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DAPI Staining (dapi + staining)
Selected AbstractsDominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organismsENVIRONMENTAL MICROBIOLOGY, Issue 6 2008Nicolai Müller Summary Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 108 cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22,28 h at 28°C. Cells were small, 0.5 × 5 ,m in size, Gram-positive, and formed terminal oval spores. At 20°C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO2. At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture. [source] Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotypeENVIRONMENTAL MICROBIOLOGY, Issue 1 2008Florin Musat Summary The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the ,candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation. [source] Effect of H. pylori on the Expression of TRAIL, FasL and their Receptor Subtypes in Human Gastric Epithelial Cells and their Role in ApoptosisHELICOBACTER, Issue 5 2004Jan Hendrik Martin ABSTRACT Background and Aims., In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. Materials and Methods., mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. Results., TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. Conclusions., Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection. [source] Novel quinolone CHM-1 induces apoptosis and inhibits metastasis in a human osterogenic sarcoma cell lineJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2009Shu-Chun Hsu Abstract Novel 2-phenyl-4-quinolone compounds have potent cytotoxic effects on different human cancer cell lines. In this study, we examined anticancer activity and mechanisms of 20-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) in human osterogenic sarcoma U-2 OS cells. CHM-1-induced apoptosis was determined by flow cytometric analysis, DAPI staining, Comet assay, and caspase inhibitors. CHM-1-inhibited cell migration and invasion was assessed by a wound healing assay, gelatin zymography, and a Transwell assay. The mechanisms of CHM-1 effects on apoptosis and metastasis signaling pathways were studied using Western blotting and gene expression. CHM-1 induced G2/M arrest and apoptosis at an IC50 (3 µM) in U-2 OS cells and caspase-3, -8, and -9 were activated. Caspase inhibitors increased cell viability after exposure to CHM-1. CHM-1-induced apoptosis was associated with enhanced ROS generation, DNA damage, decreased ,,m levels, and promotion of mitochondrial cytochrome c release. CHM-1 stimulated mRNA expression of caspase-3, -8, and -9, AIF, and Endo G. In addition, CHM-1 inhibited cell metastasis at a low concentration (<3 µM). CHM-1 inhibited the cell metastasis through the inhibition of MMP-2, -7, and -9. CHM-1 also decreased the levels of MAPK signaling pathways before leading to the inhibition of MMPs. In summary, CHM-1 is a potent inducer of apoptosis, which plays a role in the anticancer activity of CHM-1. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1637,1644, 2009 [source] MACROPHAGE INFILTRATION AND INDUCTION OF P75 NTR AND IL-1B IN THE NERVE OF DIABETIC RATSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000G. Conti Recently, inflammation has been involved in the pathogenesis of diabetic neuropathy, and activated macrophages have been found in the peripheral nervous system of diabetic rats, with a possible role in chemotaxis and regeneration. In this study, we obtained sciatic nerve specimens from diabetic rats at different time points following STZ administration. Macrophages infiltration, IL-1b and p75NTR induction were analyzed by immunocytochemistry on frozen sections and on teased nerve fibers. Apoptosis was detected on teased nerve fibers by TUNEL and DAPI staining. Cell phenotype was characterized by double-staining with antibodies specific for Schwann cells and macrophages. The nerves obtained from STZ-diabetic rats showed macrophages infiltration by day 14 following STZ administration, with complete clearance by day 35. Fifteen percent of these cells were TUNEL positive. IL-1B induction was concomitant with macrophages infiltration and not detectable by day 35. p75NTR expression began by day 21, peaking by day 35, and dropping to barely detectable levels by day 105. These findings seem to indicate that the concomitance of these processes may be crucial in the regulation of nerve damage and in promoting an attempt of regeneration at the early stages of STZ diabetic neuropathy. [source] SCHWANN CELL APOPTOSIS IN TISSUE CULTURE FOLLOWING THE ADMINISTRATION OF PRO-INFLAMMATORY CYTOKINESJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000E. Scarpini In immune-mediated demyelination of the nervous system, glial cell apoptosis has been observed recently; however, the relevance of the phenomenon and the characterization of the involved molecules are still controversial. Cytokines are secreted by many cells, including inflammatory and glial cells, and appear to play a relevant role in the peripheral nervous system (PNS) immuno-mediated demyelination, being active in promoting the damage to Schwann cells, myelin, and axons. Even though the exact role of the different cytokines is at present uncertain, they have a sequential different expression in PNS immune-mediated demyelination and could induce apoptotic death of Schwann cells in the vicinity of the inflammatory reaction via the expression of CD95 (Apo1/Fas). This study has been designed to detect in rat primary Schwann cell tissue cultures whether the administration of IL-1B and IFN-y can induce cell death. Identification of apoptotic Schwann cell was performed by morphological, immunohistochemical, and electron-microscopy analysis. Our results show that Schwann cells stimulated by proinflammatory cytokines IL-1B and IFN-y show morphological evidence of nuclear chromatin condesation at the DAPI staining and are TUNEL positive. The same features of apoptotic cell death were observed by electron microscopy. These findings provide evidence to support the hypothesis that cytokines can directly damage Schwann cells in disorders of the PNS. [source] Neuroprotective effect of luteolin on amyloid , protein (25,35)-induced toxicity in cultured rat cortical neuronsPHYTOTHERAPY RESEARCH, Issue S1 2010Hao-Yuan Cheng Abstract The present study was carried out to investigate the neuroprotective effect of luteolin on amyloid , (A,) (25,35)-induced neurotoxicity using cultured rat cortical neurons. After exposure of primary cultures of rat cortical cells to 10 ,M A, (25,35) for 48 h, cortical cell cultures exhibited marked apoptotic death. Pretreatment with luteolin (1, 10 ,M) significantly protected cortical cell cultures against A, (25,35)-induced toxicity. Luteolin (1, 10 ,M) showed a concentration-dependent inhibition on 10 ,M A, (25,35)-induced apoptotic neuronal death, as assessed by MTT assay. Furthermore, luteolin reduced apoptotic characteristics by DAPI staining. For Western blot analysis, the results showed that the protective effect of luteolin on A, (25,35)-induced neurotoxicity was mediated by preventing of ERK-p, JNK, JNK-p, P38-p and caspase 3 activations in rat primary cortical cultures. Taken together, the results suggest that luteolin prevents A, (25,35)-induced apoptotic neuronal death through inhibiting the protein level of JNK, ERK and p38 MAP kinases and caspase 3 activations. Copyright © 2009 John Wiley & Sons, Ltd. [source] | |||||||||||||