis of the heteropolysaccharide O antigen of Escherichia coli O52 requires an ABC transporter: structural and genetic evidence; Feng L et al.; The structural and genetic organization of the Escherichia coli O52 O antigen was studied . As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E . coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-manno-heptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose) . The O-antigen gene cluster of E . coli O52, which is located between the galF and gnd genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes . Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit . This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E . coli . Genes specific for E . coli O52 were also identified .

Genes Dev, 2004 Jul 1, 18(13), 1618 - 29
Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA; Duquette ML et al.; We show that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large (150-500 bp) loops . These G-loops are formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other . G-loop formation requires a G-rich nontemplate strand and reflects the unusual stability of the rG/dC base pair . G-loops and G4 DNA form efficiently within plasmid genomes transcribed in vitro or in Escherichia coli . These results establish that G4 DNA can form in vivo, a finding with implications for stability and maintenance of all G-rich genomic regions.

Clin Chem, 2004 Sep, 50(9), 1553 - 9 Epub 2004 Jul 01.
Generic scheme for independent performance assessment in the molecular biology laboratory; Birch L et al.; BACKGROUND: A variety of proficiency testing schemes are available for specific molecular analyses, but there is an acute need for more widely accessible schemes to assess and demonstrate general competence in DNA analysis . METHODS: Fifteen laboratories, including academic, clinical, and commercial organizations, were recruited into the prototype assessment exercise . A range of test samples were provided, and participants were required to extract DNA from simple matrices, perform PCR amplification, and score the samples as positive or negative by electrophoretic analysis of the amplification products . Results were requested as both gel images and a completed results table, and the performance of each laboratory was then scored on the submitted analytical results . RESULTS: Overall, laboratories performed the analysis successfully, with participants scoring a high proportion of the samples correctly in the two rounds of the scheme . However, not all of the laboratories were able to achieve amplification for all samples, and the performance of some laboratories was not consistent in the two rounds . In addition, several analytical problems were encountered at all stages of the process, including DNA extraction, PCR amplification, and correct recording of results . CONCLUSIONS: The generic approach described here has enabled effective cross-sectoral benchmarking of laboratories from a variety of analytical sectors . The problems encountered by some participating laboratories highlight the need for quality control and checks at all stages of the process to ensure accuracy of results . A statistical analysis of the results (ANOVA) allowed meaningful comparison of the consistency and sensitivity achieved by laboratories, demonstrating that an effective balance was achieved between the level of data obtained from laboratories and the time expenditure required from participants.

Bioinformatics, 2004 Jul 10, 20(10), 1500 - 5
Gene expression analysis on biochemical networks using the Potts spin model; Konig R et al.; MOTIVATION: Microarray technology allows us to profile the expression of a large subset or all genes of a cell . Biochemical research over the last three decades has elucidated an increasingly complete image of the metabolic architecture . For less complex organisms, such as Escherichia coli, the biochemical network has been described in much detail . Here, we investigate the clustering of such networks by applying gene expression data that define edge lengths in the network . RESULTS: The Potts spin model is used as a nearest neighbour based clustering algorithm to discover fragmentation of the network in mutants or in biological samples when treated with drugs . As an example, we tested our method with gene expression data from E.coli treated with tryptophan excess, starvation and trpyptophan repressor mutants . We observed fragmentation of the tryptophan biosynthesis pathway, which corresponds well to the commonly known regulatory response of the cells.

Am J Physiol Gastrointest Liver Physiol, 2004 Nov, 287(5), G954 - 61 Epub 2004 Jul 01.
Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4; Watson JL et al.; A characteristic of many enteropathies is increased epithelial permeability, a potentially pathophysiological event that can be evoked by T helper (Th)-1 (i.e., IFN-gamma) and Th2 (i.e., IL-4) cytokines and bacterial infection {e.g., enteropathogenic Escherichia coli (EPEC)} . The green tea polyphenol (-)-epigallocatechin gallate (EGCG) has immunosuppressive properties, and we hypothesized that it would ameliorate the increased epithelial permeability induced by IFN-gamma, IL-4, and/or EPEC . EGCG, but not the related epigallocatechin, completely prevented the increase in epithelial (i.e., T84 cell monolayer) permeability caused by IFN-gamma exposure as gauged by transepithelial resistance and horseradish peroxidase flux; EGCG did not alleviate the barrier disruption induced by IL-4 or EPEC . IFN-gamma-treated T84 and THP-1 (monocytic cell line) cells displayed STAT1 activation (tyrosine phosphorylation on Western blot analysis, DNA binding on EMSA) and upregulation of interferon response factor-1 mRNA, a STAT1-dependent gene . All three events were inhibited by EGCG pretreatment . Aurintricarboxylic acid also blocked IFN-gamma-induced STAT1 activation, but it did not prevent the increase in epithelial permeability . Additionally, pharmacological blockade of MAPK signaling did not affect IFN-gamma-induced epithelial barrier dysfunction . Thus, as a potential adjunct anti-inflammatory agent, EGCG can block STAT1-dependent events in gut epithelia and monocytes and prevent IFN-gamma-induced increased epithelial permeability . The latter event is both a STAT1- and MAPK-independent event.

IUBMB Life, 2004 Apr, 56(4), 215 - 9
A 29.5 kDa heat-modifiable major outer membrane protein of Rickettsia prowazekii, putative virulence factor, is a peptidyl-prolyl cis/trans isomerase; Emelyanov VV et al.; Allelic genes from three Rickettsia prowazekii strains encoding parvulin-like protein (Plp), a heat-modifiable 29.5 kDa major outer membrane protein, were earlier cloned into expression vector pQE 30 . In this work, recombinant proteins were overproduced in E . coli, purified, and found to exhibit an expected peptidyl-prolyl cis/trans isomerase activity of a parvulin type in vitro with oligopeptide substrates . Native polypeptide of prototype virulent Breinl strain is known to differ by SDS-PAGE mobility from those of both vaccine Madrid E and virulent EVir isolates . Being different in electrophoretic behavior, heat-unmodified forms of the three strains were shown to migrate apart from lipopolysaccharides . A EVir Plp gene was sequenced, and deduced protein sequence was found to be identical to previously published Breinl and Madrid E . Present data indicate that unknown post-translational modification(s) in rickettsiae are responsible for both interstrain difference and heat-modifiability of Plp.

J Vet Med B Infect Dis Vet Public Health, 2004 May, 51(4), 166 - 8
Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea; Ha SK et al.; A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR) . Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E . coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes . Twenty-three (2.3%) of the 1002 E . coli isolates carried the gene for AIDA . Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor . Other isolates carried other virulence factor genes in addition to AIDA . Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins . Sixteen isolates carried genes for enterotoxins only . The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea.

Mol Microbiol, 2004 Jul, 53(2), 665 - 74
Activation of transcription initiation from a stable RNA promoter by a Fis protein-mediated DNA structural transmission mechanism; Opel ML et al.; The leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors . Transcription from this and other stable RNA promoters is known to be affected by a cis-acting UP element and by Fis protein interactions with the carboxyl-terminal domain of the alpha-subunits of RNA polymerase . In this report, we suggest that transcription from the leuV promoter also is activated by a Fis-mediated, DNA supercoiling-dependent mechanism similar to the IHF-mediated mechanism described previously for the ilvP(G) promoter (S . D . Sheridan et al., 1998, J Biol Chem 273: 21298-21308) . We present evidence that Fis binding results in the translocation of superhelical energy from the promoter-distal portion of a supercoiling-induced DNA duplex destabilized (SIDD) region to the promoter-proximal portion of the leuV promoter that is unwound within the open complex . A mutant Fis protein, which is defective in contacting the carboxyl-terminal domain of the alpha-subunits of RNA polymerase, remains competent for stimulating open complex formation, suggesting that this DNA supercoiling-dependent component of Fis-mediated activation occurs in the absence of specific protein interactions between Fis and RNA polymerase . Fis-mediated translocation of superhelical energy from upstream binding sites to the promoter region may be a general feature of Fis-mediated activation of transcription at stable RNA promoters, which often contain A+T-rich upstream sequences.

Mol Microbiol, 2004 Jul, 53(2), 587 - 97
Delayed-relaxed response explained by hyperactivation of RelE; Christensen SK et al.; Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation . The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis . The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation . We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE . As in wild-type cells, {ppGpp} increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level . RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction . Lon protease activates RelE during amino acid starvation by degradation of RelB . We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB . Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE . Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA . The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells . Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response.

Mol Microbiol, 2004 Jul, 53(2), 485 - 95
Intragenic suppression of gain-of-function mutations in the Escherichia coli mechanosensitive channel, MscL; Li Y et al.; Mechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological 'pressure release valves' . One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli . The E . coli MscL is a 136-amino-acid protein organized as a homopentamer with each subunit containing two transmembrane segments . Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain-of-function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions . Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations . The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp . Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop . In contrast, only one mutation is found in the amino-terminal S1 domain, and none is found within the carboxyl-terminal domain . Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.

Biochem J . 2004 Jul 1; Pt {Epub ahead of print}
Characterization of the polyene macrolide P450 epoxidase from Streptomyces natalensis that converts deepoxypimaricin into pimaricin; Mendes MV et al.; The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD . Pimaricin is derived from its immediate precursor deepoxypimaricin by epoxidation of the C4-C5 double bond on the macrolactone ring . We have overproduced PimD with a N-terminal six-His affinity tag in Escherichia coli and purified the enzyme for kinetic analysis . The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 nm, indicating that it is a cytochrome P450 . Purified PimD was shown to catalyze the in vitro C4-C5 epoxidation of 4,5-deepoxypimaricin into pimaricin . The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 oC . The k cat value for the epoxidation of deepoxypimaricin was similar to the values reported for other macrolide oxidases . Enzyme activity was inhibited at high substrate concentration . This is the first time that a polyene macrolide P450 monooxygenase has been heterologously expressed and studied . The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics.

J Reprod Dev, 2004 Jun, 50(3), 323 - 31
Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA; Jayasekara WS et al.; 20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy . To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed . The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems . Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily . From the start codon to stop codon there were 323 amino acids, the same as in other species . To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria . Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity . A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy . The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

Proc Natl Acad Sci U S A, 2004 Jul 6, 101(27), 10161 - 5 Epub 2004 Jun 28.
An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating; Bartlett JL et al.; MscL is a mechanosensitive channel of large conductance that functions as an "emergency release valve," allowing bacteria to survive acute hypoosmotic stress . Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed . Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype . Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines . Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo . The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.

J Cell Sci, 2004 Jul 15, 117(Pt 16), 3473 - 80 Epub 2004 Jun 29.
Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes; Rescher U et al.; Annexin 2 is a Ca(2+)-regulated membrane protein and an F-actin-binding protein enriched at actin assembly sites both, on the plasma membrane and on endosomal vesicles . Here, we identify annexin 2 as a phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2))-interacting protein, thereby explaining this specific membrane association . Using the pleckstrin-homology (PH) domain of phospholipase Cdelta1 fused to yellow fluorescent protein as a marker for PtdIns(4,5)P(2), we show that annexin 2 and its ligand p11 (S100A10) are targeted to sites of PtdIns(4,5)P(2) enrichment where F-actin accumulates . At the plasma membrane, adhesion of pedestal-forming enteropathogenic Escherichia coli induces a recruitment of 1-phosphatidylinositol-4-phosphate 5-kinase (PtdIns4P 5-kinase) and an enrichment of PtdIns(4,5)P(2) and annexin 2-p11 at sites of bacterial adhesion . Induction of PtdIns(4,5)P(2)-enriched ruffles and PtdIns(4,5)P(2)-positive, actin-coated vacuoles by Arf6-mediated activation of PtdIns4P 5-kinase also leads to a concomitant accumulation of the annexin 2-p11 complex and the PH domain . Binding studies with immobilized phosphoinositides and phosphoinositide-containing liposomes reveal that the purified annexin 2-p11 complex directly and specifically binds to PtdIns(4,5)P(2) with an affinity comparable to that of the PH domain of phospholipase Cdelta1 . Experiments using individual subunits identify annexin 2 as the PtdIns(4,5)P(2)-binding entity . Thus, the direct interaction of annexin 2 with PtdIns(4,5)P(2) is a means of specifically recruiting the annexin 2-p11 complex to sites of membrane-associated actin assembly.

J Biol Chem, 2004 Sep 3, 279(36), 37822 - 31 Epub 2004 Jun 28.
Specific interaction between human parechovirus nonstructural 2A protein and viral RNA; Samuilova O et al.; The functional properties of the nonstructural 2A protein are variable among different picornaviruses . The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A . To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli . A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy . Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells . However, at late stages of infection some infected cells also exhibited diffuse nuclear staining . Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region . Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity . Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding . These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity . At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis . In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-) . In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.

J Biol Chem, 2004 Aug 27, 279(35), 37079 - 86 Epub 2004 Jun 28.
Identification of the spermatogenic zip protein Spz1 as a putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the PP1cgamma2 splice variant in mouse testis; Hrabchak C et al.; The spermatogenic zip protein (Spz1) was originally isolated from a mouse testis library and identified as a novel member of the basic helix-loop-helix family of transcription factors . Here we identify Spz1 as a specific binding partner of the gamma2 catalytic subunit of protein phosphatase-1 . Male mice homozygous for a null mutation in the protein phosphatase-1cgamma (PP1cgamma) gene are infertile and display a distinct impairment in spermiogenesis despite the continued presence of closely related PP1c isoforms . Yeast two-hybrid screening using the PP1cgamma2 splice variant has identified Spz1 as an interacting protein and possible mediator of the sterile PP1cgamma mutant phenotype . Spz1 was shown to interact specifically with PP1cgamma2 but did not show an interaction with PP1calpha or with a truncated version of PP1cgamma2 lacking 18 amino acids from the C terminus . Interaction between full-length Spz1 and PP1cgamma2 was verified by co-immunoprecipitation and co-localization experiments in COS-1 cells as well as gel-shift and sedimentation assays using whole testis lysates . Immunohistochemistry on wild type testis sections reveals a stage-specific expression pattern for Spz1 during spermatogenesis that appeared grossly abnormal in the testes of PP1cgamma mutant mice . Phosphatase assays using recombinant PP1c indicate that increasing concentrations of Spz1 are able to inhibit PP1cgamma2 activity while having little effect on the activity of PP1calpha . Furthermore, an interaction between PP1cgamma2 and Spz1 was shown to prevent binding of the latter to the consensus E-box promoter sequence . We propose that the interaction between Spz1 and PP1cgamma2 may be required for proper regulation of spermatogenesis and fertility in males.

J Biol Chem, 2004 Sep 17, 279(38), 40044 - 52 Epub 2004 Jun 28.
Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells; Kim SH et al.; The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent . However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria . We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli . We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction . Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor . Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL . Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL . TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure . This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity . Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.

Bioorg Med Chem Lett, 2004 Aug 2, 14(15), 4001 - 4
Aptamer selection for the inhibition of cell adhesion with fibronectin as target; Ogawa A et al.; An affinity column immobilizing a decapeptide H(2)N-RGDSPASSKP-CO(2)H was used to select RGD-binding aptamers from a pool of 86-mer single-strand oligodeoxynucleotides (ODNs) containing a random 40-mer sequence . The enriched library thus obtained was further selected against adsorbed fibronectin and individual aptamers were monocloned in E . coli and sequenced to give a couple of highly homologous ODNs, which indeed inhibited fibronectin-integrin mediated cell adhesion.

FEBS Lett, 2004 Jul 2, 569(1-3), 289 - 92
Periplasmic competition for zinc uptake between the metallochaperone ZnuA and Cu,Zn superoxide dismutase; Berducci G et al.; We have investigated the availability of zinc in the periplasmic space of Escherichia coli using a mutant Cu,Zn superoxide dismutase whose dimerization is triggered by zinc binding . This mutant enzyme accumulates in the monomeric form when wild type cells are grown in minimal medium, but assembles in the dimeric form when it is produced in the same medium by a mutant strain lacking the periplasmic zinc metallochaperone ZnuA . These results indicate that periplasmic zinc-containing proteins compete for metal binding when bacteria grow in environments where this element is present in traces . The effective ZnuA ability to sequester the available zinc ions from the periplasm suggests that zinc-containing cytoplasmic proteins are more important for bacterial viability than the periplasmic ones.

FEBS Lett, 2004 Jul 2, 569(1-3), 82 - 8
Localization of the Tat translocon components in Escherichia coli; Berthelmann F et al.; The Tat system has the ability to translocate folded proteins across the bacterial cytoplasmic membrane . In Escherichia coli, three functionally different translocon components have been identified, namely TatA, TatB, and TatC . These proteins were fused to the green fluorescent protein (GFP) and their localization was determined by confocal laser scanning fluorescence microscopy . TatA-GFP was distributed in the membrane, often with higher abundance at the poles . TatB-GFP was found in distinct spots at the poles of the cells . The fluorescence of TatC-GFP was very low and required a constitutive expression system to become higher than background, but then appearing polar . All three constructs complemented the chain-formation phenotype of corresponding mutant strains, indicating the functionality of the fusion proteins . TatB-GFP and TatC-GFP also complemented TMAO respiration deficiency and TatA-GFP the SDS-sensitivity of the mutant strains . The localization of the translocon-GFP fusions coincides with the fluorescence pattern of GFP fusions to Tat substrate signal sequences . We suggest that the active translocon complexes are mainly present at polar positions in Escherichia coli.

Mol Microbiol, 2004 Jul, 53(1), 203 - 15
Transcription activation by remodelling of a nucleoprotein assembly: the role of NarL at the FNR-dependent Escherichia coli nir promoter; Browning DF et al.; Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and NarL or NarP proteins (transcription activators triggered by nitrite and nitrate) . We found previously that FNR binds to a site centred at position - 41.5 at the nir promoter, but that FNR-dependent activation is repressed by IHF binding to a site centred at position -88 (IHF I) and Fis binding to sites centred at positions -142 (Fis I) and +23 (Fis II) . Here, we have studied the binding of purified IHF, Fis and FNR to the nir promoter in vitro . Our results show that the nir promoter contains a second IHF site at position -115 (IHF II) and a third Fis site at position -97 (Fis III), and that IHF, Fis and FNR can bind together to form multiprotein complexes . Surprisingly, IHF binding at the IHF II site increases FNR-dependent activation by decreasing the repression mediated by IHF and Fis binding at the other sites . In previous work, we found that NarL or NarP activates the nir promoter by binding to a site centred at position -69.5 and counteracting the repressive effects of IHF and Fis . We now show that NarL can displace IHF bound at the IHF I site, but IHF is unable to displace bound NarL . We suggest that NarL interferes with IHF binding at the nir promoter by distorting the minor groove at its target site, and we argue that the resulting activation by NarL results from remodelling of the local nucleoprotein structure to facilitate FNR-dependent transcription.

Mol Microbiol, 2004 Jul, 53(1), 193 - 202
Involvement of two domains with helix-turn-helix and zinc finger motifs in the binding of IS1 transposase to terminal inverted repeats; Ohta S et al.; The insertion element IS1 has two open reading frames (ORFs), insA and insB, and produces a transframe protein InsAB, known as IS1 transposase, by translational frameshifting . The transposase binds to terminal inverted repeats (IRL and IRR) to promote IS1 transposition . Unless frameshifting occurs, IS1 produces InsA protein, which also binds to IRs and therefore acts as an inhibitor of transposition, as well as a transcriptional repressor of the promoter in IRL . A helix-turn-helix (HTH) motif present in both transposase and InsA is thought to be involved in IR-specific DNA binding . A comparison of transposases encoded by IS1 family elements reveals that the N-terminal regions contain four conserved cysteine residues, which appear to constitute a C(2)C(2) zinc finger (ZF) motif . This motif is also thought to be involved in IR-specific DNA binding . In this study, we show that IS1 transposases with an amino acid substitution in the HTH or ZF motif lose the ability to promote transposition . We also show that transposases, as well as InsA proteins with the same substitution, lose the ability to repress the activity of the IRL promoter, and that purified InsA mutant proteins lose the ability to bind to the IRL-containing fragment . Furthermore, we show that InsA protein co-ordinates Zn(II) with the four cysteine residues as ligands and loses the ability to bind to the IRL-containing fragment in the presence of an agent chelating Zn(II) . These findings indicate that IS1 transposase has two domains with HTH and ZF motifs responsible for IR-specific DNA binding in promoting transposition . It is assumed that the two domains are needed for transposase to bind to each IR in an oriented manner in order to place a catalytic domain in the C-terminal region of the transposase to a region around the IR end, where the strand transfer reaction occurs in a transpososome.

