Standardisation of a new model of H9N2/Escherichia coli challenge in broilers in the Lebanon.

Primary infection by low pathogenic avian influenza (LPAI) predisposes for secondary infection by Escherichia coli in poultry, leading to significant economic losses. Future research in control of this ailment requires the establishment of a successful controlled challenge by avian influenza virus (AIV)/E. coli. Six groups of broilers (6 birds/group) were included for the standardisation of the controlled challenge by AIV/E. coli. Birds in groups 1, 2, 3, 4 and 5 received an intra-tracheal challenge of 0.5 ml of two haemagglutinating units of H9N2 virus at 20 days of age. At the age of 23 days, birds in group 1 received an intra-thoracic (right air sac)-E. coli challenge equivalent to 1.6 x 10 colony-forming units (cfu)/0.5 ml/bird, while birds in groups 2, 3, 4 and 5 received E. coli by the same route and in the following respective decreasing order of viable cells: 1.6 x 10(6), 1.6 x 10(5), 1.6 x 10(4) and 1.6 x 10(3); cfu. Birds in control group 6 were deprived of H9N2 and E. coli challenge. Results showed significant early mortality in group 1 that was challenged with the highest number of E. coli, in comparison to groups 2-6 (p<0.05); however, the average weight at 28 days of age was similar in surviving birds of groups 2-6 (p>0.05). The frequencies of four signs at 2 days and at 5 days post E. coli challenge (conjunctivitis, diarrhoea, ocular exudates and rales) in the surviving birds of groups 2-5 were most often higher than those observed in control group 6 (p<0.05). These four signs and five gross lesions (abdominal airsacculitis, left thoracic airsacculitis, pericarditis, right thoracic airsacculitis and tracheitis) had a decreasing pattern of frequency related to a decrease in the E. coli count used in the challenge.

Cytochrome P450 2C11 5'-flanking region and promoter mediate in vivo suppression by 3-methylcholanthrene.

Aromatic hydrocarbons such as 3-methylcholanthrene (MC) elicit toxic and adaptive responses through the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons act via an unknown mechanism to suppress the transcription of CYP2C11, a growth hormone-regulated gene encoding the male-specific rat hepatic cytochrome P450 2C11. We hypothesize that suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flank. Using hydrodynamics-based injections to deliver plasmid DNA to the liver of live rats, we studied the MC responsiveness of luciferase constructs containing 10.1, 5.6, and 2.4 kilobases (kb) of the CYP2C11 5'-flank. MC suppressed CYP2C11-luciferase activity of the 10.1- and 5.6-kb constructs to less than 50% of vehicle levels by 24 and 72 h. Luciferase activity of the 2.4-kb CYP2C11 construct was decreased to 63% of vehicle levels 24 h after MC treatment, but no suppression was detected by 72 h. Negative regulatory element(s) responsible for CYP2C11 reporter suppression by MC exist in the proximal 2.4 kb of the 5'-flank; however, additional cis-acting elements located between -5.6 and -2.4 kb mediate persistent reporter suppression. As a positive control for AHR activation, MC dramatically induced the luciferase activity of a Cyp1a1-driven luciferase plasmid under AHR control. Modulation of reporter gene activity by MC was accompanied by induction of endogenous CYP1A1 and suppression of endogenous CYP2C11 mRNA/protein. This is the first demonstration of aromatic hydrocarbon-mediated suppression of a CYP2C11-luciferase construct, and this finding suggests that the 5'-flanking region and promoter mediate down-regulation of this gene in the intact rat.

Cytochrome P450 2C11 5'-flanking region and promoter: regulation by aromatic hydrocarbons in vitro.

Aromatic hydrocarbons elicit toxic and adaptive responses via the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons suppress the transcription of the growth hormone-regulated, male-specific rat hepatic cytochrome P450 2C11 gene (CYP2C11) in vivo via an unknown mechanism. We hypothesize that the suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flanking region. Following bioinformatic analysis of putative transcription factor (TF) binding sites, we cloned extended lengths of the CYP2C11 5'-flanking region into a promoterless luciferase plasmid. Suppression of CYP2C11 constructs was not observed upon treatment of transfected rat 5L, BP8 or mouse Hepa-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. In human HepG2 cells, the 10.1-kb construct displayed a pronounced 6- to 8-fold induction by TCDD. Deletion analysis localized the paradoxical induction response to a region between -1.8 kb and -1.3 kb, which contains a dioxin-responsive element (DRE) previously shown by us to be capable of binding activated AHR. This was confirmed by site-directed mutagenesis of the DRE. Induction of the 10.1-kb construct by TCDD in HepG2 cells was blocked by alpha-naphthoflavone, an AHR antagonist/partial agonist. The AHR is likely involved in the induction of CYP2C11-luciferase activity by TCDD in HepG2 cells and this response is at least partly DRE-mediated. Although CYP2C11 is suppressed by aromatic hydrocarbons in vivo, CYP2C11-luciferase constructs display a potentially misleading paradoxical induction in vitro that is cell-specific. Regulation of CYP2C11-luciferase plasmids is being studied in vivo in rat liver, where an intact endocrine system and the full complement of TFs needed for CYP2C11 suppression are present.

Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1.

Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2'-PO4 from the splice junction to NAD+ to form ADP-ribose 1''-2''cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1Delta was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30 degrees C. These findings attest to the structural and functional conservation of Tpt1-like 2'-phosphotransferases and identify likely constituents of the active site.

Portability and fidelity of RNA-repair systems.

Yeast tRNA ligase (Trl1) is an essential enzyme that converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO(4), 3'-5' phosphodiester at the splice junction. Trl1 also catalyzes splicing of HAC1 mRNA during the unfolded protein response. Trl1 performs three reactions: the 2',3'-cyclic phosphate of the proximal RNA fragment is hydrolyzed to a 3'-OH, 2'-PO(4) by a cyclic phosphodiesterase; the 5'-OH of the distal RNA fragment is phosphorylated by a GTP-dependent polynucleotide kinase; and the 3'-OH, 2'-PO(4), and 5'-PO(4) ends are then sealed by an ATP-dependent RNA ligase. The removal of the 2'-PO(4) at the splice junction is catalyzed by the essential enzyme Tpt1, which transfers the RNA 2'-PO(4) to NAD(+) to form ADP-ribose 1"-2"-cyclic phosphate. Here, we show that the bacteriophage T4 enzymes RNA ligase 1 and polynucleotide kinase/phosphatase can fulfill the tRNA and HAC1 mRNA splicing functions of yeast Trl1 in vivo and bypass the requirement for Tpt1. These results attest to the portability of RNA-repair systems, notwithstanding the significant differences in the specificities, mechanisms, and reaction intermediates of the individual yeast and T4 enzymes responsible for the RNA healing and sealing steps. We surmise that Tpt1 and its unique metabolite ADP-ribose 1"-2"-cyclic phosphate do not play essential roles in yeast independent of the tRNA-splicing reaction. Our finding that one-sixth of spliced HAC1 mRNAs in yeast cells containing the T4 RNA-repair system suffered deletion of a single nucleotide at the 3' end of the splice-donor site suggests a model whereby the yeast RNA-repair system evolved a requirement for the 2'-PO(4) for RNA ligation to suppress inappropriate RNA recombination.

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

Yeast tRNA ligase (Trl1) converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO4, 3'-5' phosphodiester at the splice junction. Trl1 performs three reactions: (i) the 2',3'-cyclic phosphate of the proximal fragment is hydrolyzed to a 3'-OH, 2'-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5'-OH of the distal fragment is phosphorylated by an NTP-dependent polynucleotide kinase; and (iii) the 3'-OH, 2'-PO4, and 5'-PO4 ends are sealed by an ATP-dependent RNA ligase. Trl1 consists of an N-terminal adenylyltransferase domain that resembles T4 RNA ligase 1, a central domain that resembles T4 polynucleotide kinase, and a C-terminal CPD domain that resembles the 2H phosphotransferase enzyme superfamily. Here we show that all three domains are essential in vivo, although they need not be linked in the same polypeptide. We identify five amino acids in the adenylyltransferase domain (Lys114, Glu266, Gly267, Lys284, and Lys286) that are essential for Trl1 activity and are located within motifs I (114KANG117), IV (266EGFVI270), and V (282FFKIK286) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligases 1 and 2. Mutations K404A and T405A in the P-loop (401GXGKT405) of the central kinase-like domain had no effect on Trl1 function in vivo. The K404A and T405A mutations eliminated ATP-dependent kinase activity but preserved GTP-dependent kinase activity. A double alanine mutant in the P-loop was lethal in vivo and abolished GTP-dependent kinase activity. These results suggest that GTP is the physiological substrate and that the Trl1 kinase has a single NTP binding site of which the P-loop is a component. Two other mutations in the central domain were lethal in vivo and either abolished (D425A) or severely reduced (R511A) GTP-dependent RNA kinase activity in vitro. Mutations of the signature histidines of the CPD domain were either lethal (H777A) or conferred a ts growth phenotype (H673A).

Mutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme.

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.