Biocide susceptibility and intracellular glutathione inEscherichia coli

Summary Inhibition of growth and speed of kill by biocides with different mechanisms of action was examined with respect to intracellular glutathione levels. strain deficient in intracellular glutathione was hypersusceptible to electrophilic biocides, with the exception of an isothiazolone biocide. Growth inhibition by quaternary ammonium compounds and radical-generating biocides was unaffected by intracellular glutathione levels. Speed of kill experiments demonstrated a faster rate of killing by formaldehyde in both log and stationary phase cultures of the glutathione-deficient strain as compared to its wild-type parent. Glutathione levels had no effect on the speed of kill by hydrogen peroxide in log phase cultures, but resulted in an increased rate of killing in stationary phase cultures. Stationary phase cultures of the glutathione-deficient strain were killed by a quaternary ammonium biocide at a slower than the glutathione-replete strain. These studies provide information about both the mechanism of action of biocides as well as the role of glutathione in determining microbicide susceptibility.     

Effects of interleukin-15 on antifungal responses of human polymorphonuclear leukocytes against Fusarium spp. and Scedosporium spp

Fusarium spp. and Scedosporium spp. have emerged as important fungal pathogens that are frequently resistant to antifungal compounds. We investigated the effects of human interleukin-15 (IL-15) on human polymorphonuclear leukocyte (PMNL) activity against Fusarium solani and Fusarium oxysporum as well as Scedosporium prolificans and Scedosporium apiospermum. IL-15 (100 ng/ml) significantly enhanced PMNL-induced hyphal damage of both Fusarium spp. and S. prolificans after incubation for 22 h (P < 0.01) but not S. apiospermum. In addition, IL-15 enhanced PMNL oxidative respiratory burst evaluated as superoxide anion production in response to S. prolificans but not to the other fungi after 2 h incubation. IL-15 increased interleukin-8 (IL-8) release from PMNLs challenged with hyphae of F. solani and S. prolificans (P< or = 0.04). Release of tumor necrosis factor-alpha was not affected. The species-dependent enhancement of hyphal damage and induction of IL-8 release suggest that IL-15 plays an important role in the immunomodulation of host response to these emerging fungal pathogens.     

Localization of ERK/MAP kinase is regulated by the alphaherpesvirus tegument protein Us2

Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.     

Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.     

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.     

Carbonic anhydrase,carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO₂ (bubbled through the culture), (b) with added bicarbonate plus low added CO₂ (0.03% v/v), or (c) without added bicarbonate with high added CO₂ (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO₂ and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO₂. These results are compared with those in heterotrophic and photosynthetic microorganisms.Scientific Contribution Number 1241 from the New Hampshire Agricultural Experiment Station     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.     

Observations on graphing paleozoological data: Suggestions for better graphs

Paleozoologists have long used graphs of diverse styles to describe, analyze, and summarize their data. Some of these graphs provide excellent visual representations of complex data and are readily deciphered. Other graph styles require close study to be interpreted. Ease of visual decoding of information contained in a graph – graph perception – varies from graph style to graph style. Historical instances of graphing paleozoological data indicate some difficult to decipher graph styles have been used for at least a century. Graphs with three-dimensions, moiré effects, superimposed lines, or segmented bars, or which demand simultaneous decipherment of position and magnitude, are ill-advised. Temporal trends in data are best graphed following the principle of superposition such that data from old material is graphed at the bottom and data from younger material is graphed at the top of the diagram.     

A monoclonal antibody recognizes an epitope common to an avian-specific nuclear antigen and to cytokeratins

X3, a monoclonal antibody of unusual specificity, is described. This antibody reacts with one or more cytokeratin polypeptides and also reacts with an avian (chicken, quail) nuclear antigen that appears to be present in all cell types (chicken) tested, although with variable staining pattern and intensity. This antigen is distinct from the cytokeratins but does have an epitope in common with this class of proteins. It disappears from the nucleus during the early stages of cell division and reappears during anaphase as a granular cytoplasmic structure. In late telophase the antigen is relocated in the nucleus. This antigen, which we have designated as avian-specific nuclear antigen (AVNA), is not associated with chromatin or ribonucleoproteins. From immunoblotting experiments on chicken fibroblast nuclei, AVNA is probably a complex composed of one or several polypeptides, one of which has a molecular weight of approximately 60 kD. The proteins were identified as nuclear matrix proteins rather than pore complex-lamina proteins by immunoblotting experiments on the purified nuclear matrix of chicken erythrocytes. The major polypeptide had a molecular weight of 60 kD and the minor polypeptide a molecular weight of 69 kD.