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 immunohistochemica