Mol Microbiol, 2004 Jul, 53(1), 143 - 52
Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence; Rochman M et al.; The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA . Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA . Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS . Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region . We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence . The far upstream sequence shows no effect in the absence of an intact cluster . This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point . Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex . We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

Mol Microbiol, 2004 Jul, 53(1), 93 - 102
The role of Par proteins in the active segregation of the P1 plasmid; Li Y et al.; The parS centromere-like site promotes active P1 plasmid segregation in the presence of P1 ParA and ParB proteins . At the modest growth rate used here, time-lapse and still photomicroscopy shows that the plasmid copies are clustered as a focus at the Escherichia coli cell centre . Just before cell division, the focus is actively divided and ejects bidirectionally into opposite halves of the dividing cell . In the absence of the wild-type parS binding protein ParB, a focus was formed, but generally did not go to the cell centre . The randomly placed focus did not divide and was inherited by one daughter cell only . In the absence of ParA, foci formed and frequently fixed to the cell centre . However, they failed to divide or eject and were left at the new cell pole of one cell at division . Thus, ParB appears to be required for recognition of the plasmid and its attachment to the cell centre, and ParA is required for focus division and energetic ejection from the cell centre . The ATPase active site mutation, parAK122E, blocked ejection . Mutant parAM314I ejected weakly, and the daughter foci took two generations to reach a new cell centre . This explains the novel alternation of segregation and missegregation in successive generations seen in time-lapse images of this mutant.

Mol Microbiol, 2004 Jul, 53(1), 65 - 80
Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation; Domain F et al.; The cyanobacterial genes lexA, recA and ruvB were analysed in Synechocystis PCC6803, which is shown here to be more radiation resistant than the other unicellular model strain Synechococcus PCC7942 . We found that cyanobacteria do not have an Escherichia coli-type SOS regulon . The Synechocystis lexA and recA promoters were found to be strong and UV insensitive, unlike the ruvB promoter, which is weak and UV-C inducible . Yet, lexA and recA are regulated by UV-C, but the control is negative and occurs at the post-transcriptional level . Two novel conserved elements were characterized in the lexA promoter: (i) an unusually long crucial box 5'-TAAAATTTTGTATCTTTT-3' (-64, -47); and (ii) a negatively acting motif 5'-TAT GAT-3' (-42, -37) . These elements were not found in the recA promoter, which appeared to be unusually simple in harbouring only a single crucial element (i.e . the canonical -10 box) . RuvB, operating in recombination-dependent cellular processes, was found to be dispensable to cell growth, whereas LexA and RecA appeared to be critical to cell viability . Using DNA microarrays, we have identified 57 genes with expression that is altered, at least twofold, in response to LexA depletion . None of these genes is predicted to operate in DNA metabolism, arguing against the involvement of LexA in the regulation of DNA repair . Instead, most of the LexA-responsive genes were known to be involved in carbon assimilation or controlled by carbon availability . Consistently, the growth of the LexA-depleted strain was found to be strongly dependent on the availability of inorganic carbon.

Plant J, 2004 Jul, 39(2), 206 - 18
The calcium-dependent protein kinase HvCDPK1 mediates the gibberellic acid response of the barley aleurone through regulation of vacuolar function; McCubbin AG et al.; In the aleurone cells of the cereal grain, gibberellic acid (GA) induces the secretion of hydrolases that mobilize endosperm reserves to fuel early seedling growth . GA is known to trigger a range of cellular responses, including increases in cytoplasmic calcium, vacuolar reserve mobilization, gene transcription, and the synthesis and secretion of hydrolases . To further define elements of the Ca2+-dependent GA response machinery, we have cloned a Ca2+-dependent protein kinase (HvCDPK1) from these cells . Although expression of an inactivated (D140N) version of this kinase did not affect GA-induced gene expression or changes in cytosolic Ca2+, it did inhibit secretion, cell vacuolation, and vacuolar acidification, all responses linked to the GA response . Additionally, recombinant wild-type HvCDPK1 activated the V-type H(+)-ATPase present in isolated aleurone vacuoles . These results suggest HvCDPK1 may mediate Ca2+-dependent events of the GA response, such as control of vacuolar function, that lie downstream of transcriptional regulation.

J Endocrinol, 2004 Jul, 182(1), 133 - 44
Cloning and expression of porcine adiponectin, and its relationship to adiposity, lipogenesis and the acute phase response; Jacobi SK et al.; Adiponectin is an adipocyte-derived hormone that has been implicated recently in the regulation of inflammation in immunocytes, and in lipid metabolism and glucose homeostasis in liver, skeletal muscle and adipocytes . However, information in non-rodent models is limited . We have cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin in vivo following lipopolysaccharide (LPS) or E . coli administration . The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin respectively, and 79-83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species . Relative serum adiponectin concentrations were not altered in pigs infused with E . coli, and mRNA expression in adipose tissue was not responsive to LPS . However, analysis of serum from very lean vs a substantially fatter genotype of pig indicated that relative circulating adiponectin concentrations are higher (P<0.01) in the lean pigs than in the fatter genotype, and that the difference is established relatively early in the growth curve . Also, incubating pig adipocytes for 6 h with recombinant pig adiponectin resulted in an approximately 30% reduction (P<0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin . This is the first report in any species that adiponectin antagonizes the incorporation of glucose carbon into lipid in the adipocyte, and provides additional evidence that adiponectin acts as an autocrine regulatory factor to regulate energy metabolism.

Biochem J, 2004 Oct 1, 383(Pt 1), 149 - 58
Sputa nerve growth factor forms a preferable substitute to mouse 7S-beta nerve growth factor; Koh DC et al.; The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC . The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF . Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein . Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions . The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA-p75NTR complex of receptors . The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB gene by a corresponding down-regulation of inhibitory kappaB gene . Real-time PCR and protein profiling (by surface-enhanced laser-desorption-ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells . Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel . Hence, sputa NGF forms a new and useful NGF.

J Am Chem Soc, 2004 Jul 7, 126(26), 8320 - 8
Ligand K-edge X-ray absorption spectroscopy of {Fe4S4}1+,2+,3+ clusters: changes in bonding and electronic relaxation upon redox; Dey A et al.; Sulfur K-edge X-ray absorption spectroscopy (XAS) is reported for {Fe(4)S(4)}(1+,2+,3+) clusters . The results are quantitatively and qualitatively compared with DFT calculations . The change in covalency upon redox in both the {Fe(4)S(4)}(1+/2+) (ferredoxin) and the {Fe(4)S(4)}(2+/3+) (HiPIP) couple are much larger than that expected from just the change in number of 3d holes . Moreover, the change in the HiPIP couple is higher than that of the ferredoxin couple . These changes in electronic structure are analyzed using DFT calculations in terms of contributions from the nature of the redox active molecular orbital (RAMO) and electronic relaxation . The results indicate that the RAMO of HiPIP has 50% ligand character, and hence, the HiPIP redox couple involves limited electronic relaxation . Alternatively, the RAMO of the ferredoxin couple is metal-based, and the ferredoxin redox couple involves extensive electronic relaxation . The contributions of these RAMO differences to ET processes in the different proteins are discussed.

J Environ Qual, 2004 May-Jun, 33(3), 1088 - 97
Quantity and quality of runoff from a beef cattle feedlot in southern Alberta; Miller JJ et al.; Southern Alberta, which has a cold climate dominated by strong chinook winds, has the highest density of feedlot cattle in Canada . However, the quantity and quality of runoff from beef cattle (Bos taurus) feedlots in this unique region has not been investigated . Our objectives were to compare runoff quantity (1998-2002) with catch-basin design criteria; determine concentrations of selected inorganic chemical parameters (1998-2000) in runoff in relation to water quality guidelines and the potential implications of irrigating adjacent crop-land; and determine if total heterotrophs, total coliforms, and Escherichia coli (1998-2000) persisted in the catch-basin water and soil . Runoff (< 0.1 to 42.5 mm) for a 24-h duration that included maximum peak discharge was less than the recommended design criteria of 90 mm based on runoff from 24 h of rainfall with a 30-yr return period . We found that curve numbers between 52 and 96 (mode of 90) were required to match the USDA Natural Resources Conservation Service predicted runoff and actual runoff volumes . Total P posed the greatest threat to water quality guidelines, and K posed the greatest threat for exceeding crop fertilizer requirements if catch-basin effluent was used as irrigation water . Water in the catch basin had continually high populations of E . coli throughout the study, with values ranging between log10 2 and log10 8 100 mL(-1) . In contrast, soil in the catch basin generally had low populations of E . coli that were < log10 2 g(-1) wet wt., but at times higher populations between log10 2 and log10 6 g(-1) wet wt . were also found.

Invest Ophthalmol Vis Sci, 2004 Jul, 45(7), 2413 - 9
Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice; Pan H et al.; PURPOSE: To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization . METHODS: The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad) . Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells . 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy . The ability of recombinant 16K hPRL expressed in E . coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC) . RESULTS: 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation . Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls . r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs . CONCLUSIONS: Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy . 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells . These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies.

Scand Cardiovasc J, 2004 Jun, 38(3), 187 - 92
Increased oxygen cost of contractility in the endotoxemic porcine left ventricle; Aghajani E et al.; OBJECTIVE: Myocardial oxygen consumption (MVO) in the septic myocardium is comparatively high in relation to the sepsis-induced reduction in ventricular work . Our previous studies indicate that this energetic inefficiency is due to increased energy consumption in excitation-contraction (EC) coupling, i.e . myocardial calcium handling . DESIGN: To further confirm this observation, we assessed the oxygen cost of contractility in anesthetized pigs before and 2 h after induction of endotoxemia (1 microg/kg endotoxin infusion over 1 h, Escherichia coli toxin, n=6) . Baroreceptor reflexes were blocked by hexamethonium . Contractility was increased by stepwise dopamine infusions at baseline and 2 h after induction of endotoxemia . Oxygen cost of contractility was assessed as the relationship between myocardial contractility (E or elastance) and non-mechanical oxygen consumption (unloaded MVO), a measure of energy consumption in EC coupling or calcium handling . RESULTS: Non-mechanical oxygen consumption (unloaded MVO) was higher after endotoxin infusions than at baseline (0.641 +/- 0.05 vs 0.383 +/- 0.07 J/beat/100 g, p < 0.05) . The relationship between unloaded MVO and E, constructed by the dopamine response, was highly linear both at baseline and endotoxemia (r2 =0.76-0.99) . However, endotoxin increased oxygen cost of contractility by approximately 45% (baseline 0.06 +/- 0.03 vs endotoxin 0.09 +/- 0.04 J ml/mmHg/beat/100 g) . CONCLUSION: Acute endotoxemia increases oxygen cost of contractility, a measure of energy consumed in EC coupling or myocardial calcium handling.

Avian Pathol, 2004 Jun, 33(3), 337 - 42
Risk factors associated with colibacillosis outbreaks in caged layer flocks; Vandekerchove D et al.; Colibacillosis appears to be of increasing significance in layer flocks, but there have been no studies of the risk factors associated with outbreaks . This study aimed to investigate the possible associations between risk factors of non-infectious nature and outbreaks of mortality due to colibacillosis in flocks of caged layer hens . Information on management, biosecurity measures and housing conditions was collected in 20 flocks suffering from the disease and in 20 clinically healthy control flocks . The data were processed using multiple logistic regression . The statistical analysis demonstrated that an increase in the distance to the nearest poultry farm by 1 km was associated with a six-fold decreased risk of an outbreak of colibacillosis (odds ratio=0.16) . Furthermore, a 1 l increase in cage volume per hen was associated with a 33% decrease in the risk of an outbreak (odds ratio=0.75) . It was concluded that the distance between poultry farms and the hen density in the cages are important risk factors for outbreaks of colibacillosis in flocks of layer hens.

Avian Pathol, 2004 Jun, 33(3), 328 - 36
Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests; Johne R et al.; Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species . BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult . To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita) . Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences . No protein was detected after induction of full-length C1 expression in Escherichia coli . However, deletion of an amino-terminal arginine-rich sequence facilitated expression . C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens . The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds . Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting . The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.

Avian Pathol, 2004 Jun, 33(3), 298 - 302
Significance of interactions between Escherichia coli and respiratory pathogens in layer hen flocks suffering from colibacillosis-associated mortality; Vandekerchove D et al.; This study aimed to examine the significance of interactions between Escherichia coli and various respiratory pathogens during outbreaks of colibacillosis-associated mortality in layer hen flocks under field conditions . For this purpose, a case-control study involving 20 control flocks with baseline mortality and 20 flocks with increased mortality due to E . coli septicaemia and polyserositis, was conducted . In each colibacillosis flock, blood samples were taken from 20 hens at the onset of clinical disease and three times thereafter at 2-week intervals . Control flocks of comparable ages were sampled in the same way . Pooled sera, taken at the first and last sampling, were examined for antibody titres against infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), and the individual sera from all four samplings were examined for the presence and/or titres of antibodies against avian pneumovirus (APV), Mycoplasma gallisepticum, Mycoplasma synoviae and Ornithobacterium rhinotracheale . Titre increases were seen for IBV D274 (one control flock) and O . rhinotracheale (one control and one colibacillosis flock) . An increase in per cent reactors was seen for APV (one control flock), and for M . synoviae (one control and two colibacillosis flocks) . The study failed to detect any consistent interactions between E . coli and the aforementioned pathogens . These results indicate that, at least as observed in this study, outbreaks of increased mortality resulting from colibacillosis are not necessarily associated with IBV, NDV, APV, M . gallisepticum, M . synoviae or O . rhinotracheale infections.

J Mol Biol, 2004 Jul 16, 340(4), 891 - 907
Biochemical and structural studies of the interaction of Cdc37 with Hsp90; Zhang W et al.; The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors . In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones . Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases . These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others . Here, the biochemical details of the interaction of human Hsp90 beta and Cdc37 have been characterised . Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37 . The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.

J Mol Biol, 2004 Jul 16, 340(4), 857 - 68
Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling; Balakrishnan G et al.; The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy . The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues . Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions . However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state . Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2 . These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit . Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected . A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface . The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale . They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.

J Mol Biol, 2004 Jul 16, 340(4), 797 - 808
The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state; Butler PJ et al.; EmrE is a multidrug transporter that utilises the proton gradient across bacterial cell membranes to pump hydrophobic cationic toxins out of the cell . The structure of EmrE is very unusual, because it is an asymmetric homodimer containing eight alpha-helices, six of which form the substrate-binding chamber and translocation pathway . Despite this structural information, the precise oligomeric order of EmrE in both the detergent-solubilised state and in vivo is unclear, although it must contain an even number of subunits to satisfy substrate-binding data . We have studied the oligomeric state of EmrE, purified in a functional form in dodecylmaltoside, by high-resolution size-exclusion chromatography (hrSEC) and by analytical ultracentrifugation . The data from equilibrium analytical ultracentrifugation were analysed using a measured density increment for the EmrE-lipid-detergent complex, which showed that the purified EmrE was predominantly a dimer . This value was consistent with the apparent mass for the EmrE-lipid-detergent complex (137 kDa) determined by hrSEC . EmrE was purified under different conditions using minimal concentrations of dodecylmaltoside, which would have maintained the structure of any putative higher oligomeric states: this EmrE preparation had an apparent mass of 206 kDa by hrSEC and equilibrium analytical ultracentrifugation showed unequivocally that EmrE was a dimer, although it was associated with a much larger mass of phospholipid . In addition, the effect of the substrate tetraphenylphosphonium on the oligomeric state was also analysed for both preparations of EmrE; velocity analytical ultracentrifugation showed that the substrate had no effect on the oligomeric state . Therefore, in the detergent dodecylmaltoside and under conditions where the protein is fully competent for substrate binding, EmrE is dimeric and there is no evidence from our data to suggest higher oligomeric states . These observations are discussed in relation to the recently published structures of EmrE from two- and three-dimensional crystals.

J Mol Biol, 2004 Jul 16, 340(4), 695 - 706
Structural characterization and comparative phylogenetic analysis of Escherichia coli HemK, a protein (N5)-glutamine methyltransferase; Yang Z et al.; Protein glutamine methylation at GGQ sites of protein chain release factors plays a pivotal role in the termination of translation . We report here the crystal structure of the Escherichia coli HemK protein (N5)-glutamine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) . HemK contains two domains: a putative substrate binding domain at the N terminus consisting of a five helix bundle and a seven-stranded catalytic domain at the C terminus that harbors the binding site for AdoHcy . The two domains are linked by a beta-hairpin . Structure-guided sequence analysis of the HemK family revealed 11 invariant residues functioning in methyl-donor binding and catalysis of methyl transfer . The putative substrate-binding domains of HemK from E.coli and Thermotoga maritima are structurally similar, despite the fact that they share very little sequence similarity . When the two proteins are aligned structurally, the helical N-terminal domain is subject to approximately 10 degrees of hinge movement relative to the C-terminal domain . The apparent hinge mobility of the two domains may reflect functional importance during the reaction cycle . Comparative phylogenetic analysis of the hemK gene and its frequent neighbor gene, prfA, which encodes a major substrate, provides evidence for several examples of lateral gene transfer.

J Neuroimmunol, 2004 Jul, 152(1-2), 168 - 75
Antigen recognition properties of a Vgamma1.3Vdelta2-T-cell receptor from a rare variant of polymyositis; Dornmair K et al.; Previously we partially characterized an autoreactive human Vgamma1.3Vdelta2-T-cell receptor (TCR) that had originally been identified in muscle of a patient with an unusual form of polymyositis . This TCR recognizes a muscle-associated auto-antigen in a CDR3-dependent, MHC non-restricted way . Here we show that this TCR also recognizes an antigen from Escherichia coli . Like the muscle-associated mammalian antigen, the bacterial antigen is recognized in a CDR3-dependent, but MHC-non-restricted way . Both antigens have strikingly similar molecular characteristics suggesting that their epitopes are at least very similar . The dissociation kinetics of the bacterial antigen-TCR complexes was investigated by surface plasmon resonance using soluble single-chain TCR molecules produced in COS-7 cells . The measured dissociation rate constant (k(off)=5.7 x 10(-3) s(-1)) shows that the complexes dissociate more slowly than most previously described antigen/alphabeta-TCR complexes, but much faster than antibody/antigen pairs . These results (a) provide further insight into the molecular properties of this unusual TCR, and (b) should help in future attempts to identify the elusive target antigen(s).

Vet Microbiol, 2004 Jul 14, 101(3), 153 - 60
Enterotoxigenic K99+ Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein; Jay CM et al.; Most enterotoxigenic Escherichia coli (ETEC) isolated from neonatal cattle with diarrhea (enteric colibacillosis) exhibit the colonization factor antigen, K99 . The K99 pili are necessary for the bacteria to bind to a receptor, N-glycolylneuraminic acid-GM3 on the host cells in the small intestine where the bacteria multiply and secrete toxins that cause the diarrhea . When the attachment of the ETEC to host cell is inhibited, the bacteria do not accumulate sufficiently in the gut to cause disease . Since purified K99 pili block K99+ ETEC from binding to host epithelia, three recombinant K99 proteins of different sizes were developed and produced to demonstrate inhibition with in vitro competitive binding assays . The full-length recombinant protein, rK99-476 inhibited the binding of ETEC with an activity similar to that of the native purified K99, whereas the truncated recombinant K99 protein had no inhibitory activity . Thus this binding activity of rK99-476, which is specific and effective in blocking the receptors on the host cells, may be able to competitively inhibit K99+ ETEC infections in cattle.

Free Radic Biol Med, 2004 Aug 1, 37(3), 401 - 10
Complementation of SOD-deficient Escherichia coli by manganese porphyrin mimics of superoxide dismutase activity; Okado-Matsumoto A et al.; Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics . The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer . This ortho compound also exhibited a more positive redox potential and greater ability to facilitate the aerobic growth of a SOD-deficient Escherichia coli . Analogs with longer alkyl side chains and with methoxyethyl side chains, as well as with N,N'-diethylimidazolium and N,N'-dimethoxyethylimidazolium groups on the meso positions, have been prepared in anticipation of greater penetration of the cells due to greater lipophilicity . We now report that the more lipophilic compounds were effective at complementing the SOD-deficient E . coli at lower concentrations than were needed with the less lipophilic compounds . The greater efficacy of the more lipophilic compounds was achieved at the cost of greater toxicity that became apparent when these compounds were applied at higher concentrations.

Biochemistry, 2004 Jul 6, 43(26), 8590 - 9
Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III; Chai W et al.; Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS) . Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites . N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities . Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides . The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning . The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved . Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved . It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide . It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used . It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.

