Identification and characterization of an essential telomeric repeat binding factor in fission yeast

Whereas mammalian cells harbor two double strand telomeric repeat binding factors, TRF1 and TRF2, the fission yeast Schizosaccharomyces pombe has been thought to harbor solely the TRF1/TRF2 ortholog Taz1p to perform comparable functions. Here we report the identification of telomeric repeat binding factor 1 (Tbf1), a second TRF1/TRF2 ortholog in S. pombe. Like the Taz1p, the identified Tbf1p shares amino acid sequence similarity, as well as structural and functional characteristics, with the mammalian TRF1 and TRF2 proteins. This family of proteins shares a common architecture with two separate structural domains. An N-terminal domain is necessary and sufficient for the formation of homodimers, and a C-terminal MYB/homeodomain mediates sequence specific recognition of double-stranded telomeric DNA. The identified Tbf1p binds S. pombe telomeric DNA with high sequence specificity in vitro. Targeted deletion of the tbf1 gene reveals that it is essential for survival, and overexpression of the tbf1 gene leads to telomere elongation in vivo, which is dependent upon the MYB domain. These data suggest that fission yeast, like mammals, have two factors that bind double-stranded telomeric DNA and perform distinct roles in telomere length regulation.     

Up-regulation of vimentin expression in low-density malignant glioma cells as immediate and late effects under irradiation and temozolomide treatment

Nervous system tumors are one of the leading causes of cancer related death. Specific mechanisms facilitating the invasive behavior of gliomas remain obscure. Advanced simulation models of the in vivo response to therapy conditions should potentially improve malignant glioma treatment. Expressional profiling of vimentin––one of reliable pro-invasive tumor makers––in those simulation models was the goal of this study, in order to estimate a pro-invasive response of surviving malignant glioma cells under clinically relevant therapeutic conditions. Human U87-MG malignant glioma cells were used. These cells are characterized by the wild p53-phenotype, which is relevant for the majority of primary malignant glioblastomas. Experimental design foresaw the cells to undergo either irradiation or chemo-treatment with temozolomide alone, or combined treatment. Expression profiling of vimentin was performed by quantitative “Real-Time”-PCR under all treatment conditions simulating diverse tumor regions. Here we demonstrated that vimentin expression patterns in human malignant glioma cells strongly depend on cellular density, algorithms of drug delivery and chemo/radio treatment. Substantial differences were recognized between immediate and late therapy effects. Significant increase in vimentin expression levels was detected particularly in low-density cell cultures under durable treatment with constant concentration levels of temezolomide. Simulation of variable intratumoral regional conditions (central intratumoral regions vs. disseminated malignant cells in peripheral regions) demonstrated differential response of vimentin expression in malignant glioma cell cultures treated under clinically relevant conditions. Slight ebbing of expression levels as late effects of the treatment in confluent cultures may correspond to necrotic processes clinically observed in central intratumoral regions. Contrary, in disseminated malignant cells of peripheral regions therapy resulted in vimentin-inducing effects. This is in agreement with the clinical observations of an increased aggressiveness and malignancy grade of post-operatively chemo/radio-treated malignant gliomas.     

Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.     

RosE represses Std fimbrial expression in Salmonella enterica serotype Typhimurium

The Salmonella enterica serotype Typhimurium ( S. typhimurium ) genome contains a large repertoire of putative fimbrial operons that remain poorly characterized because they are not expressed in vitro . In this study, insertions that induced expression of the putative stdABCD fimbrial operon were identified from a random bank of transposon mutants by screening with immuno-magnetic particles for ligand expression (SIMPLE). Transposon insertions upstream of csgC and lrhA or within dam , setB and STM4463 (renamed rosE ) resulted in expression of StdA and its assembly into fimbrial filaments on the cell surface. RosE is a novel negative regulator of Std fimbrial expression as indicated by its repression of a std :: lacZ reporter construct and by binding of the purified protein to a DNA region upstream of the stdA start codon. Expression of Std fimbriae in the rosE mutant resulted in increased attachment of S. typhimurium to human colonic epithelial cell lines (T-84 and CaCo-2). A rosE mutant exhibited a reduced ability to compete with virulent S. typhimurium for colonization of murine organs, while no defect was observed when both competing strains carried a stdAB deletion. These data suggest that a tight control of Std fimbrial expression mediated by RosE is required during host pathogen interaction.     

