Selected biologically relevant ions at the air/water interface: a comparative molecular dynamics study

Interfacial behavior of selected biologically and technologically relevant ions is studied using molecular dynamics simulations employing polarizable potentials. Propensities of choline, tetraalkylammonium (TAA), and sodium cations, and sulfate and chloride anions for the air/water interface are analyzed by means of density profiles. Affinity of TAA ions for the interface increases with their increasing hydrophobicity. Tetramethylammonium favors bulk solvation, whereas cations with propyl and butyl chains behave as surfactants. The choice of counter-anions has only a weak effect on the behavior of these cations. For choline, sodium, chloride and sulfate, the behavior at the air/water interface was compared to the results of our recent study on the segregation of these ions at protein surfaces. No analogy between these two interfaces in terms of ion segregation is found.     

Glutathione Peroxidase-1 Regulates Mitochondrial Function to Modulate Redox-dependent Cellular Responses

Glutathione peroxidase-1 (GPx-1) is a selenocysteine-containing enzyme that plays a major role in the reductive detoxification of peroxides in cells. In permanently transfected cells with approximate 2-fold overexpression of GPx-1, we found that intracellular accumulation of oxidants in response to exogenous hydrogen peroxide was diminished, as was epidermal growth factor receptor (EGFR)-mediated Akt activation in response to hydrogen peroxide or EGF stimulation. Knockdown of GPx-1 augmented EGFR-mediated Akt activation, whereas overexpression of catalase decreased Akt activation, suggesting that EGFR signaling is regulated by redox mechanisms. To determine whether mitochondrial oxidants played a role in these processes, cells were pretreated with a mitochondrial uncoupler prior to EGF stimulation. Inhibition of mitochondrial function attenuated EGF-mediated activation of Akt in control cells but had no additional effect in GPx-1-overexpressing cells, suggesting that GPx-1 overexpression decreased EGFR signaling by decreasing mitochondrial oxidants. Consistent with this finding, GPx-1 overexpression decreased global protein disulfide bond formation, which is dependent on mitochondrially produced oxidants. GPx-1 overexpression, in permanently transfected or adenovirus-treated cells, also caused overall mitochondrial dysfunction with a decrease in mitochondrial potential and a decrease in ATP production. GPx-1 overexpression also decreased EGF- and serum-mediated [³H]thymidine incorporation, indicating that alterations in GPx-1 can attenuate cell proliferation. Taken together, these data suggest that GPx-1 can modulate redox-dependent cellular responses by regulating mitochondrial function.Accumulation of reactive oxygen species (ROS),² such as superoxide anion and hydrogen peroxide, is thought to contribute to cellular damage, apoptosis, and cell death (1–3); however, ROS production is part of normal cellular metabolism, and evidence is accumulating that hydrogen peroxide, in particular, may function as a signaling molecule necessary for cell growth and survival (4–8). Superoxide is generated as a byproduct of mitochondrial respiration and by cellular redox enzymes, such as NADPH oxidase, that are stimulated through receptor-mediated mechanisms (9). Hydrogen peroxide is formed from the dismutation of superoxide, which occurs spontaneously or can be catalyzed by superoxide dismutase (10) or, alternatively, is produced by the two-electron enzymatic reduction of molecular oxygen by various oxidases, such as xanthine oxidase (11). Recent studies also suggest that hydrogen peroxide may be directly generated by receptor-ligand interactions (12). One mechanism by which hydrogen peroxide may modulate signal transduction is through the reversible oxidation of proteins at redox-active cysteines, including, for example, thiols in tyrosine kinase phosphatases. Oxidation and inactivation of phosphatases, such as PTEN, have been shown to promote the activity of the pro-growth and -survival kinase, Akt (13).Antioxidant enzymes, such as glutathione peroxidase, catalase, and peroxiredoxins, serve to eliminate hydrogen peroxide, thereby regulating cellular responses to this endogenous oxidant. GPx-1 is a selenoprotein and one of a family of peroxidases that reductively inactivate peroxides using glutathione as a source of reducing equivalents (14, 15). GPx-1, in particular, is a major intracellular antioxidant enzyme that is found in the cytoplasm and mitochondria of all cell types. In cell culture models as well as in genetic mouse models, GPx-1 overexpression is associated with enhanced protection against oxidative stress (16–19); however, GPx-1-overexpressing mice can become obese and insulin-resistant, and have attenuated insulin-mediated activation of Akt (20). Thus, to study how GPx-1 modulates the effects of cellular oxidants on cell signaling and cell growth, we analyzed cellular responses to hydrogen peroxide and EGF in permanently transfected cells overexpressing GPx-1.     

Which habitats of European importance depend on agricultural practices?

The aim of this paper is to identify the habitat types listed in the Habitats Directive Annex I that require low-intensity agricultural management for their existence. We assessed the link between the Annex I habitat types and agricultural practices in order to identify habitat types that depend on the continuation of agricultural practices or whose existence is prolonged or spatially enlarged via blocking or reducing the secondary succession by agricultural activities. 63 habitat types that depend on or which can profit from agricultural activities—mainly grazing and mowing—were identified. They are classified into 2 groups: (1) habitats fully dependent on the continuation of agricultural management; (2) habitats partly dependent on the continuation of agricultural management. This paper also briefly discusses habitat types for which either doubts remain on their dependence on agricultural management, or the relation to extensive farming practices exists only in part of their area of distribution in Europe or under certain site conditions, respectively. Assessments of the conservation status of habitats of European Importance by 25 EU Member States in 2007 showed that habitats identified by us as depending on agricultural practices had a worse status than non-agricultural habitats.     

Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits

Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb of the human clotting factor VIII (hFVIII) cDNA and 4.6 kb of 3′ flanking sequences of mWAP gene were crossed for three generations. All transgenic animals showed stable transgene transmission. Transgenic females showed high level of recombinant hFVIII (rhFVIII) mRNA expression in biopsed mammary gland tissues, while marginal expression of rhFVIII mRNA was observed in the spleen, lung and brain. No adverse effects of ectopic expression on the physiology of the rabbits were observed. Expression was not detected in the liver, kidney, heart and skeletal muscle. In transgenic females derived from three generations, rhFVIII protein was secreted from the mammary gland of lactating females, as shown by Western blotting. Biological activity of rhFVIII protein, as revealed in clotting assays was ranged from 0.012 to 0.599 IU/ml corresponding to 1.2% and 59.9% of the hFVIII level in normal human plasma. No apparent effect of secreted rhFVIII on the milk performance of rabbits was observed. Our results confirm the possibility of producing a significant amount of a biologically active rhFVIII in the mammary gland of established transgenic rabbit lines.     

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.