World J Gastroenterol, 2004 Jul 1, 10(13), 1872 - 5
Primary targeting of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha against hepatocellular carcinoma; Zhang J et al.; AIM: To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC) . METHODS: Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR . MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector . This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was expressed in Escherichia coli and purified from inclusions . After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot . And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins . MTNFalpha protein and PBS were used as control at the same time . After treated for two weeks, nude mice were executed . The bulk and weight of tumors were observed . The tumor tissues were stained by immunohistochemical method with TNFalpha antibody . RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein . The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission . mTNFalpha restraining trials showed 5/5 partial remission . The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P<0.05) . The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining . The positive granules mainly existed in the cytoplasm of tumor cell . CONCLUSION: Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha . It can inhibit the cellular growth of HCC and has some potential of clinical application.

Mol Genet Genomics, 2004 Aug, 272(1), 57 - 66 Epub 2004 Jun 19.
A Rab-related small GTP binding protein is predominantly expressed in root nodules of Medicago sativa; Schiene K et al.; Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eucaryotic cells . In this paper we report the isolation of the cDNA clone MS- rab11f from Medicago sativa (alfalfa) root nodules using a combination of RT-PCR and SSCP analysis . MS- rab11f shows high homology to the Rab-related cDNA clone LJ- rab11f from Lotus japonicus root nodules . The MS-Rab11F protein expressed in Escherichia coli was found to bind GTP, confirming that the isolated cDNA indeed codes for a small GTP-binding protein . Expression analysis by RT-PCR demonstrated that MS- rab11f is preferentially expressed in root nodules of alfalfa . Using the cDNA-sequence of MS-rab11f, a peptide-specific antibody was generated . Western blot analysis with this antibody revealed that two Rab11F isoforms, designated MS-Rab11FA and MS-Rab11FB, are found in M . sativa root nodules.

Mol Genet Genomics, 2004 Aug, 272(1), 67 - 75 Epub 2004 Jun 23.
Analyses of cis -acting elements that affect the transposition of Mos1 mariner transposons in vivo; Pledger DW et al.; The left (5') inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3') ITR . The effects on the transposition frequency resulting from the use of two 3' ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti . Donor constructs that utilized two 3' ITRs transposed with greater frequency in E . coli than did donor constructs with the wild-type ITR configuration . The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor . However, the lack of these internal sequences in a donor construct that utilized two 3' ITRs resulted in a further increase in transposition frequency . Conversely, the use of a donor construct with two 3' ITRs did not result in a significant increase in transposition in Ae . aegypti . Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito . The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR . The results also indicate that host factors which are absent in E . coli, influence Mos1 transposition in Ae . aegypti.

Eur Biophys J . 2004 Jun 25; {Epub ahead of print}
The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein; Schroeder C et al.; The influenza-virus M2 protein has proton channel activity required for virus uncoating and maturation of hemagglutinin (HA) through low-pH compartments . The proton channel is cytotoxic in heterologous expression systems and can be blocked with rimantadine . In an independent, rimantadine-resistant function, M2, interacting with the M1 protein, controls the shape of virus particles . These bud from cholesterol-rich membrane rafts where viral glycoproteins and matrix (M1)/RNP complexes assemble . We demonstrate that M2 preparations from influenza virus-infected cells and from a baculovirus expression system contain 0.5-0.9 molecules of cholesterol per monomer . Sequence analyses of the membrane-proximal M2 endodomain reveal interfacial hydrophobicity, a cholesterol-binding motif first identified in peripheral benzodiazepine receptor and human immunodeficiency virus gp41, and an overlapping phosphatidylinositol 4,5-bisphosphate-binding motif . M2 induced rimantadine-reversible cytotoxicity in intrinsically cholesterol-free E . coli, and purified E . coli-expressed M2 functionally reconstituted into cholesterol-free liposomes supported rimantadine-sensitive proton translocation . Therefore, cholesterol was nonessential for M2 ion-channel function and cytotoxicity and for the effect of rimantadine . Only about 5-8% of both M2 preparations, regardless of cholesterol content, associated with detergent-resistant membranes . Cholesterol affinity and palmitoylation, in combination with a short transmembrane segment suggest M2 is a peripheral raft protein . Preference for the raft/non-raft interface may determine colocalization with HA during apical transport, the low level of M2 incorporated into the viral envelope and its undisclosed role in virus budding for which a model is presented . M2 may promote clustering and merger of rafts and the pinching-off (fission) of virus particles.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 84 - 96 Epub 2004 Jun 04.
Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements; Kabir MM et al.; An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle . Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate . The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP(+) ratio compared with those in the parent strain . However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E . coli . This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP(+)-dependent malic enzymes . The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E . coli . The appearance of the glyoxylate pathway caused lower acetate production . Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry . The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E . coli compared to the parent.

Appl Microbiol Biotechnol, 2004 Oct, 65(5), 576 - 82 Epub 2004 Jun 24.
Identification and characterization of the main beta-alanine uptake system in Escherichia coli; Schneider F et al.; In Escherichia coli, beta-alanine is a direct precursor in the biosynthesis of pantothenic acid (vitamin B5) . Although a sufficient beta-alanine supply is crucial for biotechnological vitamin B5 production, nothing was known about beta-alanine transport in E . coli until now . The aim of this work was the characterization of beta-alanine transport by E . coli and the identification and overexpression of the corresponding carrier-encoding gene for the rational improvement of pantothenic acid-producing strains . beta-Alanine uptake was found to be an active process catalyzed by the amino acid carrier CycA . The corresponding gene was cloned and overexpressed, resulting in an increase in the uptake rate, compared with the wild type . In all tested strains, this overexpression led to a strong sensitivity to beta-alanine, but not to the other CycA substrates, such as L-alanine, D-alanine, and glycine . This prevented a direct application for the improvement of pantothenic acid-producing strains by an enhanced precursor supply.

Arch Microbiol, 2004 Sep, 182(1), 1 - 6 Epub 2004 Jun 24.
Lipoprotein trafficking in Escherichia coli; Narita S et al.; Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys . Lipoproteins are involved in a wide variety of functions in bacterial envelopes . Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane . In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken . Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE) . The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined . On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins . We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.

Nat Struct Mol Biol, 2004 Aug, 11(8), 714 - 20 Epub 2004 Jun 27.
DNA binding and nucleotide flipping by the human DNA repair protein AGT; Daniels DS et al.; O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines . AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway . AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials . We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine . The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding . This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2 . Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

Methods Mol Biol, 2004, 281, 153 - 62
Assaying Cdc25 phosphatase activity; Hassepass I et al.; To determine the activity of all human Cdc25 phosphatases, two different methods are described . For assaying phosphatase activities of recombinant Cdc25 proteins produced in Escherichia coli or insect cells, a fluorimetric assay using fluorescein diphosphate (FDP) as a substrate is recommended . To analyze endogenous Cdc25 phosphatase activities of immunoprecipitates from total cellular extracts, the physiological substrate Cdk1/cyclin B1 is most sensitive.

Proc Natl Acad Sci U S A, 2004 Jul 6, 101(27), 10018 - 23 Epub 2004 Jun 25.
Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways; Segatori L et al.; In the Escherichia coli periplasm, the formation of protein disulfide bonds is catalyzed by DsbA and DsbC . DsbA is a monomer that is maintained in a fully oxidized state by the membrane enzyme DsbB, whereas DsbC is a dimer that is kept reduced by a second membrane protein, DsbD . Although the catalytic regions of DsbA and DsbC are composed of structurally homologous thioredoxin motif domains, DsbA serves only as an oxidase in vivo, whereas DsbC catalyzes disulfide reduction and isomerization and also exhibits significant chaperone activity . To reconcile the distinct catalytic activities of DsbC and DsbA, we constructed a series of chimeras comprising of the dimerization domain of DsbC, with or without the adjacent alpha-helical linker region, fused either to the first, second, third, or fifth residue of intact DsbA or to thioredoxin . The chimeras fully substituted for DsbC in disulfide-bond rearrangement and also were able to restore protein oxidation in a dsbA background . Remarkably, the chimeras could serve as a single catalyst for both disulfide-bond formation and rearrangement, thus reconciling the kinetically competing DsbB-DsbA and DsbD-DsbC pathways . This property appeared to depend on the orientation of the DsbA active-site cysteines with respect to the DsbC dimerization domain . In vitro, the chimeras had high chaperone activity and significant reductase activity but only 15-22% of the disulfide-isomerization activity of DsbC, suggesting that rearrangement of nonnative disulfides may be mediated primarily by cycles of random reduction and reoxidation.

J Virol, 2004 Jul, 78(14), 7610 - 8
Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice; Guebre-Xabier M et al.; Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination . We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines . We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice . LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses . The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone . These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination . Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.

J Virol, 2004 Jul, 78(14), 7352 - 9
Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061; Thibeault D et al.; Hepatitis C virus (HCV) displays a high degree of genetic variability . Six genotypes and more than 50 subtypes have been identified to date . In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity . In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM) . To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues . The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a . BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM . This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.

J Biol Chem, 2004 Sep 3, 279(36), 37445 - 51 Epub 2004 Jun 25.
Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam; Zhou Y et al.; Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases . To create the active form of the drug, Sb(V) must be reduced to Sb(III) . Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase . The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major . LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae . Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes . LmACR2 was purified and shown to reduce both As(V) and Sb(V) . This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania . We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.

J Inorg Biochem, 2004 Jul, 98(7), 1194 - 9
Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7; Oku Y et al.; Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity . P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution . The final R-factor was 0.194 (Rfree=0.235) . Structural comparison with cytochrome P450 from S . solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron . Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated . A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.

Biochem Biophys Res Commun, 2004 Jul 23, 320(2), 366 - 71
Optimized self-excising Cre-expression cassette for mammalian cells; Mahonen AJ et al.; We observed that overexpression of Cre recombinase in 293 T cells has toxic effects and that the chicken beta-actin promoter is active in Escherichia coli, causing expression of Cre in bacteria . This led to significant problems in the cloning of Cre/loxP constructs . Leaky Cre-expression in E . coli, and toxicity of the Cre overexpression in mammalian cells, were solved by constructing a novel silent self-inactivating Cre (SSi-Cre) expression cassette . The SSi-Cre is based on modified loxP sites flanking the Cre/Int/DsRed fusion gene containing a Cre coding sequence interrupted by an intron, which prevents leaky expression of Cre in E . coli . Additionally, this system contains a reporter gene to visualize Cre activity by fluorescent microscopy . The SSi-Cre cassette provides a universal strategy for the generation of Cre/loxP constructs, as well as a solution to the toxicity caused by the overexpression of Cre in target cells . SSi-Cre should thus provide a useful tool for various applications based on the Cre/loxP system.

Biochem Biophys Res Commun, 2004 Jul 23, 320(2), 354 - 8
The ribosome-associated inhibitor A reduces translation errors; Agafonov DE et al.; Recently we have reported about a novel stress response protein (pY or RaiA) associated with Escherichia coli ribosomes that inhibits translation at the aminoacyl-tRNA binding stage . Here we show that leucine misincorporation during in vitro poly(U) translation is inhibited by this protein much stronger than the incorporation of phenylalanine . The miscoding counteraction by RaiA is especially strong at the concentrations of magnesium ions close to those observed in vivo and diminishes at higher magnesium concentrations . The results obtained suggest that the anti-miscoding activity of RaiA could be the main function of the protein, rather than the inhibition of translation . The role of the protein in adaptation of cells to environmental stress is discussed.

Science, 2004 Jun 25, 304(5679), 1967 - 71
Computational design of a biologically active enzyme; Dwyer MA et al.; Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications . Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure . We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity . The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions . The inherent generality of the design method suggests that many enzymes can be designed by this approach.

J Biol Chem, 2004 Aug 27, 279(35), 37013 - 20 Epub 2004 Jun 24.
Identification of a nuclear localization signal in OCT4 and generation of a dominant negative mutant by its ablation; Pan G et al.; OCT4 plays a critical role in maintaining stem cell pluripotency in a dose-dependent manner by activating and repressing multiple downstream genes . The precise mechanism by which OCT4 achieves these diverse biological functions remains unknown . In this report, we identify and characterize (195)RKRKR as a nuclear localization signal responsible for its localization in the nuclei and required for the transactivation of its target genes . Point mutations within this motif yielded a mutant that localizes randomly throughout the cells and is defective in transactivating target genes . However, restoration of nuclear localization with a heterologous nuclear localization signal failed to rescue its transactivation function, suggesting that this (195)RKRKR motif has additional function in mediating transactivation function . We further demonstrate that this mutant is competent in dimerization with not only itself but also wild type OCT4 and can interfere with the activity of wild type OCT4, thus acting as a dominant negative mutant . Indeed, this mutant can induce the differentiation of P19 cells into trophoblast-like giant cells . These data suggest that this dominant negative form of OCT4 may be a useful tool for modulating the activity of OCT4 in pluripotent cells such as embryonic stem cells to achieve the desired cell types for therapeutic applications.

Adv Biochem Eng Biotechnol, 2004, 89, 163 - 95
Analysis and control of proteolysis of recombinant proteins in Escherichia coli; Rozkov A et al.; Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli . Important properties of E . coli proteases, which are relevant for the production of recombinant proteins, are reviewed . Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented . These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing . However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.

Adv Biochem Eng Biotechnol, 2004, 89, 143 - 61
Roles of heat-shock chaperones in the production of recombinant proteins in Escherichia coli; Hoffmann F et al.; Escherichia coli is a versatile organism for the production of recombinant proteins . Often, however, the recombinant protein does not reach its native, biologically active conformation within the bacterial cell but deposits as inclusion bodies . The heat-shock chaperones, a group of polypeptides omnipresent in all kingdoms of life, form a network to assist proper folding of cellular proteins, prevent their deposition and can even dissolve deposits of misfolded proteins formed during environmental stress conditions such as excessive heat . Coproduction of individual chaperones with the target protein can also reduce deposition of the recombinant protein into inclusion bodies . The selection of the suitable chaperone(s), however, is still a trial-and-error process . The wrong chaperone(s) will not lead to success, or may even negatively effect product stability or host viability . Recent progress in understanding the mechanisms and substrate specificities of the major chaperones and their roles in the chaperone network now gives some hints for a more rational choice of chaperone(s) for coproduction . Also, more specialized chaperone systems may become an alternative for application in the production of recombinant proteins.

Adv Biochem Eng Biotechnol, 2004, 89, 73 - 92
Stress induced by recombinant protein production in Escherichia coli; Hoffmann F et al.; Strong production of recombinant proteins interferes with cellular processes in many ways . Drainage of precursors and energy urges the cell to readjust metabolic fluxes and enzyme composition, stress responses are induced, and hence the cellular activity is shifted from growth to reorganisation of biomass . This may result in inhibition of growth or low level of product accumulation . The extent of the bacterial stress response is determined by the specific properties of the recombinant protein, and by the rates of transcription and translation . Taking into account the capacities of the host for protein processing and physiological adaptation, production schemes can be developed that enhance volumetric productivity and sustainability of the process.

Cell Struct Funct, 1999 Oct, 24(5), 359 - 64
Inhibition of microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein; Itoh TJ et al.; HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE . The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane . When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons . These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neurotransmitter release . Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton . HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system . Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-deltaTM), which was overexpressed in E . coli . Affinity column chromatography showed that tubulin was found to bind HPC-1 directly . Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding . In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule . Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization . Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1 . After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected . When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed . These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.

J Membr Biol, 2004 Apr 1, 198(3), 135 - 46
Control of H+/lactose coupling by ionic interactions in the lactose permease of Escherichia coli; Johnson JL et al.; A combinatorial approach was used to study putative interactions among six ionizable residues (Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325) in the lactose permease . Neutral mutations were made involving five ion pairs that had not been previously studied . Double mutants, R302L/E325Q and D240N/H322Q, had moderate levels of downhill {(14)C}-lactose transport . Mutants in which only one of these six residues was left unchanged (pentuple mutants) were also made . A Pent269(-) mutant (in which only Glu-269 remains) catalyzed a moderate level of downhill lactose transport . Pent240(-) and Pent 322(+) also showed low levels of downhill lactose transport . Additionally, a Pent240(-) mutant exhibited proton transport upon addition of melibiose, but not lactose . This striking result demonstrates that neutralization of up to five residues of the lactose permease does not abolish proton transport . A mutant with neutral replacements at six ionic residues (hextuple mutant) had low levels of downhill lactose transport, but no uphill accumulation or proton transport . Since none of the mutants in this study catalyzes active accumulation of lactose, this is consistent with other reports that have shown that each residue is essential for proper coupling . Nevertheless, none of the six ionizable residues is individually required for substrate-induced proton cotransport . These results suggest that the H(+) binding domain may be elsewhere in the permease or that cation binding may involve a flexible network of charged residues.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1388 - 92
Examining the bases of the J3/4 domain of Escherichia coli ribonuclease P; Tanaka T et al.; We prepared several mutants of the J3/4 and P4 domains of Escherichia coli ribonuclease P (RNase P): A62G, A62U, G63C/G64C, A65G, A67G, U69A, U69G, U69C, U69Delta, and U69UU . Comparison of the ribozyme and holo enzyme reactions at various concentrations of magnesium ions showed that the presence of a bulge at U69 in the P4 domain was important in the holo enzyme . The results also showed that the conserved bases G63 and G64 in the J3/4 domain were important for efficient ribozyme reactions but were replaceable in the presence of the protein component . Our data showed that the bases in the J3/4 and P4 domains displayed different responses to the metal ions that were affected by the presence of the protein component.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1306 - 14
Molecular characterization of an intracellular beta-N-acetylglucosaminidase involved in the chitin degradation system of Streptomyces thermoviolaceus OPC-520; Kubota T et al.; We purified and characterized an intracellular beta-N-acetylglucosaminidase (NagC) from a cytoplasmic fraction of Streptomyces thermoviolaceus OPC-520 . The molecular mass of NagC was estimated to be 60 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . The optimum pH and temperature of the enzyme were 6.0 and 50 degrees C respectively . Purified NagC hydrolyzed chitin oligosaccharides from N,N'-diacetylchitobiose (GlcNAc)(2) to chitopentaose (GlcNAc)(5), hydrolyzed N,N'-diacetylchitobiose especially rapidly, and showed a tendency to decrease with increases in the degree of polymerization . But, NagC didn't hydrolyze chitohexaose (GlcNAc)(6) . The gene encoding NagC was cloned and sequenced . The open reading frame of nagC encoded a protein of 564 amino acids with a calculated molecular mass of 62,076 Da . The deduced amino acid sequence of NagC showed homology with several beta-N-acetylglucosaminidases belonging to glycosyl hydrolase family 20 . The expression plasmid coding for NagC was constructed in Escherichia coli . The recombinant enzyme showed pH and temperature optima and substrate specificity similar to those of the native enzyme . The gene arrangement near the nagC gene of S . thermoviolaceus OPC-520 was compared with that of S . coelicolor A3(2) . Three genes, which appear to constitute an ABC transport system for sugar, were missing in the vicinity of the nagC gene.

Protein Sci, 2004 Jul, 13(7), 1811 - 22
Trapping a 96 degrees domain rotation in two distinct conformations by engineered disulfide bridges; Schultz-Heienbrok R et al.; Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state . We have designed two double mutants of Escherichia coli 5'-nucleotidase to trap the enzyme in both an open (S228C, P513C) and a closed (P90C, L424C) conformation by the formation of disulfide bridges . The mutant proteins have been expressed, purified, and crystallized, to structurally characterize the designed variants . The S228C, P513C is a double mutant crystallized in two different crystal forms with three independent conformers, which differ from each other by a rotation of up to 12 degrees of the C-terminal domain with respect to the N-terminal domain . This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits considerable residual domain flexibility . In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96 degrees rotation trajectory between the open and closed enzyme forms . A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface.

Plant Cell Physiol, 2004 Jun, 45(6), 723 - 33
A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor; Lindemann P et al.; A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import . PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin . The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism . Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein . A PBR-homologous Arabidopsis sequence was cloned and expressed in E . coli . The recombinant gene product showed specific high affinity benzodiazepine ligand binding . Moreover, the protein applied to E . coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX . These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.

