Rv3389C from Mycobacterium tuberculosis, a member of the (R)-specific hydratase/dehydratase family

The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C(8)-C(16)) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.     

Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.     

The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.

The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.     

Temporal distribution of PBAN‐like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior

A direct enzyme‐linked immunosorbent assay has been developed and applied to the analysis of PBAN immunoreactivity in female hemolymph of the cabbage armyworm, Mamestra brassicae. PBAN‐IR determinations have been carried out with third scotophase insects at different times of the photoperiod. The rhythm of calling and the pattern of pheromone production by third scotophase females at different times of the photoperiod have also been determined. PBAN‐IR and calling are well correlated. However, whereas pheromone titers decrease, both PBAN‐IR levels and percentage of calling females remain high in the last hours of the scotophase. These results are discussed in the context of the regulation of sex pheromone biosynthesis in M. brassicae. Arch. Insect Biochem. Physiol. 40:80–87, 1999. © 1999 Wiley‐Liss, Inc.     

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.     

Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition

The RNA-dependent RNA polymerase (NS5B) from hepatitis C virus (HCV) is a key enzyme in HCV replication. NS5B is a major target for the development of antiviral compounds directed against HCV. Here we present the structures of three thiophene-based non-nucleoside inhibitors (NNIs) bound non-covalently to NS5B. Each of the inhibitors binds to NS5B non-competitively to a common binding site in the "thumb" domain that is approximately 35 Angstroms from the polymerase active site located in the "palm" domain. The three compounds exhibit IC(50) values in the range of 270 nM to 307 nM and have common binding features that result in relatively large conformational changes of residues that interact directly with the inhibitors as well as for other residues adjacent to the binding site. Detailed comparisons of the unbound NS5B structure with those having the bound inhibitors present show that residues Pro495 to Arg505 (the N terminus of the "T" helix) exhibit some of the largest changes. It has been reported that Pro495, Pro496, Val499 and Arg503 are part of the guanosine triphosphate (GTP) specific allosteric binding site located in close proximity to our binding site. It has also been reported that the introduction of mutations to key residues in this region (i.e. Val499Gly) ablate in vivo sub-genomic HCV RNA replication. The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.     

Gender-dependent regulation of G-protein-gated inwardly rectifying potassium current in dorsal raphe neurons in knock-out mice devoid of the 5-hydroxytryptamine transporter

Agonists at G-protein-coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock-out mice devoid of the serotonin transporter (5-HTT(-/-)) exhibit lower efficacy to inhibit cellular discharge than in wild-type counterparts. Using patch-clamp whole-cell recordings, we found that a G-protein-gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5-HT1A agonists (5-carboxamidotryptamine (5-CT) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT); 50 nM-30 microM) in both wild-type and 5-HTT(-/-) female and male mice. These effects were mimicked by 5'-guanylyl-imido-diphosphate (Gpp(NH)p; 400 microM) dialysis into cells with differences between genders. The 5-HTT(-/-) knock-out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5-HT1A receptors to agonists in 5-HTT(-/-) mutants reflects notably alteration in the coupling between G-proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5-HT neurotransmission appear to depend-at least in part-on sex-related variations in corresponding receptor-G protein signaling mechanisms.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

QTL analysis of frost damage in pea suggests different mechanisms involved in frost tolerance

Key message

Avoidance mechanisms and intrinsic resistance are complementary strategies to improve winter frost tolerance and yield potential in field pea.

Abstract

The development of the winter pea crop represents a major challenge to expand plant protein production in temperate areas. Breeding winter cultivars requires the combination of freezing tolerance as well as high seed productivity and quality. In this context, we investigated the genetic determinism of winter frost tolerance and assessed its genetic relationship with yield and developmental traits. Using a newly identified source of frost resistance, we developed a population of recombinant inbred lines and evaluated it in six environments in Dijon and Clermont-Ferrand between 2005 and 2010. We developed a genetic map comprising 679 markers distributed over seven linkage groups and covering 947.1 cM. One hundred sixty-one quantitative trait loci (QTL) explaining 9–71 % of the phenotypic variation were detected across the six environments for all traits measured. Two clusters of QTL mapped on the linkage groups III and one cluster on LGVI reveal the genetic links between phenology, morphology, yield-related traits and frost tolerance in winter pea. QTL clusters on LGIII highlighted major developmental gene loci (Hr and Le) and the QTL cluster on LGVI explained up to 71 % of the winter frost damage variation. This suggests that a specific architecture and flowering ideotype defines frost tolerance in winter pea. However, two consistent frost tolerance QTL on LGV were independent of phenology and morphology traits, showing that different protective mechanisms are involved in frost tolerance. Finally, these results suggest that frost tolerance can be bred independently to seed productivity and quality.