Photocatalytic antimicrobial activity of thin surface films of TiO(2), CuO and TiO (2)/CuO dual layers on Escherichia coli and bacteriophage T4

TiO₂-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO₂, CuO and hybrid CuO/TiO₂ prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO₂ prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol–gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO₂ which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO₂/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-alpha in the mouse

Proton Nuclear Magnetic Resonance spectroscopy and Gas Chromatography Mass Spectrometry based metabolomics has been used in conjunction with multivariate statistics to examine the metabolic changes in Caenorhabditis elegans following the deletion of nuclear hormone receptor-49 (nhr-49). Deletion of the receptor produced profound changes in fatty acid metabolism, in particular an increase in the ratio of unsaturated to saturated fatty acids, a decrease in the concentration of glucose and increases in lactate and alanine. Given the proposed functional similarity between nhr-49 and the mammalian peroxisome proliferator-activated receptors (PPARs) these changes were compared with the metabolome of the PPAR-alpha null mouse. The metabolomic approach demonstrated a number of similarities including the regulation of lipid synthesis, beta-oxidation of fatty acids and changes in glycolysis/gluconeogenesis.     

Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML

We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO-binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO-dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.     

Self-recognition by an intrinsically disordered protein

The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.     

Cancer as an emergent phenomenon in systems radiation biology

Radiation-induced DNA damage elicits dramatic cell signaling transitions, some of which are directed towards deciding the fate of that particular cell, while others lead to signaling to other cells. Each irradiated cell type and tissue has a characteristic pattern of radiation-induced gene expression, distinct from that of the unirradiated tissue and different from that of other irradiated tissues. It is the sum of such events, highly modulated by genotype that sometimes leads to cancer. The challenge is to determine as to which of these phenomena have persistent effect that should be incorporated into models of how radiation increases the risk of developing cancer. The application of systems biology to radiation effects may help to identify which biological responses are significant players in radiation carcinogenesis. In contrast to the radiation biology paradigm that focuses on genomic changes, systems biology seeks to integrate responses at multiple scales (e.g. molecular, cellular, organ, and organism). A key property of a system is that some phenomenon emerges as a property of the system rather than of the parts. Here, the idea that cancer in an organism can be considered as an emergent phenomenon of a perturbed system is discussed. Given the current research goal to determine the consequences of high and low radiation exposures, broadening the scope of radiation studies to include systems biology concepts should benefit risk modeling of radiation carcinogenesis. Presented at the First International Workshop on Systems Radiation Biology, February 14–16 2007, GSF-Research Centre, Neuherberg, Germany.     

Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.     

Gonadotropin-releasing hormone stimulates prolactin release from lactotrophs in photoperiodic species through a gonadotropin-independent mechanism

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.     

Estradiol increases urethral tone through the local inhibition of neuronal nitric oxide synthase expression

Estrogens are known to modulate lower urinary tract (LUT) trophicity and neuronal nitric oxide synthase (nNOS) expression in several organs. The aim of this study was to explore the effects of endogenous and supraestrus levels of 17beta-estradiol (E2) on LUT and urethral nNOS expression and function. LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration, or castration plus chronic E2 supplementation (80 microg.kg(-1).day(-1), i.e., pregnancy level). The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher than those of ovariectomized mice. Urethral resistance was increased twofold in E2-treated mice, with no structural changes in urethra, supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression in ovariectomized mice. Long-term supraestrus levels of E2 increased urethral tone through inhibition of nNOS expression, whereas physiological levels of E2 had no effect.