Plant Cell Physiol, 2004 Jun, 45(6), 659 - 66
A novel Arabidopsis gene required for ethanol tolerance is conserved among plants and archaea; Fujishige N et al.; A novel ethanol-hypersensitive mutant, gek1, of Arabidopsis shows 10-100 times greater sensitivity to ethanol compared to the wild type, while it grows normally in the absence of ethanol, and responds normally to other alcohols and to environmental stresses such as heat shock and high salinity . Mapping of the gek1 locus indicated it is a previously unreported locus . In order to address the GEK1 function, we identified the GEK1 gene by means of map-based cloning . The GEK1 gene encodes a novel protein without any known functional motifs . Transgenic Arabidopsis plants overexpressing GEK1 displayed an enhanced tolerance to ethanol and acetaldehyde, suggesting that GEK1 is directly involved in the tolerance to those chemicals . By contrast, expression of GEK1 in E . coli and yeasts did not increase their tolerance to ethanol or acetaldehyde . Interestingly, a similarity search revealed that GEK1-related genes are conserved only in plants and archaea . These results might suggest that plants, and presumably archaea, have a novel mechanism for protection from acetaldehyde toxicity.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W606 - 9
CaspR: a web server for automated molecular replacement using homology modelling; Claude JB et al.; Molecular replacement (MR) is the method of choice for X-ray crystallography structure determination when structural homologues are available in the Protein Data Bank (PDB) . Although the success rate of MR decreases sharply when the sequence similarity between template and target proteins drops below 35% identical residues, it has been found that screening for MR solutions with a large number of different homology models may still produce a suitable solution where the original template failed . Here we present the web tool CaspR, implementing such a strategy in an automated manner . On input of experimental diffraction data, of the corresponding target sequence and of one or several potential templates, CaspR executes an optimized molecular replacement procedure using a combination of well-established stand-alone software tools . The protocol of model building and screening begins with the generation of multiple structure-sequence alignments produced with T-COFFEE, followed by homology model building using MODELLER, molecular replacement with AMoRe and model refinement based on CNS . As a result, CaspR provides a progress report in the form of hierarchically organized summary sheets that describe the different stages of the computation with an increasing level of detail . For the 10 highest-scoring potential solutions, pre-refined structures are made available for download in PDB format . Results already obtained with CaspR and reported on the web server suggest that such a strategy significantly increases the fraction of protein structures which may be solved by MR . Moreover, even in situations where standard MR yields a solution, pre-refined homology models produced by CaspR significantly reduce the time-consuming refinement process . We expect this automated procedure to have a significant impact on the throughput of large-scale structural genomics projects . CaspR is freely available at http://igs-server.cnrs-mrs.fr/Caspr/.

Nucleic Acids Res, 2004 Jun 23, 32(11), 3354 - 63 Print 2004.
Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs; Moll I et al.; It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits . In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon . In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs . In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes . Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases . These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.

J Biol Chem, 2004 Sep 17, 279(38), 39207 - 13 Epub 2004 Jun 23.
Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I; Cheng B et al.; Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R . J., and Mondragon, A . (2001) Nature 411, 1077-1081; Perry, K., and Mondragon, A . (2003) Structure 11, 1349-1358) . Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme . The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage . Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.

Development, 2004 Aug, 131(15), 3571 - 80 Epub 2004 Jun 23.
Mechanisms of HP1-mediated gene silencing in Drosophila; Danzer JR et al.; Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres . Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner . Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes . HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9 . The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber . To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E . coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats . Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin . Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes . To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated . Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.

Am J Pathol, 2004 Jul, 165(1), 331 - 40
The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia; Sharma L et al.; Tissue factor (TF) is an integral membrane protein that binds factor VIIa and initiates coagulation . The extracellular domain of TF is responsible for its hemostatic function and by implication in the dysregulation of coagulation, which contributes to death in endotoxemia . The role of the cytoplasmic domain of tissue factor in endotoxemia was studied in mice, which lack the cytoplasmic domain of TF (TF(deltaCT/deltaCT)) . These mice develop normally and have normal coagulant function . Following i.p injection with 0.5 mg of lipopolysaccharide (LPS), TF(deltaCT/deltaCT) mice showed significantly greater survival at 24 hours compared to the wt mice (TF(+/+)) . The serum levels of TNF-alpha and IL-1beta were significantly lower at 1 hour after LPS injection and IL-6 levels were significantly lower at 24 hours in TF(deltaCT/deltaCT) mice compared to TF(+/+)mice . Neutrophil recruitment into the lung was also significantly reduced in TF(deltaCT/deltaCT) mice . Nuclear extracts from tissues of endotoxemic TF(deltaCT/deltaCT) mice also showed reduced NFkappaB activation . LPS induced leukocyte rolling, adhesion, and transmigration in post-capillary venules assessed by intravital microscopy was also significantly reduced in TF(deltaCT/deltaCT) mice . These results indicate that deletion of the cytoplasmic domain of TF impairs the recruitment and activation of leukocytes and increases survival following endotoxin challenge.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2604 - 9
Absorption of ester prodrugs in Caco-2 and rat intestine models; He X et al.; The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs . Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs . The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when p-nitrophenyl acetate was used as a substrate . Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer . When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation . On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed . The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract . The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model . It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium . Broader evaluation of such a system is warranted.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2437 - 47
Model system for high-throughput screening of novel human immunodeficiency virus protease inhibitors in Escherichia coli; Cheng TJ et al.; Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS . To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system . This E . coli-based system involves coexpression of an engineered beta-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain . Autoprocessing of the HIV protease precursor releases the mature HIV protease . Subsequently, the HIV protease cleaves beta-galactosidase, resulting in a loss of the beta-galactosidase activity, which can be detected in high-throughput screens . Using Food and Drug Administration-approved HIV protease inhibitors, this E . coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity . The usefulness of the E . coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores . A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E . coli-based system . This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains . This simple yet efficient E . coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.

J Vet Med A Physiol Pathol Clin Med, 2004 Apr, 51(3), 106 - 12
Effect of Escherichia coli heat-labile enterotoxin on the myoelectric activity of the duodenum in weaned pigs; Yao G et al.; The objective of the present study was to elucidate the effect of subclinical doses of Escherichia coli heat-labile enterotoxin (LT) on the antro-duodenal myoelectric activities of weaned pigs . Twelve weaned pigs were surgically implanted with three pairs of electrodes on the antrum 3 cm before the pylorus, 5 and 20 cm after the pylorus on the duodenum, respectively . An infusion cannula was inserted into the duodenum between duodenal electrodes . Using a wireless telemetry recording system, an electromyography (EMG) tracing lasting at least 24 h was recorded as the control, then another 24-h EMG recording was performed with a bolus intraduodenal infusion of LT (0.1 and 0.5 microg/kg b.w.) . After a 1- to 2-day break, a 5-fold higher dose of LT was administered using the same protocol . In the antrum, LT administration barely modified the EMG signal . However, in the duodenum it prolonged the duration of phase II and the migrating myoelectric complex (MMC) cycle when compared with the control . The number of duodenal MMC cycles was also significantly diminished . Moreover, the migrating velocity of phase III was increased . The migrating action potential complex (MAPC) was present both without and with LT, but occurred more frequently following LT administration . In conclusion, LT caused a dose-dependent, lagged alteration in the duodenal MMC in weaned pigs, involving a reduction of the MMC number by lengthening phase II, increased phase III migration velocity, and increased MAPC frequency . The disturbances did not, however, result in diarrhoea and may reflect the induction of a local protection mechanism of the gut to expel unwanted foreign content from the lumen of the upper gut.

J Biomol Struct Dyn, 2004 Aug, 22(1), 13 - 23
Evolutionary forces in shaping the codon and amino acid usages in Blochmannia floridanus; Banerjee T et al.; Endosymbiotic relationship has great effect on ecological system . Codon and amino acid usages bias of endosymbiotic bacteria Blochmannia floridanus (whose host is an ant Camponotus floridanus) was investigated using experimentally known genes of this organism . Correspondence Analysis on RSCU values show that there exists only one single explanatory major axis that is linked to the strand specific mutational biases . Majority of the genes have a tendency to concentrate on the leading strand, which may be related to the adaptive property related to the replication mechanisms . Amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes . Comparative analyses of the orthologous genes of Escherichia coli and Blochmannia floridanus show that highly expressed genes are significantly more conserved than lowly expressed genes . Based on our results we concluded that strand specific mutational bias is strongly operational in selecting the codon usage in this organism . Replicational-transcriptional selection can be invoked from the presence of majority of highly expressed genes in the leading strand . Conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism . Hydrophobicity of the genes is the second major source in differentiating the genes according to their amino acid usages in this organism .

Protein J, 2004 May, 23(4), 239 - 45
Functional characterization of the purified holo form of hemoglobin I from Lucina pectinata overexpressed in Escherichia coli; Collazo E et al.; The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico . It contains three different hemoglobins . Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state . Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA) . Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography . Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively . The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin . Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI . The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively . The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier . This effect is attributed mostly to the first of two missense mutations {Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101} and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations . The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics . A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.

Thromb Haemost, 2004 Jul, 92(1), 47 - 53
Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide; Hagemeyer CE et al.; We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)) . Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target . In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa . Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor . Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule.Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity . The recombinant fusion did not destroy the function of the fusion partners . Antibody binding function was on a par with the parent molecule . TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency . In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all . In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition . The production in E . coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

Ann Surg, 2004 Jul, 240(1), 142 - 50
Tissue- and time-dependent upregulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome; Volman TJ et al.; OBJECTIVE: We sought to quantitate the course of specific cytokine mRNA expression in tissues that exhibit increasing histopathological changes in time in an animal model for the multiple organ dysfunction syndrome (MODS) . SUMMARY BACKGROUND DATA: The development of treatment protocols for MODS requires elucidation of the mechanisms and mediators involved . To devise logical interventions, it is necessary to collect data on cytokine expression at tissue level during the development of MODS . METHODS: Ninety-four C57BL/6 mice were given an intraperitoneal injection of 40 microg of lipopolysaccharide (LPS), followed by zymosan at a dose of 0.8 mg/g body weight 6 days later (day 0) . Six additional animals did not receive zymosan and acted as controls . At several time points after zymosan injection, 6 randomly assigned, zymosan-treated animals were killed, and their livers, lungs, spleens, and kidneys were collected . mRNA expression of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, macrophage migration inhibiting factor, IL-12, interferon-gamma, and IL-10 was measured using a real-time reverse transcription-polymerase chain reaction assay . RESULTS: The injection of zymosan induced an acute peritonitis, followed by an apparent recovery . From approximately day 6 onwards, animals started to display MODS-like symptoms . During the peritonitis phase, up-regulation of cytokine mRNA was limited . During the period of apparent recovery, cytokine mRNA expression strongly increased, mostly reaching its maximum at day 9 when deterioration of the clinical condition had already set in . The up-regulation of tumor necrosis factor-alpha mRNA was most pronounced, especially in the lungs and liver . CONCLUSIONS: Interventions should preferentially be targeted against multiple cytokines and, at least in this model, there may be a treatment window well after the initial challenge.

J Biomol NMR, 2004 Jul, 29(3), 351 - 61
Site-specific labelling with a metal chelator for protein-structure refinement; Pintacuda G et al.; A single free Cys sidechain in the N-terminal domain of the E . coli arginine repressor was covalently derivatized with S-cysteaminyl-EDTA for site-specific attachment of paramagnetic metal ions . The effects of chelated metal ions were monitored with (15)N-HSQC spectra . Complexation of Co(2+), which has a fast relaxing electron spin, resulted in significant pseudocontact shifts, but also in peak doubling which was attributed to the possibility of forming two different stereoisomers of the EDTA-Co(2+) complex . In contrast, complexation of Cu(2+) or Mn(2+), which have slowly relaxing electron spins, did not produce chemical shift changes and yielded self-consistent sets of paramagnetic relaxation enhancements of the amide protons . T (1) relaxation enhancements with Cu(2+) combined with T (2) relaxation enhancements with Mn(2+) are shown to provide accurate distance restraints ranging from 9 to 25 A . These long-range distance restraints can be used for structural studies inaccessible to NOEs . As an example, the structure of a solvent-exposed loop in the N-terminal domain of the E . coli arginine repressor was refined by paramagnetic restraints . Electronic correlation times of Cu(2+) and Mn(2+) were determined from a comparison of T (1) and T (2) relaxation enhancements.

J Biomol NMR, 2004 Jul, 29(3), 289 - 97
Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background; Fiaux J et al.; A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E . coli GroE chaperone proteins by solution NMR . In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling . The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa . The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns . Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1319 - 22 Epub 2004 Jun 22.
Crystallization and preliminary X-ray diffraction study of mammalian mitochondrial seryl-tRNA synthetase; Chimnaronk S et al.; The mitochondrial seryl-tRNA synthetase (mt SerRS) from Bos taurus was overexpressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion method . Crystals grew in a very narrow range of conditions using PEG 8000 as precipitant at room temperature . An appropriate concentration of lithium sulfate was critical for crystal nucleation . Crystals diffracted well beyond a resolution of 1.6 A and were found to belong to the orthorhombic space group C222(1), with unit-cell parameters a = 79.89, b = 230.42, c = 135.60 A . There is one dimer (M(r) approximately 113 kDa) in the asymmetric unit, with a solvent content of 55% . Efforts to solve the phase problem by molecular replacement are under way.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1301 - 3 Epub 2004 Jun 22.
Expression, purification and crystallization of a functional core of the voltage-dependent calcium channel beta subunit; Opatowsky Y et al.; Two versions of the functional core of the rabbit voltage-dependent calcium channel beta2a subunit were expressed in Escherichia coli . These proteins were purified to homogeneity and screened for crystallization . Crystallization conditions were refined using the hanging-drop vapour-diffusion method and two crystal forms were pursued . Crystal form I is represented by thick rods with tetragonal symmetry, unit-cell parameters a = b = 75, c = 165 A and a diffraction limit of 3.4 A which were obtained using ammonium sulfate as a precipitant . Crystal form II gives rise to plates with orthorhombic symmetry, unit-cell parameters a = 35, b = 75, c = 165 A and a diffraction limit of 2.3 A which were grown using polyethylene glycol 20K as a precipitant.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1292 - 4 Epub 2004 Jun 22.
Protein preparation, crystallization and preliminary X-ray analysis of human adrenal gland protein AD-004; Ren H et al.; The adrenal gland protein AD-004 was identified in the human adrenal gland . Full-length AD-004 contains 172 amino acids, with a predicted molecular weight of about 20 kDa . In attempts to crystallize human AD-004, the gene was subcloned into a modified pET vector, pET21-DEST, with an N-terminal His(5) tag using the Gateway cloning system, followed by protein expression in Escherichia coli strain BL21(DE3) . The protein was purified in two steps to near-homogeneity and was crystallized . The crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 99.56, c = 57.19 A . A complete 2.0 A data set has been collected at a rotating-anode X-ray source and structure determination is under way.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1284 - 5 Epub 2004 Jun 22.
Crystallization and preliminary X-ray analysis of Escherichia coli K12 YgjK protein, a member of glycosyl hydrolase family 63; Tonozuka T et al.; Processing alpha-glucosidase I, which is classified into glycosyl hydrolase (GH) family 63, hydrolyzes an oligosaccharide precursor of eukaryotic N-linked glycoproteins . Recently, many bacteria have been reported to possess genes for proteins that are homologous to the GH family 63 glucosidases . In this paper, Escherichia coli K12 YgjK protein, a member of GH family 63, was overexpressed, purified and crystallized using the vapour-diffusion method . Diffraction data were collected to 1.8 A resolution and the crystal was found to belong to the monoclinic space group P2(1), with unit-cell parameters a = 88.5, b = 137.1, c = 60.9 A, beta = 98.1 degrees . The V(M) value was determined to be 2.1 A(3) Da(-1), which corresponds to the presence of two protein molecules in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1272 - 4 Epub 2004 Jun 22.
SOS3 (salt overly sensitive 3) from Arabidopsis thaliana: expression, purification, crystallization and preliminary X-ray analysis; Sanchez-Barrena MJ et al.; The salt-tolerance gene SOS3 (salt overly sensitive 3) of Arabidopsis thaliana encodes a calcium-binding protein that is able to sense the cytosolic calcium signal elicited by salt stress . SOS3 activates the SOS2 protein kinase, which activates various ion transporters . SOS3 was cloned into a plasmid and expressed in Escherichia coli, allowing purification of the protein to homogeneity . Two crystals with different additive contents were grown . Both diffract to 3.2 A resolution and belong to space group I4(1), with unit-cell parameters a = 93.65, c = 80.08 A and a = 91.79, c = 85.78 A, respectively . A promising molecular-replacement solution has been found using neuronal calcium-sensor 1 as the search model . Interestingly, no solution was found using AtCBL2 (A . thaliana calcineurin B-like protein) structure as a search model, although this protein belongs to the same family and displays 50% sequence identity.

J Physiol Pharmacol, 2004 Jun, 55(2), 409 - 21
Involvement of platelet activating factor in immediate heart response to lipopolysaccharide; Jakubowski A et al.; Although lipopolysaccharide (LPS) is recognized to induce a biphasic cardiovascular response its mechanism is not fully elucidated . In this study we analysed the involvement of PAF, TXA(2) and cysteinyl leukotrienes (cysLTs) in the acute cardiovascular effects of LPS in the isolated rat heart as well as in delayed phase of LPS response using a surrogate cellular model of the induction of NOS-2 by LPS in mouse macrophages . Perfusion of rat hearts with LPS resulted, in an immediate fall in heart contractility and coronary flow by 2.5 +/- 0.59 ml x min(-1) and 560 +/- 81 mmHg x sec(-1), respectively . This response was fully blocked by platelet activating factor (PAF) antagonist - WEB 2170 and partially inhibited, by inhibitor of cyclooxygenase (indomethacin) or by inhibitor of thromboxane synthase (camonagrel) . The inhibition of leukotriene synthesis (BAY x1005) or cysLTs receptors (BAY x7195) was without effect . Administration of stable PAF analog (methylcarbamyl-PAF - MC-PAF) alone, mimicked heart response to LPS . In cultured mouse macrophages, MC-PAF did not induce NOS-2 expression and when given with LPS it slightly potentiated NOS-2 induction by LPS . However, in presence of WEB 2170 NOS-2 induction by LPS was inhibited in a dose-dependent manner . Inhibition of cyclooxygenase and leukotriene pathways had no effect on NOS-2 induced by LPS . These results indicate that PAF and TXA(2) but not cysLTs mediate the instant heart response induced by LPS, while PAF alone mediates a delayed NOS-2 induction by LPS . Accordingly, PAF may constitute the mediator that links acute and delayed phases of LPS-induced cardiovascular response.

J Biochem (Tokyo), 2004 Jun, 135(6), 663 - 71
Mass spectrometry on hydrogen/deuterium exchange of dihydrofolate reductase: effects of ligand binding; Yamamoto T et al.; To address the effects of ligand binding on the structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the hydrogen/deuterium (H/D) exchange kinetics of its binary and ternary complexes formed with various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP(+), and methotrexate) were examined using electrospray ionization mass spectrometry . The kinetic parameters of H/D exchange reactions, which consisted of two phases with fast and slow rates, were sensitively influenced by ligand binding, indicating that changes in the structural fluctuation of the DHFR molecule are associated with the alternating binding and release of the cofactor and substrate . No additivity was observed in the kinetic parameters between a ternary complex and its constitutive binary complexes, indicating that ligand binding cooperatively affects the structural fluctuation of the DHFR molecule via long-range interactions . The local H/D exchange profile of pepsin digestion fragments was determined by matrix-assisted laser desorption/ionization mass spectrometry, and the helix and loop regions that appear to participate in substrate binding, largely fluctuating in the apo-form, are dominantly influenced by ligand binding . These results demonstrate that the structural fluctuation of kinetic intermediates plays an important role in enzyme function, and that mass spectrometry on H/D exchange coupled with ligand binding and protease digestion provide new insight into the structure-fluctuation-function relationship of enzymes.

J Biol Chem, 2004 Sep 3, 279(36), 37324 - 33 Epub 2004 Jun 22.
The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase; Morgan-Kiss RM et al.; We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources . This beta-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD . The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain . The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components . We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms) . The enzymatic mechanism of FadK is similar to other acyl-CoA synthetases in that it forms an acyl-AMP intermediate prior to the formation of the final acyl-CoA product . Expression of FadK is repressed during aerobic growth and is maximally expressed under anaerobic conditions in the presence of the terminal electron acceptor, fumarate.

Infect Immun, 2004 Jul, 72(7), 4072 - 80
Nasal delivery of antigen with the B subunit of Escherichia coli heat-labile enterotoxin augments antigen-specific T-cell clonal expansion and differentiation; Apostolaki M et al.; Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice . These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest . However, the inherent toxicity mediated by the A subunit precludes its widespread use . This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit . The ability of the B subunit of E . coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry . Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens . The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production . Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation . Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained . Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation . Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.

