Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.     

Identification of a potential bottleneck in branched chain fatty acid incorporation into triacylglycerol for lipid biosynthesis in agronomic plants

In plant, unusual fatty acids are produced by a limited number of species. The industrial benefits of these unusual structures have led several groups to study their production in transgenic plants. Their research results led to very modest accumulation in seeds which was largely due to a limited knowledge of the lipid metabolism and fatty acid transfer in plants. More specifically we need to better understand the substrate specificity and selectivity of acyltransferases which are required for the incorporation of these unusual fatty acids into storage triacylglycerols. In our studies we have compared the incorporation of [¹⁴C] Oleoyl-CoA and Branched Chain Acyls-CoA into [³H] LPA-C18:1 by the Lysophosphatidic acid Acyltransferase (LPAAT) from developing seeds of agronomic plants (flax (Linum usitatissimum) and rape (Brassica napus)) and from a plant capable of producing high amounts of hydroxy fatty acids (castor bean (Ricinus communis)). Our assays demonstrate that LPAATs of the three studied species (1) incorporated preferentially oleyl-CoA, (2) could incorporate cyclopropane acyl-CoA when added alone as a substrate, however very weakly for rapeseed and castor bean seeds, (3) presented a low capacity to incorporate methyl branched acyl-CoA when added alone as a substrate (4) weakly incorporated cyclopropane acyl-CoA and was unable to incorporate methyl branched acyl-CoA when presented with an equimolar mix of oleyl-CoA and branched chain acyl-CoA. In all cases, the LPAAT had a low affinity for branched chain acyl-CoAs. The results show that LPAAT activity from agronomic plants constitutes a bottleneck for the incorporation of branched Chain acyl-CoA into PA.     

Mycobacterial Phosphatidylinositol Mannosides Negatively Regulate Host Toll-like Receptor 4, MyD88-dependent Proinflammatory Cytokines, and TRIF-dependent Co-stimulatory Molecule Expression

Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM₆ or PIM₂ exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon β (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFκB translocation. Synthetic PIM₁ and a PIM₂ mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.Multifold interactions between Mycobacterium tuberculosis and host phagocytes determine immune responses to M. tuberculosis and tuberculosis pathogenesis (for review, see Refs. 1 and 2). Alveolar macrophages, the primary host cells for M. tuberculosis, and dendritic cells that carry mycobacterial antigens from the infection site to the draining lymph nodes to establish a T cell-mediated immune response contribute to modulate the innate immune response by secreting cytokines after recognition of microbial motives. Among them, TNF² is an essential mediator for granuloma formation and containment of M. tuberculosis infection. Similarly, IL-12, interferon γ, but also IL-1, IL-18, IL-23, and nitric oxide are required for host defense (1–6). Phagocytes are also a source of immuno-modulatory cytokines, such as IL-10 and transforming growth factor-β, which dampen the immune response and inflammation. Mycobacteria-derived molecules down-modulating the immune system have been described, including the protein ESAT-6, mannose-capped lipoarabinomannan (ManLAM), and lipomannans (LM) (7–12). Here, we report that phosphatidyl-myo-inositol mannosides (PIM), the glycosylphosphatidylinositol (GPI) anchor structure of LAM and LM, exert strong anti-inflammatory activities.Mycobacterial cell wall LAM, LM, and PIM are recognized by macrophages and dendritic cells through various pattern recognition receptors, including Toll-like receptors (TLRs) (13–16) and C-type lectins such as mannose receptor (MR/CD206) and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN/CD209), central to M. tuberculosis binding and internalization by human dendritic cells (17–20). DC-SIGN and mannose receptor were proposed to mediate ManLAM inhibition of LPS-induced IL-12 production in dendritic cells, an activity ascribed to the mannosylated cap (8, 9). We showed recently that mycobacterial LM have a dual potential for pro-inflammatory and anti-inflammatory effects (11), tri- and tetra-acylated LM fractions exerting stimulatory effects through TLR2, TLR4, and MyD88 (21), whereas diacylated LM inhibit LPS-induced cytokine response independently of TLR2, SIGN-R1, and mannose receptor (12).PIM are biosynthetic precursors of LM and LAM (22–25). Dimannoside (PIM₂) and hexamannoside (PIM₆) PIM are the two most abundant classes of PIM found in M. tuberculosis H37Rv and Mycobacterium bovis BCG (see Fig. 1). PIM purification and molecular chemical characterization revealed four major acyl forms, mono- to tetra-acylated (lyso-PIM for one acyl, PIM for two acyl, Ac₁PIM for three acyl, and Ac₂PIM for four acyl, respectively; see Fig. 1) for both PIM₂ and PIM₆ (26–29). Higher order PIM with mannose cap-like structures were found to preferentially associate with human MR and to contribute to phagosome-lysosome fusion (20). The degree of acylation influenced higher order PIM association with the MR, whereas PIM₂ was recognized by DC-SIGN independently of its acylation degree. The complete synthesis of the different PIM has recently been reported (30–33).Open in a separate windowFIGURE 1.Natural PIM and synthetic PIM₁ and PIM₂ mimetics used in the study. Shown is a schematic representation of natural lyso-PIM₆, PIM₆, Ac₁PIM₆, Ac₂PIM₆, and PIM₂ (A) and synthetic PIM₁ (B) showing the C16 and C18 acyl groups on glycerol chain positions sn-2 and sn-1, the precursor PI, a synthetic mimetic of PIM₂ (PIM₂ mimetic) bearing C16 and C18 acyl chains, the de-acylated control molecule precursor of the PIM₂ mimetic (de-AcPIM₂ mimetic), and a PIM₂ mimetic with replacement of the phosphodiester moiety by a carbonate.Here, we analyzed isolated acyl forms of PIM and identified PIM₂ and PIM₆ but also synthetic PIM₁ and a mimetic of PIM₂ as strong inhibitors of endotoxin-induced proinflammatory responses in vitro and in vivo. Using macrophages from genetically modified mice, we characterized PIM inhibitory effects on MyD88, TRIF, and NFκB signaling pathways. Hence, not only complex mycobacterial lipoglycans like ManLAM and LM but also small molecular weight PIM analogues are potent inhibitors of host inflammatory responses.     

Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin

To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-alpha-[SD]-beta. This vector allows the co-expression of alpha-Hb as a fusion protein with Glutathione S-Transferase (GST-alpha-Hb) and beta-Hb with an additional methionine at the N-terminal extremity (rbeta-Hb). These proteins were solubilized as GST-alpha-Hb/rbeta-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-alpha-Hb/rbeta-Hb complex and recombinant Hb A exhibit the same allosteric behavior as for native Hb A. The GST moiety, which does not modify the function of the complex, can be easily eliminated by cleavage by the PreScission Protease. After cleavage during the rapid purification procedure, over 20mg of recombinant Hb per liter of culture were obtained, more than double the yield of previous co-expression systems. This polycistronic vector system, which offers the additional advantage of a very rapid purification, is especially well suited for the study of abnormal, unstable globins in order to better understand the associated pathology.     

The importance of clonal growth to the recovery of Gaultheria procumbens L. (Ericaceae) after forest disturbance

We investigated the importance of clonal growth to the recovery of a common eastern North American sub-shrub, Gaultheria procumbens L. (Ericacea), after clearcut logging. Changes in vegetative growth and development of G. procumbens clones and clonal populations were examined in a chronosequence of logged stands representing different stages of successional development after clearcutting (open habitat, young regenerating forest, closed regenerating forest) and in neighboring undisturbed late-successional forests representative of presettlement conditions. We specifically quantified seedling presence and above-ground ramet production, demographic condition (e.g., sexual vs. vegetative stems), belowground rhizome growth and spread, and assessed the degree of intraspecific variation in clonal morphology and biomass allocation in stands differing in their disturbance history and degree of successional development. Recovery in G. procumbens was largely driven by the “release growth” of pre-existing clonal bud-banks in response to canopy removal. Release growth was expressed as greater ramet initiation, rhizome branching and clonal spread. Conversely, we found no evidence of sexual establishment in the species, although production of reproductive biomass (e.g., inflorescence mass, number of flowering shoots) was significant. These findings support a deterministic model of vascular resistance and resilience to catastrophic disturbance, in which recovery of forest plant communities derives from the life-history characteristics of constituent species.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Use of response surface methodology to optimise environmental stress conditions on Penicillium glabrum, a food spoilage mould

Fungi are ubiquitous microorganisms often associated with spoilage and biodeterioration of a large variety of foods and feedstuffs. Their growth may be influenced by temporary changes in intrinsic or environmental factors such as temperature, water activity, pH, preservatives, atmosphere composition, all of which may represent potential sources of stress. Molecular-based analyses of their physiological responses to environmental conditions would help to better manage the risk of alteration and potential toxicity of food products. However, before investigating molecular stress responses, appropriate experimental stress conditions must be precisely defined. Penicillium glabrum is a filamentous fungus widely present in the environment and frequently isolated in the food processing industry as a contaminant of numerous products. Using response surface methodology, the present study evaluated the influence of two environmental factors (temperature and pH) on P. glabrum growth to determine 'optimised' environmental stress conditions. For thermal and pH shocks, a large range of conditions was applied by varying factor intensity and exposure time according to a two-factorial central composite design. Temperature and exposure duration varied from 30 to 50 °C and from 10 min to 230 min, respectively. The effects of interaction between both variables were observed on fungal growth. For pH, the duration of exposure, from 10 to 230 min, had no significant effect on fungal growth. Experiments were thus carried out on a range of pH from 0.15 to 12.50 for a single exposure time of 240 min. Based on fungal growth results, a thermal shock of 120 min at 40 °C or a pH shock of 240 min at 1.50 or 9.00 may therefore be useful to investigate stress responses to non-optimal conditions.     

Adiponectin, type 2 diabetes and the metabolic syndrome: lessons from human genetic studies

Adiponectin, a protein exclusively secreted by adipose tissue but present at low levels in obesity, is now widely recognised as a key determinant of insulin sensitivity and of protection against obesity-associated metabolic syndrome. In this review we explain how genetic findings have contributed to a better understanding of the physiological role of adiponectin in humans. The adiponectin-encoding gene, ADIPOQ (ACDC), is very polymorphic: many frequent exonic synonymous, intronic and promoter single-nucleotide polymorphisms (SNPs) have been identified, as well as a few rare exonic amino acid substitutions. Several of these variations additively contribute to the modulation of adiponectin level and function, and associate with insulin sensitivity, type 2 diabetes and vascular complications of obesity.