Leukotrienes and 8-isoprostane in exhaled breath condensate in bronchoprovocation tests with occupational allergens

Exhaled breath condensate (EBC) contains many substances, which could help in diagnosis of occupational asthma. The aim of the study is to monitor leukotrienes (LT) and 8-isoprostane from EBC in bronchoprovocation tests with allergens in 47 patients with suspected occupational asthma. Forty-one patients were tested negative. In negative bronchoprovocation tests, no significant differences (P<0.05) were seen between the five measurements during and after the test. In control measurements (without provocation), significant differences were found among four measurements done within 24h for 8-isoprostane (P=0.0138). The relationship between the log transformed ratios of the EBC parameters and FEV(1) was never significant at the 5% level in control measurements, while in negative tests, statistical significance was recorded for LTB(4) (P=0.0299) before and 5h after the test. Six of 47 patients were tested positive. Such a small number of patients did not allow proper statistical analysis and therefore, the results are described separately for each patient.     

SRC-dependent signalling regulates actin ruffle formation induced by glycerophosphoinositol 4-phosphate

The glycerophosphoinositols are diffusible phosphoinositide metabolites reported to modulate actin dynamics and tumour cell spreading. In particular, the membrane permeant glycerophosphoinositol 4-phosphate (GroPIns4P) has been shown to act at the level of the small GTPase Rac1, to induce the rapid formation of membrane ruffles. Here, we have investigated the signalling cascade involved in this process, and show that it is initiated by the activation of Src kinase. In NIH3T3 cells, exogenous addition of GroPIns4P induces activation and translocation of Rac1 and its exchange factor TIAM1 to the plasma membrane; in addition, in in-vitro assays, GroPIns4P favours the formation of a protein complex that includes Rac1 and TIAM1. Neither of these processes involves direct actions of GroPIns4P on these proteins. Thus, through the use of specific inhibitors of tyrosine kinases and phospholipase C (and by direct evaluation of kinase activities and inositol 1,4,5-trisphosphate production), we show that GroPIns4P activates Src, and as a consequence, phospholipase Cgamma and Ca(2+)/calmodulin kinase II, the last of which directly phosphorylates TIAM1 and leads to TIAM1/Rac1-dependent ruffle formation.     

Calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells

The aminoglycoside antibiotic gentamicin elicits proximal tubular toxicity and cell death. In calcium-sensing receptor (CaR)-transfected HEK-293 (CaR-HEK) cells and CaR-expressing proximal tubule-derived opossum kidney (OK) cells, chronic gentamicin treatment elicits dose-dependent, caspase-mediated apoptotic cell death. Here we investigated whether the renal cell toxicity of the CaR agonist gentamicin could be prevented by CaR antagonism or by lithium cotreatment which may interfere with receptor-mediated signalling. Chronic treatment of OK and CaR-HEK cells with low concentrations of gentamicin elicited cell death, an effect that was ameliorated by cotreatment with the CaR negative allosteric modulator (calcilytic) NPS-89636. This calcilytic also attenuated CaR agonist-induced ERK activation in these cells. In addition, 1 mM LiCl, equivalent to its therapeutic plasma concentration, also inhibited gentamicin-induced toxicity in both cell types. This protective effect of lithium was not due to the disruption of phosphatidylinositol-mediated gentamicin uptake as the cellular entry of Texas red-conjugated gentamicin into OK and CaR-HEK cells was unchanged by lithium treatment. However, the protective effect of lithium was mimicked by glycogen synthase 3beta inhibition. Together, these data implicate CaR activation and a lithium-inhibitable signalling pathway in the induction of cell death by gentamicin in renal epithelial cells in culture.