Infect Immun, 2004 Jul, 72(7), 3914 - 24
Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins; Berberov EM et al.; Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood . Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins . LT(-) (DeltaeltAB) and complemented mutants of an F4(+) LT(+) STb(+) EAST1(+) ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4(+) fimbria . Sixty percent of the piglets inoculated with the LT(-) mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal(r)) parent and 100% of those inoculated with the complemented mutant strain . Compared to piglets inoculated with the Nal(r) parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT(-) mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001) . Similarly, piglets inoculated with the LT(-) mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05) . These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

Infect Immun, 2004 Jul, 72(7), 3907 - 13
Identification of an I-Ed-restricted T-cell epitope of Escherichia coli outer membrane protein F; Williams KM et al.; A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2(d) mice . BALB/c-derived T-cell hybridomas generated against this region were CD3(+), CD4(+), CD8(-), and T-cell receptor alphabeta(+) and secreted TH-1-associated cytokines (interleukin-2 {IL-2} and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314 . Class II(+) mouse lymphoma (A20) cells, but not class II(-) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs) . Antibodies specific for I-E(d) blocked IL-2 secretion by hybridomas, but I-A(d)-specific antiserum did not . When transfected L cells expressing I-A(d) (AalphaAbeta(d)), I-E(d) (EalphaEbeta(d)), or the hybrid molecule I-EalphaAbeta(d) were used as APCs, hybridomas recognized peptide only when presented by the I-E(d)-transfected cells . When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined . Critical residues were determined by using alanine-substituted peptide analogues . T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion . Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302 . This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E(d) and variability in the amino acid composition of the C and N termini of these peptides.

Infect Immun, 2004 Jul, 72(7), 3733 - 42
Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion; Kansau I et al.; We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells . Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements . We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells) . Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the alpha5beta1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria . We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positive bacteria . We also observed that extracting membrane cholesterol with methyl-beta-cyclodextrin (MBCD) did not affect the recruitment of CD55, GM1, or beta1 integrin to adhering Dr-positive bacteria . In contrast, extracting or changing membrane-bound cholesterol by means of drugs that modify lipid rafts (MBCD, filipin III, or mevalonate plus lovastatin plus MBCD) inhibited the entry of Dr-positive IH11128 both into cells that expressed VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) and into those that did not (parental Caco-2 cells) . Finally, restoring cholesterol within the cell membrane of MBCD-treated cells restored Dr-positive IH11128 internalization.

Exp Cell Res, 2004 Jul 15, 297(2), 495 - 507
Morphological variation of individual Escherichia coli 30S ribosomal subunits in vitro and in situ, as revealed by cryo-electron tomography; Zhao Q et al.; Cryo-electron tomography has been used to reconstruct the structures of individual ribosomal 30S subunits in Escherichia coli cells treated with rifampicin . Rifampicin inhibits transcription initiation, thus giving depletion of mRNA and accumulation of free 30S and 50S subunits in the cell . Here, we present the 3D morphologies of reconstructed individual 30S ribosomal subunits both in vitro and in situ from E . coli . The head, the platform, and the body of the structures show large conformational movements relative to each other . The particles were grouped into three conformational groups according to the ratio between width and height in the subunit solvent side view . Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit . The results demonstrate a considerable morphological heterogeneity and structural variability among 30S ribosomal subunits.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 129 - 36
Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737; Bin Y et al.; A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737 . The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized . Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail . The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 109 - 14
Promoted proliferation of an SOD-deficient mutant of Escherichia coli under oxidative stress induced by photoexcited TiO2; Kim SY et al.; An SOD null mutant of Escherichia coli (IM303) and its wild-type strain (MM294) were cultivated with or without sublethal oxidative stress generated from photoexcited TiO2 . Concerning maximum specific growth rate of the cells, mum, measured under various conditions, the mum value of IM303 cells increased notably in the presence of TiO2 illuminated with light (I = 12.5 W m(-2)), being about two times higher than that of the cells grown in the absence of TiO2 and light . The mum value of IM303 cells under the oxidative condition restored to a level comparable to that of wild-type MM294 cells, which coincided with the finding that the content of reactive oxygen species lowered in IM303 cells under the oxidative stress . Colony isolation was conducted to obtain the cells prevailing in the early culture phase of IM303 cells in the presence of TiO2 and light . It was found that the isolates exhibited the outgrowing properties with the increased mum values under both the conditions with and without TiO2 and light . It was also indicated that in the culture of typically selected isolate, the cells started to grow with a relatively short lag in a threonine-minus medium.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 97 - 102
A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7; Suzuki Y et al.; We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7 . The ORF was cloned and expressed as a fusion protein in Escherichia coli . The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration . The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0 . The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide . From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 61 - 4
Transformation of colonial Escherichia coli on solid media; Maeda S et al.; We report that colonial Escherichia coli cells on various solid media can develop modest genetic competence . Using an on-filter culture system, we found that E . coli colonies on CaCl2-containing agar were transformed in the presence of plasmid DNA . Interestingly, transformation also occurred on LB-agar, various moist foods and even on H2O-agar . These results suggest that some populations of colonial E . coli in various environments could become transformable regardless of the surrounding Ca2+ concentration.

J Agric Food Chem, 2004 Jun 30, 52(13), 4192 - 6
Glutathione-dependent biotransformation of the fungicide chlorothalonil; Kim YM et al.; A gene responsible for the chlorothalonil biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, capable of efficiently dissipating the chlorothalonil . The gene encoding glutathione S-transferase (GST) of O . anthropi SH35B was expressed in Escherichia coli, and the GST was subsequently purified by affinity chromatography . The fungicide chlorothalonil was rapidly transformed by the GST in the presence of glutathione . LC-MS analysis supported the formation of mono-, di-, and triglutathione conjugates of chlorothalonil by the GST . The monoglutathione conjugate was observed as an intermediate in the enzymatic reaction . The triglutathione conjugate has not been previously reported and seems to be the final metabolite in the biotransformation of chlorothalonil . The glutathione-dependent biotransformation of chlorothalonil catalyzed by the bacterial GST is reported.

Proteins, 2004 Aug 1, 56(2), 376 - 83
YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays; Lehmann C et al.; The Escherichia coli open reading frame YbdK encodes a member of a large bacterial protein family of unknown biological function . The sequences within this family are remotely related to the sequence of gamma-glutamate-cysteine ligase (gamma-GCS), an enzyme in the glutathione biosynthetic pathway . A gene encoding gamma-GCS in E . coli is already known . The 2.15 A resolution crystal structure of YbdK reveals an overall fold similar to that of glutamine synthetase (GS), a nitrogen metabolism enzyme that ligates glutamate and ammonia to yield glutamine . GS and gamma-GCS perform related chemical reactions and require ATP and Mg2+ for their activity . The Mg2+-dependent binding of ATP to YbdK was confirmed by fluorescence spectroscopy employing 2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate, and yielding a dissociation constant of 3 +/- 0.5 microM . The structure of YbdK contains a crevice that corresponds to the binding sites of ATP, Mg2+ and glutamate in GS . Many of the GS residues that coordinate the metal ions and interact with glutamic acid and the phosphoryl and ribosyl groups of ATP are also present in YbdK . GS amino acids that have been associated with ammonia binding have no obvious counterparts in YbdK, consistent with a substrate specificity that is different from that of GS . Ligase activity between glutamic acid and each of the twenty amino acid residues was tested on high performance liquid chromatography (HPLC) by following the hydrolysis of ATP to ADP . Catalysis was observed only with cysteine . A pyruvate kinase/lactic acid dehydrogenase coupled assay was used to rule out GS activity and to determine that YbdK exhibits gamma-GCS activity . The catalytic rate was found to be approximately 500-fold slower than that reported for authentic gamma-GCS .

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9573 - 7 Epub 2004 Jun 21.
Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer; Hishida T et al.; The Escherichia coli RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks . The RuvB protein belongs to the AAA(+) (ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner . Studies on the oligomeric AAA(+) class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis . This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer . RuvBR174A shows a dominant negative phenotype for DNA repair in vivo and inhibits the branch migration catalyzed by wild-type RuvB . A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation) . RuvB K68A-R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A . Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio . These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers . This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB . This role may be a general feature of the oligomeric AAA/AAA(+) ATPases.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9612 - 7 Epub 2004 Jun 21.
A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly; Medintz IL et al.; The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed . It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials . As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core-shell QDs . Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD . In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface . Results indicate that MBP has a preferred orientation on the QD surface . The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size . The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein-nanoparticle materials . To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.

J Biol Chem, 2004 Aug 27, 279(35), 36876 - 83 Epub 2004 Jun 21.
The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases; Jones RJ et al.; Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains . This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail . Smaller sequence elements also appear to correlate with control . Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information . In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element . Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions . Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors . Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.

J Biol Chem, 2004 Aug 6, 279(32), 33047 - 50 Epub 2004 Jun 20.
Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli beta clamp with DNA polymerase IV; Bertram JG et al.; Replication forks often stall at undamaged or damaged template sites in Escherichia coli . Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V . The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities . DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E . coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo . Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends . Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress . We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.

J Biol Chem, 2004 Aug 6, 279(32), 33043 - 6 Epub 2004 Jun 20.
Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication; Meyer AS et al.; Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication . In particular, Arg668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus . We investigated the interaction between Arg668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP . Hydrogen bonds from Arg668 were probed with the site-specific mutant R668A . Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus . Hydrogen bonds from the incoming dNTP were probed with (1 'R,3 'R,4 'R)-1-{3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl}uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group . We found that the pre-steady-state parameter kpol was decreased 1,600 to 2,000-fold with each of the single substitutions . When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in kpol . These results are consistent with a hydrogen-bonding fork from Arg668 to the primer terminus and incoming dNTP . These interactions may play an important role in fidelity as well as catalysis of DNA replication.

J Biol Chem, 2004 Aug 27, 279(35), 36819 - 27 Epub 2004 Jun 20.
The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities; Yakunin AF et al.; In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25) . The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain . The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes . The C-terminal HD domain in E . coli tRNA nucleotidyltransferase demonstrated Ni(2+)-dependent phosphatase activity toward pyrophosphate, canonical 5'-nucleoside tri- and diphosphates, NADP, and 2'-AMP . Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2',3'-cAMP, -cGMP, and -cCMP . Without metal or in the presence of Mg(2+), the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni(2+), the protein also produced some 3'-nucleotides . Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain . Low concentrations of the E . coli tRNA (10 nm) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities . The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities . This inhibition was completely abolished by the addition of Mg(2+), Mn(2+), or Ca(2+), but not Ni(2+) . The data suggest that the phosphohydrolase activities of the HD domain of the E . coli tRNA nucleotidyltransferase are involved in the repair of the 3'-CCA end of tRNA.

J Biol Chem, 2004 Aug 27, 279(35), 37049 - 60 Epub 2004 Jun 20.
Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex; Hopkins BB et al.; We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer . Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding . This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs . The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent . We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex . The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment . Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes . Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental . Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.

J Biol Chem, 2004 Aug 27, 279(35), 36898 - 905 Epub 2004 Jun 21.
Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin; Cellini B et al.; In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination . Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine . In this investigation, two site-directed mutants, K238A and Y123F, have been characterized . The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine . However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination . For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization . Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine . Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124 . All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor . Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.

J Mol Biol, 2004 Jul 9, 340(3), 469 - 75
Purification and characterisation of a soluble N-terminal fragment of the breast cancer susceptibility protein BRCA1; Sturdy A et al.; The BRCA1 gene encodes a large multidomain protein of 1863 residues, mutations in which lead to breast cancer . Studies to elucidate the mechanisms by which BRCA1 prevents tumour formation have been restricted by the size of the protein . Unable to purify large amounts of the full-length protein, we have identified a fragment of BRCA1, amino acid residues 230-534, that when cloned into the expression vector pET 22b and expressed in Escherichia coli is found predominantly in the soluble portion of the cell lysate . The resulting protein was purified to homogeneity and studies reveal that BRCA1 230-534 binds specifically to four-way junction DNA when compared to duplex and single-stranded DNA .

Bioorg Chem, 2004 Aug, 32(4), 244 - 62
The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase; Jiang YL et al.; The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA . We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached . We find that the bipartite mimics of TS2 are superior to the TS1 mimics . These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.

Biochim Biophys Acta, 2004 Jul 1, 1700(1), 105 - 16
P22 tailspike trimer assembly is governed by interchain redox associations; Danek BL et al.; Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro . The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation . We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation . Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged . In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7 . Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer . These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction . Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide . This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway . Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.

Biochim Biophys Acta, 2004 Jul 1, 1700(1), 85 - 91
Active site mutants of the "non-hydrolyzing" UDP-N-acetylglucosamine 2-epimerase from Escherichia coli; Samuel J et al.; The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc) . This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that one subunit plays a regulatory role, while that of the other plays a catalytic role . In this work, five active site mutants were prepared (D95N, E117Q, E131Q, K15A, and H213N) and analyzed in terms of their effects on binding, catalysis, and allosteric regulation . His213 appears to play a role in UDP binding and may also assist in catalysis and/or regulation, but is not a key catalytic residue . Lys15 appears to be quite important for binding . All three of the carboxylate mutants showed dramatic decreases in the value of k(cat) but relatively unaffected values of K(M) . Thus, these residues are playing key roles in catalysis and/or regulation . In the case of E117Q, the reaction intermediates are released into solution at a rate comparable to that of the overall catalysis . This may indicate that Glu117 plays the role as an acid/base catalyst in the second step of the UDP-GlcNAc epimerization reaction . All three carboxylate mutants were found to exhibit impaired allosteric control.

Biochemistry, 2004 Jun 29, 43(25), 8247 - 55
Tyrphostins are inhibitors of guanylyl and adenylyl cyclases; Jaleel M et al.; Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues . The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain . Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD) . In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro . Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM . The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor . Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM . As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP . Inhibition was reversible, ruling out a covalent modification of the receptor . Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25 . Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity . Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.

Biochemistry, 2004 Jun 29, 43(25), 8178 - 83
Repair of oxidized abasic sites by exonuclease III, endonuclease IV, and endonuclease III; Greenberg MM et al.; 2-Deoxyribonolactone (L) and the C4'-oxidized abasic site (C4-AP) are produced by a variety of DNA-damaging agents . If not repaired, these lesions can be mutagenic . Exonuclease III and endonuclease IV are the major enzymes in E . coli responsible for 5'-incision of abasic sites (APs), the first steps in AP repair . Endonuclease III efficiently excises AP lesions via intermediate Schiff-base formation . Incision of L and C4-AP lesions by exonuclease III and endonuclease IV was determined under steady-state conditions using oligonucleotide duplexes containing the lesions at defined sites . An abasic lesion (AP) in an otherwise identical DNA sequence was incised by exonuclease III or endonuclease IV approximately 6-fold more efficiently than either of the oxidized abasic sites (L, C4-AP) . Endonuclease IV incision efficiency of 2-deoxyribonolactone or C4-AP was independent of whether the lesion was opposite dA or dG . 2-Deoxyribonolactone is known to cross-link to endonuclease III (Hashimoto, M . (2001) J . Am . Chem . Soc . 123, 3161.) . However, the C4-AP lesion is efficiently excised by endonuclease III . Oxidized abasic site repair by endonuclease IV and endonuclease III (C4-AP only) is approximately 100-fold less efficient than repair by exonuclease III . These results suggest that the first step of C4-AP and L oxidized abasic site repair will be the same as that of regular AP lesions in E . coli.

Biochemistry, 2004 Jun 29, 43(25), 8077 - 83
Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations; Choi MY et al.; Polar residues comprise about 15% of the transmembrane (TM) domains of proteins, where they can stabilize structure via native side chain-side chain interhelical hydrogen bonds between TM helices . However, non-native H-bonds may be implicated in disease states, through limiting protein dynamics during transport and/or misfolding the protein by inducing non-native rotational positions about TM helical axes . Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G(194)LALAHFVWIAPLQ(207)VALLMGLIWELLQASAFAGLGFLIV(232)LALFQ(237)AGLG(241)) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 {Therien, A . G., Grant, F . E., and Deber, C . M . (2001) Nat . Struct . Biol 8, 597-601} . In the present work, a library of 21 TM3/4 constructs was prepared, where Asp residues were placed individually at TM4 positions 221-241 . Using gel shift assays-in which H-bond-linked hairpins (closed conformation) migrate faster than the elongated forms (open conformation)-we found that Q207 in TM3 is able to "capture" all 21 TM4 D mutations into measurable populations of interhelical H-bonds . A similar library of TM4 D mutants-but also containing Q207L-reverted to wild-type migration rates, confirming Q207 as the polar partner for TM4 D residues . In view of the broad capture range of Q207, these results emphasize the potential consequences to folding and dynamics of introducing polar mutations into the TM domains of membrane proteins in the vicinity of a native polar TM residue.

Biochemistry, 2004 Jun 29, 43(25), 8055 - 66
Thermodynamic stability of domain 2 of epithelial cadherin; Prasad A et al.; Cadherin is a cell adhesion molecule that participates in ordered calcium-dependent self-association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions) . Cadherin is a transmembrane protein that has 3-7 independently folded beta-barrel extracellular domains . Both types of self-association interactions are mediated through the most N-terminal domain (Domain 1) . Although the structural nature of the trans-interactions is clear, the nature of the cis-interactions is ambiguous despite several high-resolution structural studies . From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory . Hence, our first steps are to study the energetic driving forces for the cis-interactions . We have simplified the approach by first examining participating extracellular domains individually . We report here our initial experiments into the stability of Domain 2 of E-cadherin (ECAD2) . ECAD2 appears monomeric, according to results from mass spectrometry and sedimentation equilibrium studies . We report denaturation data from differential scanning calorimetric experiments, and temperature and denaturant-induced unfolding experiments monitored by circular dichroism . These studies give a unified picture of the energetics of ECAD2-folding and stability, for which DeltaG degrees is 6.6 kcal/mol, T(m) is 54 degrees C, DeltaH(m) is 90 kcal/mol, and DeltaC(p) is 1300 cal/Kmol . These parameters are independent of calcium up to 5 mM, indicating that ECAD2 does not bind calcium at physiological calcium levels.

Biochemistry, 2004 Jun 29, 43(25), 8038 - 47
Nickel superoxide dismutase structure and mechanism; Barondeau DP et al.; The 1.30 A resolution crystal structure of nickel superoxide dismutase (NiSOD) identifies a novel SOD fold, assembly, and Ni active site . NiSOD is a hexameric assembly of right-handed 4-helix bundles of up-down-up-down topology with N-terminal hooks chelating the active site Ni ions . This newly identified nine-residue Ni-hook structural motif (His-Cys-X-X-Pro-Cys-Gly-X-Tyr) provides almost all interactions critical for metal binding and catalysis, and thus will likely be diagnostic of NiSODs . Conserved lysine residues are positioned for electrostatic guidance of the superoxide anion to the narrow active site channel . Apo structures show that the Ni-hook motif is unfolded prior to metal binding . The active site Ni geometry cycles from square planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (His1 and Cys2) ligands, to square pyramidal Ni(III) with an added axial His1 side chain ligand, consistent with electron paramagentic resonance spectroscopy . Analyses of the three NiSOD structures and comparisons to the Cu,Zn and Mn/Fe SODs support specific molecular mechanisms for NiSOD maturation and catalysis, and identify important structure-function relationships conserved among SODs.

Biochemistry, 2004 Jun 29, 43(25), 8029 - 37
Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein; Thomsen MS et al.; Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain . The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect . Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1 . To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used . These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain . The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions . In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA . Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.

J Matern Fetal Neonatal Med, 2004 Feb, 15(2), 129 - 31
Meningitis in a newborn infant with urosepsis, negative blood cultures and initially normal cerebrospinal fluid findings; Behera S et al.; This case presentation supports the observation that initial cerebrospinal fluid findings can be normal in newborn infants with sepsis syndrome who then develop evidence for meningeal involvement . Therefore, if initial lumbar puncture results are negative, a repeat lumbar puncture is recommended to look for meningitis in newborns that are critically ill with sepsis syndrome.

Nat Biotechnol, 2004 Jul, 22(7), 841 - 7 Epub 2004 Jun 20.
Engineered riboregulators enable post-transcriptional control of gene expression; Isaacs FJ et al.; Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell . Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression . We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene . Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5'-untranslated region of the mRNA . The stem-loop structure interferes with ribosome binding, silencing gene expression . A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression . Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.

