Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller)

Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602+/-1 Da for the major subunit and 23289+/-1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.     

Methylation of imidazoline related compounds leads to loss of α₂-adrenoceptor affinity. Synthesis and biological evaluation of selective I₁ imidazoline receptor ligands

Methylated analogues of imidazoline related compounds (IRC) were prepared; their abilities to bind I(1) imidazoline receptors (I(1)Rs), I(2) imidazoline binding sites (I(2)BS) and α(2)-adrenoceptor subtypes (α(2)ARs) were assessed. Methylation of the heterocyclic moiety of IRC resulted in a significant loss of α(2)AR affinity. Amongst the selective ligands obtained, LNP 630 (4) constitutes the first highly selective I(1)R agent showing hypotensive activity after intravenous administration.     

Glycogen loading alters muscle glycogen resynthesis after exercise.

This study compared muscle glycogen recovery after depletion of approximately 50 mmol/l (DeltaGly) from normal (Nor) resting levels (63.2 +/- 2.8 mmol/l) with recovery after depletion of approximately 50 mmol/l from a glycogen-loaded (GL) state (99.3 +/- 4.0 mmol/l) in 12 healthy, untrained subjects (5 men, 7 women). To glycogen load, a 7-day carbohydrate-loading protocol increased muscle glycogen 1.6 +/- 0.2-fold (P < or = 0.01). GL subjects then performed plantar flexion (single-leg toe raises) at 50 +/- 3% of maximum voluntary contraction (MVC) to yield DeltaGly = 48.0 +/- 1.3 mmol/l. The Nor trial, performed on a separate occasion, yielded DeltaGly = 47.5 +/- 4.5 mmol/l. Interleaved natural abundance (13)C-(31)P-NMR spectra were acquired and quantified before exercise and during 5 h of recovery immediately after exercise. During the initial 15 min after exercise, glycogen recovery in the GL trial was rapid (32.9 +/- 8.9 mmol. l(-1). h(-1)) compared with the Nor trial (15.9 +/- 6.9 mmol. l(-1). h(-1)). During the next 45 min, GL glycogen synthesis was not as rapid as in the Nor trial (0.9 +/- 2.5 mmol. l(-1). h(-1) for GL; 14.7 +/- 3.0 mmol. l(-1). h(-1) for Nor; P < or = 0.005) despite similar glucose 6-phosphate levels. During extended recovery (60-300 min), reduced GL recovery rates continued (1.3 +/- 0.5 mmol. l(-1). h(-1) for GL; 3.9 +/- 0.3 mmol. l(-1). h(-1) for Nor; P < or = 0.001). We conclude that glycogen recovery from heavy exercise is controlled primarily by the remaining postexercise glycogen concentration, with only a transient synthesis period when glycogen levels are not severely reduced.     

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

HGF/SF Induces Mesothelial Cell Migration and Proliferation by Autocrine and Paracrine Pathways

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.     

Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.     

A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

The prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats harbor diverse viruses, some of which are of particular interest because they cause severe human diseases. However, little is known about the diversity of the global population of viruses found in bats (virome). We determined the viral diversity of five different French insectivorous bat species (nine specimens in total) in close contact with humans. Sequence-independent amplification, high-throughput sequencing with Illumina technology and a dedicated bioinformatics analysis pipeline were used on pooled tissues (brain, liver and lungs). Comparisons of the sequences of contigs and unassembled reads provided a global taxonomic distribution of virus-related sequences for each sample, highlighting differences both within and between bat species. Many viral families were present in these viromes, including viruses known to infect bacteria, plants/fungi, insects or vertebrates, the most relevant being those infecting mammals (Retroviridae, Herpesviridae, Bunyaviridae, Poxviridae, Flaviviridae, Reoviridae, Bornaviridae, Picobirnaviridae). In particular, we detected several new mammalian viruses, including rotaviruses, gammaretroviruses, bornaviruses and bunyaviruses with the identification of the first bat nairovirus. These observations demonstrate that bats naturally harbor viruses from many different families, most of which infect mammals. They may therefore constitute a major reservoir of viral diversity that should be analyzed carefully, to determine the role played by bats in the spread of zoonotic viral infections.