Hydroxylation and deglycosylation of 2'-deoxyguanosine in the presence of magnesium and nickel

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2'-deoxyguanosine (dG) that has been caused by ascorbic acid and H2O2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H2O2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H2O2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2-7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2'-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.     

A mammalian in vitro model to study gangliogenesis from neural crest cells

In spite of considerable advances towards understanding lineages derived from neural crest cells using amphibian and avian embryos, the molecular mechanisms involved in the formation of mammalian peripheral ganglia remain largely unknown, mainly because of the lack of experimental systems that will allow their in vitro manipulation. Here, we present a novel mammalian in vitro model permitting to study gangliogenesis from neural crest cells. This model allowed us to manipulate molecules involved in cell-cell interactions. Our data are in favour of the existence of a hierarchy among adhesion molecules.     

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.     

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

Expression of the mammalian mitochondrial genome. Role for membrane potential in the production of mature translation products

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.     

Separation of poly(ADP-ribosylated) nuclear proteins by polyacrylamide gel electrophoresis at acidic pH and low temperature

A method for the extraction and electrophoresis of poly(ADP-ribosylated) nuclear proteins is described. An extraction method using lithium dodecyl sulfate as detergent at pH 2.4 and room temperature is shown to fully extract nuclear proteins under conditions where full stability of protein-linked polymer is ensured. The polyacrylamide gel electrophoresis is performed again under conditions where full stability is ensured. This work provides a technique whereby misinterpretation of relative ADP ribosylation of nuclear proteins can be avoided.     

Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.     

An increased requirement for methionine by transformed rat liver epithelial cells in vitro

The growth of two normal and four transformed rat liver epithelial cell lines in a methionine-containing medium and a methionine-deficient medium supplemented with homocysteine was examined. The growth rates of the normal cells on the homocysteine-supplemented medium were approximately one-half the growth rates shown by the same cells in the methionine-containing medium. In contrast, three of the four transformed cell lines studied showed virtually no growth on the homocysteine-supplemented medium, although they grew quite rapidly on the methionine-containing medium. The fourth, transformed by N-methyl-N-nitrosourea, was able to grow on the homocysteine-supplemented medium at about one-third the rate as on the methionine-containing medium. Thus, transformed rat liver epithelial cells resemble other malignant cells in their reduced capacity to grow on homocysteine in the absence of methionine.     

Older than New Caledonia emergence? A molecular phylogenetic study of the eneopterine crickets (Orthoptera: Grylloidea)

Aim A New Caledonian insect group was studied in a world‐wide phylogenetic context to test: (1) whether local or regional island clades are older than 37 Ma, the postulated re‐emergence time of New Caledonia; (2) whether these clades show evidence for local radiations or multiple colonizations; and (3) whether there is evidence for relict taxa with long branches in phylogenetic trees that relate New Caledonian species to geographically distant taxa. Location New Caledonia, south‐west Pacific. Methods We sampled 43 cricket species representing all tribes of the subfamily Eneopterinae and 15 of the 17 described genera, focusing on taxa distributed in the South Pacific and around New Caledonia. One nuclear and three mitochondrial genes were analysed using Bayesian and parsimony methods. Phylogenetic divergence times were estimated using a relaxed clock method and several calibration criteria. Results The analyses indicate that, under the most conservative dating scenario, New Caledonian eneopterines are 5–16 million years old. The largest group in the Pacific region dates to 18–29 Ma. New Caledonia has been colonized in two phases: the first around 10.6 Ma, with the subsequent diversification of the endemic genus Agnotecous, and the second with more recent events around 1–4 Ma. The distribution of the sister group of Agnotecous and the lack of phylogenetic long branches in the genus refute an assumption of major extinction events in this clade and the hypothesis of local relicts. Main conclusions Our phylogenetic studies invalidate a simple scenario of local persistence of this group in New Caledonia since 80 Ma, either by survival on the New Caledonian island since its rift from Australia, or, if one accepts the submergence of New Caledonia, by local island‐hopping among other subaerial islands, now drowned, in the region during periods of New Caledonian submergence.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla _OXA-23 was the most frequently detected (82%), followed by bla _OXA-24 (11%) and bla _OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.