J Allergy Clin Immunol, 2004 Jun, 113(6), 1079 - 85
Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis; Hsu CH et al.; BACKGROUND: Sublingual-swallow immunotherapy in house dust mite-related asthma has a good safety profile and improves respiratory function and bronchial hyperreactivity . Zucchini yellow mosaic virus (ZYMV) is envisaged as a promising viral vector for expressing large quantity of foreign proteins in cucurbit species . OBJECTIVE: We sought to investigate whether oral feeding of dust mite allergen expressed by ZYMV in a cucurbit species can suppress allergen-induced inflammation and IgE synthesis . METHODS: An infectious plant virus clone, p35SZYMV2-26, that contains the full-length cDNA to the genomic RNA of a Taiwan isolate of ZYMV, driven by the cauliflower mosaic virus 35S promoter, was engineered as an in vivo viral vector to express Dermatophagoides pteronyssinus group 5 allergen (Der p 5) in cucurbit species . Female BALB/c mice were intraperitoneally sensitized with Escherichia coli bacteria-expressed Der p 5 and orally treated with the virus-expressed Der p 5 (vDer p 5) extracted from the recombinant virus-infected squash plants . Der p 5-specific immunoglobulins were measured by ELISA, and bronchoalveolar lavage assays were used to measure airway inflammation . RESULTS: Infectivity assays and immunoblotting revealed that large quantities of free-form vDer p 5 are produced in the recombinant virus-infected squash plants . The recombinant virus carried and expressed the Der p 5 allergen in squash plants for at least 1 year after numerous passages . In animal tests, squash extract containing vDer p 5 inhibited Der p 5-specific IgE synthesis and airway inflammation . CONCLUSION: Our results suggest that oral feeding with allergen produced by the plant viral vector provides a novel approach for the therapy of allergic asthma.

RNA, 2004 Jul, 10(7), 1097 - 107
A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity; Odom OW et al.; The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea . Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp . from Antarctica that self-splices accurately in vitro . Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E . myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria . In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+) . Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription . Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability . Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity . This work provides the first description of a ribozyme from a psychrophilic organism . It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.

RNA, 2004 Jul, 10(7), 1026 - 33
Crystal structure of the highly divergent pseudouridine synthase TruD reveals a circular permutation of a conserved fold; Hoang C et al.; The pseudouridine (Psi) synthases Pus7p and TruD define a family of RNA-modifying enzymes with no sequence similarity to previously characterized Psi synthases . The 2.2 A resolution structure of Escherichia coli TruD reveals a U-shaped molecule with a catalytic domain that superimposes closely on that of other Psi synthases . A domain that appears to be unique to TruD/Pus7p family enzymes hinges over the catalytic domain, possibly serving to clasp the substrate RNAs . The active site comprises residues that are conserved in other Psi synthases, although at least one comes from a structurally distinct part of the protein . Remarkably, the connectivity of the structural elements of the TruD catalytic domain is a circular permutation of that of its paralogs . Because the sequence of the permuted segment, a beta-strand that bisects the catalytic domain, is conserved among orthologs from bacteria, archaea and eukarya, the permutation likely happened early in evolution.

J Biol Chem, 2004 Aug 27, 279(35), 36884 - 91 Epub 2004 Jun 17.
The three isoforms of the light-harvesting complex II: spectroscopic features, trimer formation, and functional roles; Standfuss J et al.; The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus . Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers . All three isoforms are highly conserved among different species, suggesting distinct functional roles . We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments . Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b . Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization . The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE . Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms . We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes . The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.

J Biol Chem, 2004 Oct 8, 279(41), 42907 - 15 Epub 2004 Jun 17.
Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase; Barabas O et al.; dUTPase is essential to keep uracil out of DNA . Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions . A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution . Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue . Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site . Results provide an understanding for the catalytic role of conserved residues in dUTPases.

J Biol Chem, 2004 Aug 27, 279(35), 36708 - 14 Epub 2004 Jun 18.
Phosphorylation-independent dimer-dimer interactions by the enhancer-binding activator NtrC of Escherichia coli: a third function for the C-terminal domain; Yang XF et al.; The response regulator NtrC transcriptionally activates genes of the nitrogen-regulated (Ntr) response . Phosphorylation of its N-terminal receiver domain stimulates an essential oligomerization of the central domain . Deletion of the central domain reduces, but does not eliminate, intermolecular interactions as assessed by cooperative binding to DNA . To analyze the structural determinants and function of this central domain-independent as well as phosphorylation-independent oligomerization, we randomly mutagenized DNA coding for an NtrC without its central domain and isolated strains containing NtrC with defective phosphorylation-independent cooperative binding . The alterations were primarily localized to helix B of the C-terminal domain . Site-specific mutagenesis that altered surface residues of helix B confirmed this localization . The purified NtrC variants, with or without the central domain, were specifically defective in phosphorylation-independent cooperative DNA binding and had little defect, if any, on other functions, such as non-cooperative DNA binding . We propose that this region forms an oligomerization interface . Full-length NtrC variants did not efficiently repress the glnA-ntrBC operon when NtrC was not phosphorylated, which suggests that phosphorylation-independent cooperative binding sets the basal level for glutamine synthetase and the regulators of the Ntr response . The NtrC variants in these cells generally, but not always, supported wild-type growth in nitrogen-limited media and wild-type activation of a variety of Ntr genes . We discuss the differences and similarities between the NtrC C-terminal domain and the homologous Fis, which is also capable of intermolecular interactions.

Res Microbiol, 2004 Jun, 155(5), 342 - 51
Amplification-mutagenesis--how growth under selection contributes to the origin of genetic diversity and explains the phenomenon of adaptive mutation; Roth JR et al.; The behavior of a particular bacterial genetic system has been interpreted as evidence that selective stress induces general mutagenesis or even preferentially directs mutations to sites that improve growth (adaptive mutation) . It has been proposed that changes in mutability are a programmed response to stress in non-growing cells . In contrast, the amplification-mutagenesis model suggests that stress has no direct effect on the mutation rate and that mutations arise in cells growing under strong selection . In this model, stress serves only as a selective pressure that favors cells with multiple copies of a growth-limiting gene . Mutations are made more probable because more target copies are added to the selection plate-more cells with more mutational targets per cell . The amplification-mutagenesis process involves standard genetic events and therefore should apply to all biological systems . Idiosyncrasies of the particular system described here accelerate this process, allowing an evolutionary series of events to be completed in only a few days.

Res Microbiol, 2004 Jun, 155(5), 337 - 41
Survival versus maintenance of genetic stability: a conflict of priorities during stress; Matic I et al.; Bacteria are constantly facing many different environmental assaults, which may be of such severity that numerous survivors have important alterations in their genetic material . Some genetic systems induced in response to such stresses, for example the SOS system and the sigmaS regulon, actively participate in the generation of genetic alterations . The key priority of those genetic systems during stress is to ensure survival . Therefore, the repair of lethal DNA lesions is an absolute necessity, while perfect restoration of original genetic information is not . Furthermore, the nature of DNA lesions might render error-free repair too costly, or even impossible for stressed bacterial cells . Although the majority of these genetic alterations are deleterious, the rare advantageous alterations may have long-term evolutionary consequences independently of whether the selection of molecular mechanisms involved in their generation is linked to survival strategies or not.

Tuberculosis (Edinb), 2004, 84(5), 283 - 92
Interleukin (IL)-8 (CXCL8) induces cytokine expression and superoxide formation by guinea pig neutrophils infected with Mycobacterium tuberculosis; Lyons MJ et al.; SETTING: Interleukin (IL)-8, a neutrophil attracting chemokine, is known to be made by a variety of leukocyte populations following stimulation by Mycobacterium tuberculosis . OBJECTIVE: The effect of recombinant guinea pig IL-8 on the ability of neutrophils to generate a cytokine response after infection with M . tuberculosis H37Ra was examined . DESIGN: Recombinant gpIL-8 was produced by subcloning the gene into Escherichia coli and purification over a nickel column . The identity of the rgpIL-8 was confirmed by sequencing . Neutrophils were harvested from the blood of non-vaccinated or M . bovis BCG-vaccinated guinea pigs and tested for their ability to migrate toward media alone, 10 microg/ml PPD, f-Met-Leu-Phe (f-MLP), or rgpIL-8 in 96-well chemotactic chambers . Neutrophils were also pre-stimulated with rgpIL-8 then restimulated with LPS (10 microg/ml) or infected in vitro with M . tuberculosis H37Ra (MOI 1:1) . RESULTS: Recombinant gpIL-8 and f-MLP induced significant chemotaxis in neutrophils from both non-vaccinated and BCG-vaccinated guinea pigs, with the best chemotaxis occurring at a concentration of 10(-7)M . Real-time PCR analysis revealed that pre-treatment of neutrophils induced elevated levels of IL-8 and TNF-alpha mRNA and protein as well as superoxide, but not mRNA for MCP-1, IFN-gamma, or TGF-beta when compared to neutrophils pre-stimulated with media alone . CONCLUSIONS: The presence of IL-8 early in the host response to M . tuberculosis infection may be an important contributor to a successful immune response . How essential a role IL-8 plays remains unknown and merits further study.

Biochem Biophys Res Commun, 2004 Jul 16, 320(1), 156 - 64
Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1; Sawada N et al.; Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein . Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3 . Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3 . As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1 . The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2 . These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not . The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jul, 20(4), 481 - 3
{Cloning and expression of fusion gene of transmembrane domain of human CD20 and g3pN in Escherichia coli}; Zhang XY et al.; AIM: To construct the expression vector containing transmembrane domain gene of human CD20 and g3pN1 gene and express the fusion gene high-efficiently in E.coli . METHODS: The human CD20 gene and g3pN1 domain gene were amplified by RT-PCR and PCR from Daudi cells and M13K07 phage antibody library, respectively, and then cloned into expression vector pTIG-Trx.The constructed expression vector was expressed in E.coli . RESULTS: Western blot analysis showed that expressed product could bind to anti-CD20 mAb . CONCLUSION: The pTIG-GS has been constructed and expressed successfully in E.coli, which lays the foundation for further screening anti-CD20 antibody from phage antibody library.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jul, 20(4), 444 - 8
{Expression and bioactivity analysis of the fusion protein GFP-V(L) of the neutralizing monoclonal antibody MA18/7 against HBV}; Guan BQ et al.; AIM: To express the fusion protein of enhanced green fluorescent protein (EGFP) with the light chain variable domain of the neutralizing monoclonal antibody MA18/7 (mAb) against hepatitis B virus in E.coli, and determine its bioactivity . METHODS: The EGFP gene was cloned into vector pTO-T7 to construct an expression vector . And then according to ORF gene, MA18/7-V(L) was inserted into the 5' terminal of EGFP gene free of terminal code TAA to construct expression vector of fusion protein . The fusion protein was expressed in E.coli and its bioactivity was detected with ELISA and relative fluorescence intensity . RESULTS: The expression vector EGFP-V(L) was constructed . SDS-PAGE analysis showed that expressed fusion protein was mainly in the form of inclusion body . The fusion protein retained the property of EGFP and it could bind to V(H) to form Fv which had binding activity to pre-S1 . CONCLUSION: The obtained fusion protein had good bioactivity and could be applied to further studies.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jul, 20(4), 422 - 4
{The expression and activity detection of a variant N protein of SARS-CoV}; Qi Y et al.; AIM: To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV and GST in E.coli . METHODS: The N region gene of SARS-CoV was cloned by RT-PCR . The expression vector was constructed by DNA recombination . The recombinant plasmid was transformed into E.coli BL21(DE3) . The expression of the fusion protein was detected by Western blot . RESULTS: (1) As compared with the sequences in GenBank, 20 bp were deleted in DNA sequence of the cloned N protein . (2) The fusion protein GST-N was soluble . Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive . CONCLUSION: The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, which lays the foundation for further study of SARS-CoV N protein.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jul, 20(4), 402 - 5
{Cloning, expression, renaturation and purification of soluble hSCF}; Wang LL et al.; AIM: To construct an expression vector for soluble recombinant human stem cell factor (rhSCF) gene, and optimize culture conditions for high-level expression of rhSCF in E.coli . METHODS: hSCF cDNA was amplified by PCR with total cDNA from human lymph node as a template, followed by cloning into pMD18-T vector . The 5' terminal of the hSCF cDNA was modified by degenerative PCR to obtain high-level expression in E.coli . The biological activity of the refolded rhSCF, purified with high performance hydrophobic interaction chromatography, was examined by MTT colorimetry . RESULTS: hSCF cDNA was amplified and cloned, and was inserted into an E.coli expression vector pBV220 . The expressed rhSCF accounted for about 20% of total bacterial proteins and reached 40% of total bacterial proteins under optimal culture conditions . The expressed rhSCF appeared in bacterial lysates in the form inclusion body . The rhSCF with biological activity was obtained after solubilization of the inclusion body with 8 mol/L urea or 7 mol/L guanidine chloride, followed by preliminary refolding and purification . CONCLUSION: hSCF was cloned and expressed in E.coli successfully . The E.coli strain expressing rhSCF can be used to produce rhSCF with biological activity on large-scale production.

BMC Struct Biol . 2004 Jun 18;4(1):8.
Improved protein structure selection using decoy-dependent discriminatory functions; Wang K et al.; BACKGROUND: A key component in protein structure prediction is a scoring or discriminatory function that can distinguish near-native conformations from misfolded ones . Various types of scoring functions have been developed to accomplish this goal, but their performance is not adequate to solve the structure selection problem . In addition, there is poor correlation between the scores and the accuracy of the generated conformations . RESULTS: We present a simple and nonparametric formula to estimate the accuracy of predicted conformations (or decoys) . This scoring function, called the density score function, evaluates decoy conformations by performing an all-against-all Calpha RMSD (Root Mean Square Deviation) calculation in a given decoy set . We tested the density score function on 83 decoy sets grouped by their generation methods (4state_reduced, fisa, fisa_casp3, lmds, lattice_ssfit, semfold and Rosetta) . The density scores have correlations as high as 0.9 with the Calpha RMSDs of the decoy conformations, measured relative to the experimental conformation for each decoy.We previously developed a residue-specific all-atom probability discriminatory function (RAPDF), which compiles statistics from a database of experimentally determined conformations, to aid in structure selection . Here, we present a decoy-dependent discriminatory function called self-RAPDF, where we compiled the atom-atom contact probabilities from all the conformations in a decoy set instead of using an ensemble of native conformations, with a weighting scheme based on the density scores . The self-RAPDF has a higher correlation with Calpha RMSD than RAPDF for 76/83 decoy sets, and selects better near-native conformations for 62/83 decoy sets . Self-RAPDF may be useful not only for selecting near-native conformations from decoy sets, but also for fold simulations and protein structure refinement . CONCLUSIONS: Both the density score and the self-RAPDF functions are decoy-dependent scoring functions for improved protein structure selection . Their success indicates that information from the ensemble of decoy conformations can be used to derive statistical probabilities and facilitate the identification of near-native structures.

Chem Res Toxicol, 2004 Jun, 17(6), 742 - 52
O6-alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate; Liu L et al.; O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O(6)-alkylguanine adducts . The interaction of dibromomethane (CH(2)Br(2)) and bromomethyl acetate (BrCH(2)OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant . Both CH(2)Br(2) and BrCH(2)OAc reacted with AGT at its cysteine acceptor site, abolishing its DNA repair activity with the latter agent being much more potent . The formation of AGT-Cys(145)S-CH(2)OAc by BrCH(2)OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys(145)S-CH(2)Br adduct from CH(2)Br(2) was too unstable for such characterization . In the presence of CH(2)Br(2), AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5'-d(AG)(8)-3', but no cross-link was formed by BrCH(2)OAc . Survival of cells exposed to CH(2)Br(2) was reduced, and the number of mutants was greatly increased when wild-type AGT was present . The cytotoxicity of CH(2)Br(2) was similar to that of BrCH(2)CH(2)Br(2), but the mutagenicity was about four times less . Virtually all of the AGT-mediated mutants induced by CH(2)Br(2) in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A . In contrast, BrCH(2)OAc was more than 10-fold less genotoxic than CH(2)Br(2) and the survival of cells exposed to BrCH(2)OAc was not affected by AGT . The number of mutations (almost all G:C to A:T transitions) induced by BrCH(2)OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant . These results with CH(2)Br(2) are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys(145)S-CH(2)Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity . The results with BrCH(2)OAc suggest that it reacts directly with DNA to form O(6)-(CH(2)OAc)guanine, which, if unrepaired, causes G:C to A:T transitions . Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys(145)S-CH(2)-DNA adducts) by which CH(2)Br(2) may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.

Chem Res Toxicol, 2004 Jun, 17(6), 717 - 30
Structure of a site specific major groove (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl DNA adduct of butadiene diol epoxide; Scholdberg TA et al.; The solution structure of the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct arising from the alkylation of adenine N(6) at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), by butadiene diol epoxide, was determined . This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene . This oligodeoxynucleotide, containing the adenine N(6) adduct butadiene triol (BDT) adduct at the second position of codon 61, was named the ras61 S,S-BDT-(61,2) adduct . NMR spectroscopy revealed modest structural perturbations localized to the site of adduction at X(6).T(17), and its nearest-neighbor base pairs C(5).G(18) and A(7).T(16) . All sequential NOE connectivities arising from DNA protons were observed . Torsion angle analysis from COSY data suggested that the deoxyribose sugar at X(6) remained in the C2'-endo conformation . Molecular dynamics calculations using a simulated annealing protocol restrained by a total of 442 NOE-derived distances and J coupling-derived torsion angles refined structures in which the BDT moiety oriented in the major groove . Relaxation matrix analysis suggested hydrogen bonding between the hydroxyl group located at the beta-carbon of the BDT moiety and the T(17) O(4) of the modified base pair X(6).T(17) . The minimal perturbation of DNA induced by this major groove adduct correlated with its facile bypass by three Escherichia coli DNA polymerases in vitro and its weak mutagenicity {Carmical, J . R., Nechev, L . V., Harris, C . M., Harris, T . M., and Lloyd, R . S . (2000) Environ . Mol . Mutagen . 35, 48-56} . Overall, the structure of this adduct is consistent with an emerging pattern in which major groove adenine N(6) alkylation products of styrene and butadiene oxides that do not strongly perturb DNA structure are not strongly mutagenic.

Pediatr Surg Int, 2004 Jul, 20(7), 534 - 7 Epub 2004 Jun 16.
Treatment strategy when using intraoperative peritoneal lavage for perforated appendicitis in children: a preliminary report; Ohno Y et al.; We attempt to quantify the amount of peritoneal irrigation required to significantly decrease the intraperitoneal bacteria in children with perforated appendicitis, as no ideal volume of peritoneal lavage has yet been determined . A series of 11 children who were operated on for peritonitis caused by perforated appendicitis were reviewed retrospectively . All children were treated with our treatment protocol that included intraoperative peritoneal lavage using a large volume of saline . Peritoneal fluid samples were taken before and after peritoneal lavage and then were cultured to determine the colony counts . Twenty of 24 bacteria were available for evaluation of the changes in the flora counts . We found 85% of species to be resistant to peritoneal lavage when 3-5 l of saline per square meter of body surface area (l/m2) were used . In contrast, 5.8+/-1.54 l/m2 of peritoneal lavage fluid was necessary to completely eradicate the intraperitoneal bacterial flora . The residual bacteria showed a greater decrease when lavage fluid in excess of 6 l/m2 was used . Although this is only a preliminary report, these findings could be used to justify a true prospective randomized trial in the future.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9562 - 7 Epub 2004 Jun 17.
Strand exchange activity of human recombination protein Rad52; Kumar JK et al.; Repair of double-strand breaks is essential for the maintenance of genome integrity and cell survival . In eukaryotes, double-strand-break repair by homologous recombination requires the Rad52 group of proteins . Human Rad52 protein (HsRad52)-mediated annealing of complementary strands has been studied in detail, but little has been reported on the recombinase activities of HsRad52 . For this study, we purified HsRad52 from Escherichia coli . DNase I protection experiments indicated that HsRad52 binds preferentially to single-stranded DNA and protects it against digestion by DNase I . HsRad52 catalyzed D-loop formation in superhelical DNA, as well as strand exchange among oligonucleotide substrates . The formation of a stoichiometric complex between HsRad52 and single-stranded DNA was found to be critical for strand exchange activity, and the coating of both the single- and double-stranded oligonucleotides inhibited the exchange reaction.

