Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.     

A targeted proteomics-based pipeline for verification of biomarkers in plasma

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.     

Multiple ATR-Chk1 pathway proteins preferentially associate with checkpoint-inducing DNA substrates

The ATR-Chk1 DNA damage checkpoint pathway is a critical regulator of the cellular response to DNA damage and replication stress in human cells. The variety of environmental, chemotherapeutic, and carcinogenic agents that activate this signal transduction pathway do so primarily through the formation of bulky adducts in DNA and subsequent effects on DNA replication fork progression. Because there are many protein-protein and protein-DNA interactions proposed to be involved in activation and/or maintenance of ATR-Chk1 signaling in vivo, we systematically analyzed the association of a number of ATR-Chk1 pathway proteins with relevant checkpoint-inducing DNA structures in vitro. These DNA substrates included single-stranded DNA, branched DNA, and bulky adduct-containing DNA. We found that many checkpoint proteins show a preference for single-stranded, branched, and bulky adduct-containing DNA in comparison to undamaged, double-stranded DNA. We additionally found that the association of checkpoint proteins with bulky DNA damage relative to undamaged DNA was strongly influenced by the ionic strength of the binding reaction. Interestingly, among the checkpoint proteins analyzed the checkpoint mediator proteins Tipin and Claspin showed the greatest differential affinity for checkpoint-inducing DNA structures. We conclude that the association and accumulation of multiple checkpoint proteins with DNA structures indicative of DNA damage and replication stress likely contribute to optimal ATR-Chk1 DNA damage checkpoint responses.     

Mesenchymal stem cells restore frataxin expression and increase hydrogen peroxide scavenging enzymes in Friedreich ataxia fibroblasts

Dramatic advances in recent decades in understanding the genetics of Friedreich ataxia (FRDA)--a GAA triplet expansion causing greatly reduced expression of the mitochondrial protein frataxin--have thus far yielded no therapeutic dividend, since there remain no effective treatments that prevent or even slow the inevitable progressive disability in affected individuals. Clinical interventions that restore frataxin expression are attractive therapeutic approaches, as, in theory, it may be possible to re-establish normal function in frataxin deficient cells if frataxin levels are increased above a specific threshold. With this in mind several drugs and cytokines have been tested for their ability to increase frataxin levels. Cell transplantation strategies may provide an alternative approach to this therapeutic aim, and may also offer more widespread cellular protective roles in FRDA. Here we show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. We demonstrate that normal human mesenchymal stem cells (MSCs) induce both an increase in frataxin gene and protein expression in FRDA fibroblasts via secretion of soluble factors. Finally, we show that exposure to factors produced by human MSCs increases resistance to hydrogen peroxide-mediated toxicity in FRDA fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest, for the first time, that stem cells may increase frataxin levels in FRDA and transplantation of MSCs may offer an effective treatment for these patients.     

Olfactory cues and nest recognition in the solitary bee Osmia lignaria

Abstract.  The use of olfactory cues for nest recognition by the solitary bee Osmia lignaria is studied in a greenhouse environment. Glass tubes are provided as nesting cavities to allow the in-nest behaviour of bees to be observed. In addition, each glass tube is cut into three sections for experimental manipulation and for subsequent chemical analysis. Nesting females drag their abdomen along the tube before exiting, spiral inside the tube, and sometimes deposit fluid droplets from the tip of the abdomen. For the manipulation, the outer section, the middle section, or both sections are removed and replaced with similar clean glass tube sections, and the behaviour exhibited by test females is recorded upon arrival in front of the nesting site and inside the nesting tubes. The resulting hesitation behaviour displayed by females after treatments appears to indicate the loss of some olfactory cues used for nest recognition inside the entire nest. Chemical analysis of the depositions inside the nesting tube, as well as analysis of the cuticular lipids of the nesting bees, reveals the presence of free fatty acids, hydrocarbons and wax esters.     

The histone deacetylase inhibitor trichostatin A alters the pattern of DNA replication origin activity in human cells

Eukaryotic chromatin structure limits the initiation of DNA replication spatially to chromosomal origin zones and temporally to the ordered firing of origins during S phase. Here, we show that the level of histone H4 acetylation correlates with the frequency of replication initiation as measured by the abundance of short nascent DNA strands within the human c-myc and lamin B2 origins, but less well with the frequency of initiation across the β-globin locus. Treatment of HeLa cells with trichostatin A (TSA) reversibly increased the acetylation level of histone H4 globally and at these initiation sites. At all three origins, TSA treatment transiently promoted a more dispersive pattern of initiations, decreasing the abundance of nascent DNA at previously preferred initiation sites while increasing the nascent strand abundance at lower frequency genomic initiation sites. When cells arrested in late G₁ were released into TSA, they completed S phase more rapidly than untreated cells, possibly due to the earlier initiation from late-firing origins, as exemplified by the β-globin origin. Thus, TSA may modulate replication origin activity through its effects on chromatin structure, by changing the selection of initiation sites, and by advancing the time at which DNA synthesis can begin at some initiation sites.     

Uterine activity, sperm transport, and the role of boar stimuli around insemination in sows

This paper describes changes in spontaneous myometrial activity around estrus, factors that affect myometrial activity, and the possible role of uterine contractions in the process of (artificial) insemination, sperm transport and fertilization. Myometrial activity in the sow increases during estrus. The activity is myogenic in origin, but several factors have been shown to affect myometrial activity. Natural mating stimulates uterine contractions through several mechanisms. The presence of a boar, rather than the act of mating, induces central oxytocin release in the sow and thus increases uterine activity. Estrogens in the ejaculate of a boar can trigger prostaglandin release by the endometrium and thus increase uterine activity. Tactile stimulation of the genital tract (cervix) or tactile stimulation of the back and flanks of the sow during artificial insemination does not cause a release of oxytocin. There is hardly any evidence for the effects of these latter stimuli on uterine activity, and if they are present at all, the effects are very small. Evidence for the effects of synthetic boar odor on oxytocin release and/or uterine activity is inconsistent. The mere presence of a boar during insemination, in contrast, clearly stimulates uterine activity through the release of oxytocin. Hormonal stimulation (intrauterine) of uterine activity with estrogens, prostaglandins, or oxytocins before, during or after insemination generally improves fertilization rate, especially in situations with reduced fertility. Therefore, uterine contractions are believed to play an important role in the transport of sperm cells to the oviducts after insemination. Whether uterine contractions are absolutely necessary for sperm transport through the uterine horns, however, is not clear. Intensive stimulation of uterine contractions using hormones can also reduce the fertilization rate, probably by increasing the reflux of sperm cells during insemination. In this respect, the presence of a boar during AI seems more adequate, as only sows with a low level of uterine activity show an increase in uterine activity in response to this stimulus.     

New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin

In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.