J Bacteriol, 2004 Jul, 186(13), 4402 - 6
Point mutations in transmembrane helices 2 and 3 of ExbB and TolQ affect their activities in Escherichia coli K-12; Braun V et al.; Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli . Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB . E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

J Bacteriol, 2004 Jul, 186(13), 4399 - 401
Independent regulation of two genes in Escherichia coli by tetracyclines and Tet repressor variants; Kamionka A et al.; We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities . One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline . The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline . We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

J Bacteriol, 2004 Jul, 186(13), 4326 - 37
P pilus assembly motif necessary for activation of the CpxRA pathway by PapE in Escherichia coli; Lee YM et al.; P pilus biogenesis occurs via the highly conserved chaperone-usher pathway, and assembly is monitored by the CpxRA two-component signal transduction pathway . Structural pilus subunits consist of an N-terminal extension followed by an incomplete immunoglobulin-like fold that is missing a C-terminal seventh beta strand . In the pilus fiber, the immunoglobulin-like fold of each pilin is completed by the N-terminal extension of its neighbor . Subunits that do not get incorporated into the pilus fiber are driven "OFF-pathway." In this study, we found that PapE was the only OFF-pathway nonadhesin P pilus subunit capable of activating Cpx . Manipulation of the PapE structure by removing, relocating within the protein, or swapping its N-terminal extension with that of other subunits altered the protein's self-associative and Cpx-activating properties . The self-association properties of the new subunits were dictated by the specific N-terminal extension provided and were consistent with the order of the subunits in the pilus fiber . However, these aggregation properties did not directly correlate with Cpx induction . Cpx activation instead correlated with the presence or absence of an N-terminal extension in the PapE pilin structure . Removal of the N-terminal extension of PapE was sufficient to abolish Cpx activation . Replacement of an N-terminal extension at either the amino or carboxyl terminus restored Cpx induction . Thus, the data presented in this study argue that PapE has features inherent in its structure or during its folding that act as specific inducers of Cpx signal transduction.

J Bacteriol, 2004 Jul, 186(13), 4238 - 45
Multiple paths for nonphysiological transport of K+ in Escherichia coli; Buurman ET et al.; Mutants of Escherichia coli lacking all of the known saturable K+ transport systems, "triple mutants," require elevated K+ concentrations for growth . K+ transport activity in such mutants, called TrkF activity, has low substrate specificity and a low rate that increases with increasing external pH . Attempts to isolate mutants requiring even higher concentrations of K+ failed, implying that either TrkF is essential or is composed of multiple minor K+ transport activities . Instead, we sought mutations that allowed triple mutants to grow at lower K+ concentrations . Mutations so identified include ones altering MscL, the large mechanosensitive channel, or Opp, the oligopeptide permease . However, a possible contribution of wild-type Opp and MscL to TrkF activity was not proven . In contrast, expression of wild-type ProP, TrkG, and TrkH proteins increased uptake when encoded on multicopy plasmids . In all of these situations, the driving force for K+ appeared to be the transmembrane electric potential, and in most cases substrate specificity was low; these are characteristics of TrkF activity . These results support the view that TrkF is composed of multiple, "aberrant" K+ transport activities, i.e., paths that, regardless of their physiological function, allow K+ to cross the cell membrane by a uniport process.

J Bacteriol, 2004 Jul, 186(13), 4192 - 8
Dps protects cells against multiple stresses during stationary phase; Nair S et al.; Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli . Dps homologs are found throughout the bacteria and in at least one archaeal species . Dps has been shown to protect cells from oxidative stress during exponential-phase growth . During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal . We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells . We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock . The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression.

Anal Biochem, 2004 Jul 15, 330(2), 251 - 6
A fusion protein expression analysis using surface plasmon resonance imaging; Jung JM et al.; A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli . With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E . coli were analyzed . The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin . After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins . Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained . The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.

Acta Biochim Biophys Sin (Shanghai), 2004 Mar, 36(3), 184 - 90
Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli; Yan HL et al.; To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method . The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E . coli BL21-RIL, and then induced to express under the control of T7 promoter . The AnxB1ScuPA protein expressed amounted to 22% of the total bacterial proteins . The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column . HPLC analysis revealed that the final purity is about 95% . The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg . It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chimera fully retained the components of enzymatic and membrane-binding activities of the parent molecules . In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfusion ratio, and less bleeding effects than those with urokinase . These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.

Methods Mol Biol, 2004, 277, 19 - 28
Analysis of triplet repeat replication by two-dimensional gel electrophoresis; Krasilnikova MM et al.; Expansions of triplet repeats are responsible for more than 15 hereditary neurological disorders in humans . Triplet repeats are fairly stable when the number of elementary units is under approx 30, but become polymorphic in length with a clear bias for expansions when this threshold is exceeded . This results in the rapid addition of hundreds or even thousands of extra repeats and, ultimately, disease . The mechanisms of triplet repeat expansions are not yet understood . The role of several genetic processes, including replication, recombination, and repair, was suggested . However, given the swift accumulation of extra DNA material, DNA replication seems to be an intuitive candidate for generating expansions . Numerous data point to the aberrant replication of triplet repeats as a cause of triplet repeat expansions . Direct experimental proof of aberrant replication through triplet repeats was lacking . This encouraged us to study the mode of replication fork progression through triplet repeats in vivo . We analyzed the effects of triplet repeats on replication of bacterial or yeast plasmids using an approach called two-dimensional neutral/neutral gel electrophoresis of replication intermediates . This technique, originally developed for mapping replication origins, is also instrumental in defining replication pause sites . Using this technique, we were able to unambiguously demonstrate that expandable triplet repeats attenuate replication fork progression in vivo and get some insights into the mechanisms of repeat expansions.

J Biol Chem, 2004 Aug 27, 279(35), 36803 - 8 Epub 2004 Jun 16.
Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-alpha; Soriano ME et al.; We studied the effects of cyclosporin A (CsA) administration 1) on the properties of the permeability transition pore (PTP) in mitochondria isolated from the liver and 2) on the susceptibility to hepatotoxicity induced by lipopolysaccharide of Escherichia coli (LPS) plus D-galactosamine (D-GalN) in rats . CsA exerted a marked PTP inhibition ex vivo, with an effect that peaked between 2 and 9 h of drug treatment and decayed with an apparent half-time of about 13 h . Administration of LPS plus D-GalN to naive rats caused the expected increased serum levels of tumor necrosis factor (TNF)-alpha, liver inflammation with BID cleavage, activation of caspase 3, appearance of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive nuclei, and release of alanine aminotransferase and aspartate aminotransferase into the bloodstream . Treatment with CsA before or within 5 h of the administration of LPS plus D-GalN protected rats from hepatotoxicity despite the normal increase of serum TNF-alpha and BID cleavage . These results indicate that CsA prevents the hepatotoxic effects of TNF-alpha by blocking the mitochondrial proapoptotic pathway through inhibition of the PTP and provides a viable strategy for the treatment of liver diseases that depend on increased production and/or liver sensitization to TNF-alpha.

J Biol Chem, 2004 Sep 3, 279(36), 37298 - 303 Epub 2004 Jun 16.
Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones; Ben-Zvi A et al.; Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell . Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear . Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation . We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates . The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide . This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Jun, 24(6), 628 - 30, 635
{Construction and expression of the fusion vector of His-tagged human ARPC2 gene}; Gong XW et al.; OBJECTIVE: To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E . coli . METHODS: ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures . After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography . RESULTS: The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E . coli . With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained . CONCLUSION: The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

Plant J, 2004 Jul, 39(1), 84 - 97
Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues; Garcia-Ortiz MV et al.; Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates . Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants . We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family . This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily . We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs . The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain . Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template . We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs . Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.

Biotechnol Lett, 2004 Apr, 26(8), 649 - 53
Production of a biologically active growth hormone from giant catfish (Pangasianodon gigas) in Escherichia coli; Promdonkoy B et al.; Giant catfish growth hormone (gcGH) cDNA was cloned and expressed in E . coli . The expected 20.5 kDa protein corresponded to the mature gcGH and was efficiently expressed . This protein was produced as inclusion bodies and comprised about 20% of total cellular proteins . The recombinant hormone promoted growth when injected intramuscularly or intraperitoneally into goldfish (Carassius auratus) at 0.1 or 1 microg soluble gcGH per g fish body wt per week . In addition, the recombinant gcGH inclusions had growth-promoting activity similar to that of the soluble form when the fish was received either by intraperitoneal injection or by oral administration.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9654 - 9 Epub 2004 Jun 15.
Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress; Kantorow M et al.; Age-related cataract, an opacity of the eye lens, is the leading cause of visual impairment in the elderly, the etiology of which is related to oxidative stress damage . Oxidation of methionine to methionine sulfoxide is a major oxidative stress product that reaches levels as high as 60% in cataract while being essentially absent from clear lenses . Methionine oxidation results in loss of protein function that can be reversed through the action of methionine sulfoxide reductase A (MsrA), which is implicated in oxidative stress protection and is an essential regulator of longevity in species ranging from Escherichia coli to mice . To establish a role for MsrA in lens protection against oxidative stress, we have examined the levels and spatial expression patterns of MsrA in the human lens and have tested the ability of MsrA to protect lens cells directly against oxidative stress . In the present report, we establish that MsrA is present throughout the human lens, where it is likely to defend lens cells and their components against methionine oxidation . We demonstrate that overexpression of MsrA protects lens cells against oxidative stress damage, whereas silencing of the MsrA gene renders lens cells more sensitive to oxidative stress damage . We also provide evidence that MsrA is important for lens cell function in the absence of exogenous stress . Collectively, these data implicate MsrA as a key player in lens cell viability and resistance to oxidative stress, a major factor in the etiology of age-related cataract.

Nucleic Acids Res, 2004 Jun 15, 32(10), 3240 - 7 Print 2004.
Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress; Rachek LI et al.; Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation . Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases . In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII) . To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element . Successful integrations of MTS-EndoIII or MTS-EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot . Western blots of mitochondrial extracts from MTS-EndoIII and MTS-EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent . Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS-EndoIII and MTS-EndoVIII are functional, and both MTS-EndoIII and MTS-EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA . This enhanced repair led to increased cellular resistance to oxidative stress.

Mol Cell Biol, 2004 Jul, 24(13), 6084 - 93
The single-strand DNA binding activity of human PC4 prevents mutagenesis and killing by oxidative DNA damage; Wang JY et al.; Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function . We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity . Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability . PC4 expression suppresses the peroxide sensitivity of the yeast sub1Delta mutant, suggesting that the human protein has a similar function . A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG . We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex . We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

J Biol Chem, 2004 Aug 20, 279(34), 35616 - 21 Epub 2004 Jun 15.
Rotor/Stator interactions of the epsilon subunit in Escherichia coli ATP synthase and implications for enzyme regulation; Bulygin VV et al.; The H(+)-translocating F(0)F(1)-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F(0) to the synthesis of ATP by F(1) . Although the epsilon subunit appears to be tightly associated with the gamma subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of epsilon can undergo significant conformational change as part of a regulatory process . Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of epsilon with an F(0) component of the rotor (subunit c) and with an F(1) component of the stator (subunit beta) . Oxidation of the engineered F(0)F(1) causes formation of two disulfide bonds, betaD380C-S108C epsilon and epsilonE31C-cQ42C, to give a beta-epsilon-c cross-linked product in high yield . The results demonstrate the ability of epsilon to span the central stalk region from the surface of the membrane (epsilon-c) to the bottom of F(1) (beta-epsilon) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated epsilon (Uhlin, U., Cox, G . B., and Guss, J . M . (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the gamma subunit (Rodgers, A . J., and Wilce, M . C . (2000) Nat . Struct . Biol . 7, 1051-1054) . The kinetics of beta-epsilon and epsilon-c cross-linking were studied separately using F(0)F(1) containing one or the other matched cysteine pair . The rate of cross-linking at the epsilon/c (rotor/rotor) interface is not influenced by the type of nucleotide added . In contrast, the rate of beta-epsilon cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP . This is consistent with a regulatory role for a reversible beta/epsilon (stator/rotor) interaction that blocks rotation and inhibits catalysis . Furthermore, the rate of beta-epsilon cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.

Transgenic Res, 2004 Apr, 13(2), 119 - 34
Transgenic mice over-expressing endothelin-1 in testis transactivated by a Cre/loxP system showed decreased testicular capillary blood flow; Lo AC et al.; It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals . Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner . In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1alpha (hEF1alpha) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system . In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression . To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos . These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology . Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.

Bioelectromagnetics, 2004 Jul, 25(5), 352 - 5
Effects of high static magnetic field exposure on different DNAs; Potenza L et al.; The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro . Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target . The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40) . The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations . We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals . This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses .

Parasitol Res, 2004 Aug, 93(5), 339 - 43 Epub 2004 Jun 09.
Expression and cross-species reactivity of fatty acid-binding protein of Clonorchis sinensis; Lee JS et al.; Clonorchis sinensis is a Chinese liver fluke that chronically resides in the biliary tract . The fatty acid-binding protein (FABP) is known to play an important role in the intracellular transport of long-chain fatty acids that are obtained by the fluke from the host . Although FABP has stimulated considerable interest as a vaccine target candidate, the nature of FABP from C . sinensis (CsFABP) remains unclear . In this paper, we describe the cloning and expression of recombinant FABP and immune cross-reaction by Western blot analysis . Sequence analysis revealed that the CsFABP cDNA contained a single open reading frame (ORF) coding for 134 amino acids with an estimated molecular mass of a 15.2 kDa . The DNA sequence of CsFABP cDNA showed significant homology to schistosome cytosolic FABPs, with a 49% amino acid sequence identity and 89% similarity to Schistosoma japonicum . This DNA also showed a high sequence similarity at the amino acid level to S . mansoni (Sm14; 83%) and Fasciola hepatica (80%) . The CsFABP cDNA was cloned into expression vector pET28a, expressed in Escherichia coli and the recombinant protein purified by affinity chromatography . The recombinant CsFABP was cross-reacted with sera obtained from patients with fascioliasis and paragonimiasis . These results suggest that CsFABP may be useful as a vaccine for clonorchiasis.

Methods Mol Biol, 2004, 283, 255 - 66
Conjugation of glycopeptide thioesters to expressed protein fragments: semisynthesis of glycosylated interleukin-2; Tolbert TJ et al.; This method describes the conjugation of a synthetic glycopeptide to the N-terminus of a recombinant human interleukin-2 (IL-2) protein fragment . The IL-2 protein fragment is produced as an affinity-tagged fusion protein in Escherichia coli and then cleaved with the highly selective TEV protease to remove the affinity tag and uncover an N-terminal cysteine . The N-terminal cysteine is then used in native chemical ligation reaction to join the IL-2 protein fragment to a glycosylated tripeptide thioester that had been previously synthesized to produce a glycosylated form of IL-2.

Methods Mol Biol, 2004, 283, 137 - 44
High-density labeling of DNA for single molecule sequencing; Brakmann S; Two unusual enzymatic activities are required for the realization of a single molecule sequencing: a polymerase for copying a deoxyribonuclease (DNA) target into complementary flurophore-labeled DNA, and an exonuclease for the successive hydrolysis of the completely dye-labeled DNA . Recently, we found that the wild-type Klenow fragment of Escherichia coli DNA polymerase I is well-suited for the synthesis of DNA in a reaction set-up that contains exclusively specific rhodamine-labeled analogs of the natural pyrimidine nucleotides (dCTP and dTTP) . This protocol describes the procedure used for the preparation of DNA that is labeled at all pyrimidine bases of one strand, as well as an example of enzymatic downstream processing of the DNA product.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9211 - 6 Epub 2004 Jun 14.
Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA; Jiang X et al.; RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli . Like its E . coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end . To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before . Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases . Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA . Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers . Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9223 - 8 Epub 2004 Jun 14.
Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex; Kiel C et al.; Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex . Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering . The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT . A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced . The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state . This conclusion is backed up by Phi analysis of the encounter complex and transition state of the RalGDS(M26K,D47K,E54K)/Ras complex, with both values being close to 1 . Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged . This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry . The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9423 - 8 Epub 2004 Jun 14.
TorI, a response regulator inhibitor of phage origin in Escherichia coli; Ansaldi M et al.; The torI gene has been identified by using a genetic multicopy approach as a negative regulator of the torCAD operon that encodes the trimethylamine N-oxide reductase respiratory system in Escherichia coli . The negative effect was due to a previously unidentified small ORF (66 aa) of phage origin that we called torI for Tor inhibition . Overexpression of torI led to an 8-fold decrease of the torCAD operon transcription . This operon is positively regulated, in the presence of trimethylamine N-oxide, by a four-step phosphorelay involving the TorS sensor and the TorR response regulator . Epistatic experiments showed that TorI acts downstream of TorS and needs the presence of TorR . In vitro experiments showed that it is neither a TorR phosphatase nor a histidine kinase inhibitor and that it binds to the effector domain of TorR . Unexpectedly, TorI did not impede TorR DNA binding, and we propose that it may prevent RNA polymerase recruitment to the torC promoter . This study thus reveals a previously uncharacterized class of response regulator inhibitors.

FEBS Lett, 2004 Jun 18, 568(1-3), 171 - 7
Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists; Urbinati C et al.; The HIV-1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium . Here, we report about the Tat-antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide . O-sulfated K5 with high sulfation degree (K5-OS(H)) and N,O-sulfated K5 with high (K5-N,OS(H)) or low (K5-N,OS(L)) sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV-LTR-transactivation . Also, K5-OS(H) and K5-N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor-2 on endothelial cell (EC) surface . Finally, K5-OS(H) inhibits alphav beta3 integrin/Tat interaction and EC adhesion to immobilized Tat . Consequently, K5-OS(H) and K5-N,OS(H) inhibit the angiogenic activity of Tat in vivo . In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities . These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti-AIDS therapies.

J Biotechnol, 2004 Jul 1, 111(1), 51 - 7
A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides; Peng CC et al.; An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins . A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage . Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP . After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation . Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.

Neurosci Lett, 2004 Jul 1, 364(2), 119 - 23
ATP and cytochrome c-dependent inhibition of caspase-9 activity in the cerebral cortex of newborn piglets; Mishra OP et al.; The present study investigates the mechanism of activation of caspase-9 during hypoxia and tests the hypothesis that ATP and cytochrome c regulate the activity of caspase-9 in the cerebral cortex of newborn piglets . Cerebral tissue hypoxia was documented by decreased levels of high energy phosphates, ATP and phosphocreatine (PCr) . Cytosolic fractions were prepared from cerebral cortices and passed through a G50 column, to remove endogenous ATP and cytochrome c . Caspase-9 activity was determined spectrofluorometrically using a specific fluorogenic substrate for caspase-9 at increasing concentrations of ATP (0-1.0 mM) or cytochrome c (0-3.0 microM) . Caspase-9 activity (nmol/mg protein/h) was 1.26 +/- 0.15 in the normoxic and 2.13 +/- 0.14 in the hypoxic group (P < 0.05) . The enzyme activity was inhibited by ATP or cytochrome c in both normoxic and hypoxic groups . The IC50 for ATP and cytochrome c increased 5-fold and 1.5-fold, respectively, following hypoxia, suggesting a hypoxia-induced modification of the ATP and cytochrome binding sites . The data demonstrate that ATP (1 mM) and cytochrome c (3.0 microM) inhibit caspase-9 activity by approximately 70% . On the basis of these observations, we propose a new and novel concept that the caspase-9 activity remains inhibited under the normoxic conditions and during hypoxia the decrease in ATP and decreases in the affinity for ATP and cytochrome c release the inhibitory block to activate the enzyme . Results of ATP- and cytochrome c-dependent inhibition of purified caspase-9 human recombinant show that the inhibitory effect by ATP and cytochrome c does not require Apaf-1 . To our knowledge, this is a completely new concept and a new mechanism of regulation of caspase-9 activity that may lead to hypoxia-induced programmed cell death .

FEMS Immunol Med Microbiol, 2004 Jul 1, 41(3), 243 - 8
The interaction of F4 fimbriae with porcine enterocytes as analysed by surface plasmon resonance; Verdonck F et al.; Fimbriae often play a prominent role in anchoring bacterial cells to host tissue and mediate the first step in pathogenesis . As a consequence, there is a continuous development of new strategies to block the binding of fimbriae to their specific receptor on host cells . The present study demonstrates the specific interaction of F4 (K88) fimbriae and porcine enterocytes using a real-time biomolecular interaction analysis system (BIAcore 3000), based on the principles of surface plasmon resonance (SPR) . This method offers new opportunities to screen therapeutics for prevention of adhesion and subsequent disease without receptor purification.

Biochemistry, 2004 Jun 22, 43(24), 7834 - 42
Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity; Kerdpin O et al.; Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8 . The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6alpha-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate . Within SRS1, the single mutation S114F abolished paclitaxel 6alpha-hydroxylation, while the I113V substitution resulted in modest parallel reductions in K(m) and V(max) . Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (V(max)/K(m)) by 88-100% . Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity . However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8 . The mutations significantly increased (28-968%) K(m) values for both torsemide methyl and ring hydroxylation but had variable effects on V(max) . The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations . Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities . The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.

Biol Chem, 2004 May, 385(5), 381 - 8
Identification of arginine residues important for the activity of Escherichia coli signal peptidase I; Kim YT et al.; Escherichia coil signal peptidase I (leader peptidase, SPase I) is an integral membrane serine protease that catalyzes the cleavage of signal (leader) peptides from pre-forms of membrane or secretory proteins . We previously demonstrated that E . coil SPase I was significantly inactivated by reaction with phenylglyoxal with concomitant modification of three to four of the total 17 arginine residues in the enzyme . This result indicated that several arginine residues are important for the optimal activity of the enzyme . In the present study, we have constructed 17 mutants of the enzyme by site-directed mutagenesis to investigate the role of individual arginine residues in the enzyme . Mutation of Arg127, Arg146, Arg198, Arg199, Arg226, Arg236, Arg275, Arg282, and Arg295 scarcely affected the enzyme activity in vivo and in vitro . However, the enzymatic activity toward a synthetic substrate was significantly decreased by replacements of Arg77, Arg222, Arg315, or Arg318 with alanine/lysine . The kcat values of the R77A, R77K, R222A, R222K, R315A, R318A, and R318K mutant enzymes were about 5.5-fold smaller than that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type . Moreover, the complementing abilities in E . Arg222, Arg315, coil IT41 were lost completely when Arg77, or Arg318 was replaced with alanine/lysine . The circular dichroism spectra and other enzymatic properties of these mutants were comparable to those of the wild-type enzyme, indicating no global conformational changes . However, the thermostability of R222A, R222K, R315A, and R318K was significantly lower compared to the wild type . Therefore, Arg77, Arg222, Arg315, and Arg318 are thought to be important for maintaining the proper and stable conformation of SPase I.

Biotechnol Lett, 2004 May, 26(9), 753 - 6
Chemical modification of L-asparaginase from Escherichia coli with a modified polyethyleneglycol under substrate protection conditions; Zhang JF et al.; L-Asparaginase was chemically modified with 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-S-triazine (mPEG2) in the presence of L-asparagine . Optimal modification was performed under the condition that the molar ratio of mPEG2/-NH2 is 10 . The modified enzyme retained 33% of initial enzymatic activity with complete abolishment of immunogenicity . In vitro half-life increment from 4.6 h to 33 h has been obtained.

Biotechnol Lett, 2004 May, 26(9), 717 - 21
Enhanced expression of an antibody-targeted plasminogen activator in Escherichia coli by fusion to decorsin; Yu M et al.; To improve the thrombolytic specificity of plasminogen activators, an antibody-targeted plasminogen activator was constructed consisting of a single-chain variable fragment of a monoclonal antibody SZ-51 raised specifically against human P-selectins on activated platelets and a low molecular weight single-chain urokinase . After fusion to the 3' end of the gene coding for decorsin, originally isolated from the leech Macrobdella decora, expression of the antibody-targeted plasminogen activator gene in E . coli strain Rosetta (DE3) pLysS was greatly enhanced.

Biotechnol Lett, 2004 May, 26(9), 689 - 93
Production of D(-)-lactate from sucrose and molasses; Shukla VB et al.; Escherichia coli W3110 derivatives, strains SZ63 and SZ85, were previously engineered to produce optically pure D(-) and L(+)-lactate from hexose and pentose sugars . To expand the substrate range, a cluster of sucrose genes (cscR' cscA cscKB) was cloned and characterized from E . coli KO11 . The resulting plasmid was functionally expressed in SZ63 but was unstable in SZ85 . Over 500 mM D(-)-lactate was produced from sucrose and from molasses by SZ63(pLOI3501).

Yi Chuan Xue Bao, 2004 Mar, 31(3), 251 - 6
Screening specifically expressed ESTs related to piglet diarrhea resistance; Qi XH et al.; RNA was extracted from spleens of diarrhea and non-diarrhea piglets of Jiuyang Pig, Jianbai Pig and Landrace . RNA pools were established and DDRT-PCR using three anchored and seven arbitrary primers combined with silver staining was conducted to identify ESTs differentially expressed in diarrhea resistance to E . coli K88 . Five cDNA were identified in the non-diarrhea RNA pools . Among them two are new sequences and three are highly identical to the ESTs in the GeneBank, of which two have known functions . One is the mammalian Nck adaptor protein 1 and the other is the human LINE-1 reverse transcriptase.

J Virol, 2004 Jul, 78(13), 7217 - 26
An exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies; Zhou T et al.; Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV) . A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli . The properly refolded protein fragment specifically bound to the surface of Vero cells . Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms . These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity . These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids . Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

J Virol, 2004 Jul, 78(13), 6855 - 63
Vaccinia virus G1 protein, a predicted metalloprotease, is essential for morphogenesis of infectious virions but not for cleavage of major core proteins; Ansarah-Sobrinho C et al.; Genes encoding orthologs of the vaccinia virus G1 protein are present in all poxviruses for which sequence information is available, yet neither the role of the protein nor its requirement for virus replication is known . G1 was predicted to be involved in the cleavage of core proteins, based on a transfection study and the presence of an HXXEH motif found in a subset of metallopeptidases . In the present study, we engineered a recombinant vaccinia virus containing a single copy of the G1L gene with a C-terminal epitope tag that is stringently regulated by the Escherichia coli lac repressor . In the absence of inducer, expression of G1 was repressed and virus replication was inhibited . Rescue of infectious virus was achieved by expression of wild-type G1 in trans, but not when the putative protease active site residues histidine-41, glutamate-44, or histidine-45 were mutated . Nevertheless, the synthesis and proteolytic processing of major core and membrane proteins appeared unaffected under nonpermissive conditions, distinguishing the phenotype of the G1L mutant from one in which the gene encoding the I7 protease was repressed . Noninfectious virus particles, assembled in the absence of inducer, did not attain the oval shape or characteristic core structure of mature virions . The polypeptide composition of these particles, however, closely resembled that of wild-type virus . Full-length and shorter forms of the G1 protein were found in the core fraction of virus particles assembled in the presence of inducer, suggesting that G1 is processed by self-cleavage or by another protease.

J Biol Chem, 2004 Aug 27, 279(35), 36809 - 18 Epub 2004 Jun 12.
The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis; Clarke TA et al.; Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus . These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis . Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction . All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane . Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4 . A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

J Biol Chem, 2004 Aug 13, 279(33), 34931 - 7 Epub 2004 Jun 11.
tRNA recognition by glutamyl-tRNA reductase; Randau L et al.; During the first step of porphyrin biosynthesis in Archaea, most bacteria, and in chloroplasts glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde . Elements in tRNA(Glu) important for utilization by Escherichia coli GluTR were determined by kinetic analysis of 51 variant transcripts of E . coli Glu-tRNA(Glu) . Base U8, the U13*G22**A46 base triple, the tertiary Watson-Crick base pair 19*56, and the lack of residue 47 are required for GluTR recognition . All of these bases contribute to the formation of the unique tertiary core of E . coli tRNA-(Glu) . Two tRNA(Glu) molecules lacking the entire anticodon stem/loop but retaining the tertiary core structure remained substrates for GluTR, while further decreasing tRNA size toward a minihelix abolished GluTR activity . RNA footprinting experiments revealed the physical interaction of GluTR with the tertiary core of Glu-tRNA(Glu) . E . coli GluTR showed clear selectivity against mischarged Glu-tRNA(Gln) . We concluded that the unique tertiary core structure of E . coli tRNA(Glu) was sufficient for E . coli GluTR to distinguish specifically its glutamyl-tRNA substrate.

Biochem Biophys Res Commun, 2004 Jul 9, 319(4), 1276 - 80
A cleavable signal peptide is required for the full function of the polytopic inner membrane protein FliP of Escherichia coli; Pradel N et al.; FliP is a rare bacterial polytopic membrane protein synthesized with a cleavable highly hydrophobic signal peptide . It is essential for flagellum assembly and for bacterial motility . In this study, we assessed specificity of signal peptide for the FliP function . Like the wild type FliP, two altered FliPs with more hydrophilic Tat- or Sec-dependent signal peptides were both able to restore the motility of the DeltafliP mutant . Therefore, the Tat- and the Sec-dependent signal peptides seemed to be compatible with the FliP function . Moreover, deletion of the FliP signal peptide or replacing it with the transmembrane segment of MotA severely impaired the FliP function . Together these results showed that a cleavable signal peptide is required for the full function of FliP.

Biochimie, 2004 Apr-May, 86(4-5), 327 - 33
Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active; Wang LC et al.; Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy . To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR . The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E . coli . The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture . The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro . The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0 . For the first time, ABRaA expressed as soluble form in E . coli from a PCR-synthesized gene is catalytically and functionally active.

Placenta, 2004 Aug, 25(7), 608 - 22
Placental protein 13 (PP-13): effects on cultured trophoblasts, and its detection in human body fluids in normal and pathological pregnancies; Burger O et al.; Placental tissue protein 13 (PP-13), one of the 56 known placental proteins identified till today, was purified from placentas obtained from women at delivery, and used to evoke antibodies against it . The purified PP-13 was lysed to peptides, which were sequenced, leading to the full-length cDNA sequencing and its expression in Escherichia coli . Sequence analysis in databases showed homology to the galectin family . Of the various antibody preparations developed, a pair of monoclonal antibodies (MAbs) coupled to the recombinant PP-13 (PP-13-R) was used for the immunodetection of PP-13 in pregnant women's serum with the solid-phase ELISA format . With a dynamic range of 25-500 pg/mL with no background in non-pregnant women's serum and men's serum, the ELISA test was suitable for the detection of PP-13 in the 1st, 2nd, and 3rd trimesters . PP-13 levels slowly increase during pregnancy . In the 1st trimester, lower than normal PP-13 levels were found in fetal growth restriction (IUGR), preeclampsia (PE), and particularly in early PE (<34 weeks of gestation) . In the 2nd and 3rd trimesters, higher than normal concentrations were found in PE, IUGR and in preterm delivery (PTD) . Application of PP-13 to cultured trophoblasts elicited depolarization carried by calcium ions, followed by liberation of linoleic and arachidonic acids from the trophoblast membrane, and a subsequent elevation of prostacyclin and thromboxane . These effects were negligible when PP-13 derived from the placentas of patients with IUGR, PE or PTD was used . The results are discussed in view of the potential utilization of PP-13 for early serum screening to assess the risk to develop placental insufficiency, coupled to a differential analysis of the various pathologies by analyzing cultured trophoblasts.

J Pharm Biomed Anal, 2004 Jun 29, 35(4), 817 - 28
Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis; Deng G et al.; An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme . A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed . The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay . Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively) . The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated . In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated .

J Struct Biol, 2004 Aug, 147(2), 146 - 58
Studies on the compaction of isolated nucleoids from Escherichia coli; Zimmerman SB; The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids . Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure . The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies . Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids . These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C . The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA . Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation . Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions . Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 356 - 9
{Preliminary study on regulable DNA vaccines against Plasmodium falciparum}; Lei JC et al.; AIM: To construct regulable DNA vaccine against Plasmodium falciparum by using tetracycline(Tet) regulable system . METHODS: Eukaryotic expression vectors pTL-8/apical membrane antigen 1 (AMA-1) (tTA) and pTL-8/AMA-1(rtTA) gene which express trans-activator (tTA) or reverse trans-activator(rtTA), respectively, and AMA-1 gene of Plasmodium falciparum were constructed . BALB/c mice were immunized with these plasmids and doxycycline (dox) was administered to regulate the expression of AMA-1 . For some mice immunized with pTL-8/AMA-1(rtTA), pUHS6-1, a plasmid containing trans-silencer (tTS) to suppress basal expression of AMA-1 from pTL-8/AMA-1(rtTA), was injected into these mice together with pTL-8/AMA-1(rtTA) . The sera of the mice were isolated at 2,4,6 and 8 weeks post-immunization and the antibodies specific to AMA-1 were measured by ELISA . RESULTS: pTL-8/AMA-1 and pTL-8/AMA-1(rtTA) were constructed successfully . The mice immunized by pTL-8/AMA-1(tTA) with dox or by pTL-8/AMA-1(rtTA) without dox (at these conditions, AMA-1 was expressed at basal level)developed significant antibodies against AMA-1 . Mice immunized by pTL-8/AMA-1(rtTA) and pUHS6-1 without dox did not develop significantly antibodies against AMA-1 . In contrast, the mice immunized by pTL-8/AMA-1(rtTA) and pUHS6-1 with dox produced high level of antibodies . CONCLUSION: pTL-8/AMA-1(rtTA) combined with pUHS6-1 is a good regulable DNA vaccine candidate against Plasmodium falciparum.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 352 - 5
{Prediction of T-cell epitopes from schistosoma japonicum 28 kDa glutathione-S-transferase and identification of their Th1 type T-cell epitopes}; Li GF et al.; AIM: To predict T-cell epitopes of recombinant schistosoma japonicum 28 kDa glutathione-S-transferase(GST) with software and identify the Th1 type T-cell epitopes by experiments . METHODS: T-cell epitopes of recombinant schistosoma japonicum 28kDa GST were predicted with software and several epitopic candidates were screened from them according to their scores . Some of the epitopic candidates were synthesized and the other epitope peptides fused with thioredoxin(Trx) were expressed in E.coli BL21(DE3) and purified by Ni(+) column affinity chromatography . C57BL/6 (H-2(b)) mice's were immunized via peritoneal infection with ultraviolet ray irradiated-cercariae and then boosted with recombinant schistosoma japonicum 28 kDa GST . The immunized mice splenocytes were prepared, cultured and stimulated with synthesized epitope peptides and epitope peptide fusion proteins, respectively . Stimulation activity of synthesized epitope peptides and epitope peptides fusion proteins were assayed by lymphocyte proliferation assay . Levels of IFN-gamma and IL-2 were measured by ELISA . CD4(+) T cells and T cells secreting IFN-gamma and IL-4 were detected by flow cytometry . RESULTS: Epitope P6(73-86aa) among 9 epitopic candidates could generate the strongest stimulation effect on splenocytes, stimulate secretion of higher levels of IFN-gamma and IL-2, and induce more IFN-gamma(+) and IL-4 (+) T cells . CONCLUSION: The recombinant 28 kDa GST possesses functional Th1 type T-cell epitope.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 340 - 3
{Cloning and expression of the extracellular domain of calcium-activated chloride channel in mouse airway goblet cells}; Song LQ et al.; AIM: To clone and express the extracellular domain of murine calcium-activated chloride channel (mCLCA3) in airway goblet cell of mouse . METHODS: According to the gene sequence of mCLCA3 the PCR primers for N-terminal, middle and C-terminal extracellular domains were designed . Using recombinant plasmid pcDNA3.1(-)/mCLCA3 as template, the DNAs coding for the three extracellular domains were amplified . And then the DNAs encoding N-terminal and C-terminal extracellular domains were inserted into expression vector pRSET-A, while the middle extracellular domain DNA was inserted into pGEX-T1 . E.coli . BL21(DE3) were transformed with the three recombinant plasmids, respectively, and were induced with IPTG for expression . RESULTS: DNA sequencing showed that the cloned DNAs encoding extracellular domains were identical with those in GenBank (GenBank accession No . NM-017474 ) . The 3 domains were expressed in E.coli and most of the expressed products existed in the form of inclusion body . CONCLUSION: The expression of three extracellular domains of mCLCA3 lays the foundation for further preparing anti-mCLCA3 antibody and exploring the mechanism of modulation of mCLCA3.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 265 - 8
{In-vitro refolding and biotinylation of soluble HLA-A2-peptide complex}; Wong XF et al.; AIM: To refold and biotinylate HLA-A2-peptide complex in-vitro . METHODS: The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(2m) were expressed highly as insoluble aggregates in E.coli, and then the two subunits were refolded to form an HLA-A2-peptide complex by dilution method in the presence of an antigenic peptide (NH(2)-CLGGLLTMV-COOH of EB virus latent membrane protein 2A LMP2A) . Then the BirA enzyme was used to biotinylate the refolded complex . The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti-human beta(2m) antibody and streptavidin . RESULTS: The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(2m) . Both HLA-A2-peptide complex and the H chain aggregate could be biotinylated . CONCLUSION: The refolding and biotinylation of HLA-A2-peptide complex were successfully performed and the products were confirmed by our practical immunological method . This study laid the foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cells.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 257 - 60
{The cloning, expression, purification and identification of SARS virus S2 gene and study on its immunological characteristics}; Dong XY et al.; AIM: To express S2 protein of SARS virus fused with Trx and then detect its reactivity to the sera from convalescent SARS patients . METHODS: The Trx-S2 fusion protein was expressed in E.coli . After purification, the Trx-S2 fusion protein was detected by Western blot with 6 serum samples of convalescent SARS patients and 6 serum samples of healthy donors . RESULTS: According to the SDS-PAGE analysis, the relative molecular mass (M(r)) of the Trx-S2 fusion protein is about 76 x 10(3) . The fusion protein could react with all the sera from convalescent SARS patients but not with the sera from healthy donors . CONCLUSION: The Trx-S2 fusion protein provides a basis for the research on its role in the course of SARS virus infection of host cells and preparation of recombinant vaccine against SARS virus.

Biochem J, 2004 Sep 1, 382(Pt 2), 441 - 9
Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast; Morgan CP et al.; The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase {Ferber, Munder, Fischer and Gerisch (1970) Eur . J . Biochem . 14, 253-257} . We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine . We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus . Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation . From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function . The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product . Expression of the gene in Escherichia coli confirmed that it encodes a PLB . Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine . The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals . There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast . We therefore have identified a novel family of intracellular PLBs.

Apoptosis, 2004 Jul, 9(4), 485 - 93
Involvement of MIP-2 and CXCR2 in neutrophil infiltration following injection of late apoptotic cells into the peritoneal cavity; Iyoda T et al.; Apoptotic cells are cleared by phagocytes, such as macrophages, as soon as they appear in vivo . If apoptosis occurs acutely, however, macrophages may be outnumbered by apoptotic cells, which causes late apoptosis . We previously showed that injection of late apoptotic cells into the peritoneal cavity led to transient infiltration of neutrophils . In this study, we examined the involvement of MIP-2 and CXCR2 in the neutrophil infiltration . We first produced a recombinant MIP-2 protein, and a fusion protein between CXCR2 and GST in E . coli, and then generated anti-MIP-2 antibodies and anti-CXCR2 antibodies in rabbits . We then confirmed their specificity by Western blotting analysis and flow cytometry . Injection of late apoptotic cells, such as P388 cells treated with etoposide for 24 hours and CTLL-2 cells cultured in IL-2-free medium for 28 hours, induced neutrophil infiltration into the peritoneal cavity, as expected . The antibodies, but not control antibodies against GST, suppressed the neutrophil infiltration to the level caused by injection of normal (viable) cells, suggesting that MIP-2 and CXCR2 are mainly involved in the neutrophil infiltration caused by late apoptotic cells .

J Vasc Res, 2004 Jul-Aug, 41(4), 305 - 13 Epub 2004 Jun 10.
Adenovirus-encoded hammerhead ribozyme to PDGF A-chain mRNA inhibits neointima formation after arterial injury; Lin ZH et al.; To develop a strategy for gene therapy of restenosis following coronary angioplasty, we examined the effects of a recombinant adenovirus vector encoding a hammerhead ribozyme specific for rat platelet-derived growth factor (PDGF) A-chain mRNA (Ad.Ribozyme) and a control recombinant adenovirus vector encoding the Escherichia coli LacZ gene (Ad.LacZ) on neointima formation in rat carotid artery after balloon injury . Ad.Ribozyme (10(8) PFU/ml) markedly reduced the increased expression of PDGF A-chain mRNA and protein . Ad.Ribozyme significantly decreased the intima/media ratio (68%) of the injured artery, whereas Ad.LacZ had no effect on the intima/media ratio . Most carotid arteries developed thrombi by 14 days after balloon injury, whereas Ad.Ribozyme completely inhibited thrombus formation . Expression of thromboxane A2 (TXA2) receptor mRNA was significantly increased after balloon injury . Ad.Ribozyme significantly decreased the levels of TXA2 receptor . Expression of prostaglandin I2 (PGI2) synthase mRNA was significantly decreased after balloon injury . Ad.Ribozyme significantly increased levels of PGI2 synthase mRNA after balloon injury . The observation that adenovirus-encoded ribozyme to PDGF A-chain inhibits neointima formation may serve as a novel strategy to prevent restenosis after coronary angioplasty . Inhibition of growth factors by genetic approaches may yield new insights into the mechanisms underlying responses to vascular injury and lead to new therapeutic applications.

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