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Days Incubation (day + incubation)
Selected AbstractsDetecting active methanogenic populations on rice roots using stable isotope probingENVIRONMENTAL MICROBIOLOGY, Issue 3 2005Yahai Lu Summary Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/13CO2 or N2/13CO2 with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system. [source] Studies on the cell treatment conditions to elicit lipolytic responses from 3T3-L1 adipocytes to TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Wen Li Abstract Wasting syndrome is one of the hallmark symptoms of poisoning by TCDD (=dioxin), which is associated with the massive loss of adipose tissue and serum hyperlipidemia in vivo. Yet, the most widely used in vitro cell model 3T3-L1 adipocyte has not been useful for studying such an action of TCDD because of the difficulty of inducing their mature adipocytes to respond to TCDD to go through lipolysis. Here, we made efforts to find the right cell culture and treatment conditions to induce mature 3T3-L1 adipocytes to go through lipolysis, which is defined as events leading to reduction of lipids in adipocytes. The optimum condition was found to require 7-day differentiated adipocytes being subjected to DMEM medium containing TCDD (but without insulin) for 5 day incubation with two medium changes (the same composition) on incubation days 2 and 4. After 24 h, the early effect of TCDD on adipocytes was predominantly on inflammation, particularly induction of COX-2 and KC (IL-8), which is accompanied by upregulation of C/EBP, and ,. The sign of TCDD-induced lipolysis starts slowly and by incubation day 3, a few markers showed modestly significant changes. By day 5 of incubation, however, many markers show highly significant signs of lipolytic changes. Although this process could take place without exogenous macrophages or their cytokines, addition of exogenous TNF, considerably synergized this action of TCDD. In conclusion, under a right condition, 3T3-L1 adipocytes were found to respond to TCDD to go through lipolysis. The early trigger of such a response appears to be activation of COX-2, which is amplified by TNF,. J. Cell. Biochem. 102: 389,402, 2007. © 2007 Wiley-Liss, Inc. [source] Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's StabilityBIOTECHNOLOGY PROGRESS, Issue 6 2002Rodrigo Torres Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme. [source] Reproducibility of a microbial river water community to self-organize upon perturbation with the natural chemical enantiomers, R - and S -carvoneFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Katja Lehmann Abstract A river water microbial community was studied in response to perturbation with the monoterpene enantiomers R - and S -carvone. The microbial community structure and function was also evaluated after enantiomers exposure was switched. Microbial communities were evaluated by length heterogeneity PCR. The addition of R - and S -carvone enriched for a range of functionally different communities: enantiomer-selective, racemic and ones that contain both. After 5 days incubation, the R - and S -carvone treatments developed a range of dominant microbial communities, which were increasingly dissimilar from the ones in which no carvone degradation had taken place (R -values: R -carvone 0.743, S -carvone 0.5007). There was an increase in the evenness of the microbial community structure upon carvone depletion. After the cross-over, the rate of carvone utilization was significantly faster than after the first carvone addition (P=0.008) as demonstrated by concomitant carvone and oxygen depletion. The main R -degrading community (450,456 bp) appeared enantioselective and largely unable to degrade S -carvone, whereas the S -carvone-degrading community (502,508 bp) appeared to have racemic catabolic capacity. In conclusion, chemical perturbations, such as enantiomers, might generate a significant shift in the river microbial ecology that can have implications for the function of a river in both a spatial and temporal context. [source] Cytotoxic effects of ,, T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapyINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Kiyoshi Sato Abstract Nitrogen containing-bisphosphonates (N-BPs), widely used to treat bone diseases, have direct antitumor effects via the inactivation of Ras proteins. In addition to the direct antitumor activities, N-BPs expand gd,,T cells, which exhibit major histocompatibility complex-unrestricted lytic activity. BPs accumulate intermediate metabolites which may be tumor antigens in target cells. The purpose of our study was to clarify the cytotoxicity of gd,, T cells expanded ex vivo by the most potent N-BP, zoledronate (ZOL). Especially, we focused on the importance of pretreatment against target cells also with ZOL; 1 m,M ZOL plus IL-2 increased the absolute number of gd,,T cells 298,768 fold for 14 days incubation. The small cell lung cancer and fibrosarcoma cell lines pretreated with 5 m,M ZOL showed a marked increase in sensitivity to lysis by gd,,T cells. While, untreated cell lines were much less sensitive to lysis by gdT cells. Video microscopy clearly demonstrated that gd,,T cells killed target cells pre-treated with ZOL within 3 hr. Pretreatment with 80 m,g/kg ZOL also significantly enhanced the antitumor activity of gd,,T cells in mice xenografted with SBC-5 cells. These findings show that ZOL significantly stimulated the proliferation of gd,,T cells and that gd,,T cells required pre-treatment with ZOL for cytotoxic activity against target cells. © 2005 Wiley-Liss, Inc. [source] African horse sickness epidemiology: vector competence of South African Culicoides species for virus serotypes 3, 5 and 8MEDICAL AND VETERINARY ENTOMOLOGY, Issue 3 2000G. J. Venter Summary The oral susceptibilities of 17 Culicoides species to infection with African horse sickness virus (AHSV) serotypes 3, 5 and 8 were determined by feeding field-collected midges on AHSV infected horse blood. The mean titres of virus in the bloodmeals for the three serotypes of AHSV were between 5.7 and 6.5 log10TCID50/ml. Virus was detected, after 10 days incubation at 23.5°C, in the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) that had fed on blood containing AHSV 5 (8.5%) and 8 (26.8%), and in the Culicoides bolitinos Meiswinkel that had fed on AHSV 3 (3.8%), 5 (20.6%) and 8 (1.7%). Although 44.4% of the C. imicola were shown to have ingested AHSV 3 immediately after feeding, no virus was detected in 96 C. imicola after incubation. The relatively high titres of virus recorded in individual midges of both species after 10 days incubation suggested a fully disseminated infection. Previously, C. imicola was considered to be the only field vector of AHSV in Africa. Identifying C. bolitinos as a potential vector for AHSV is an important finding, which if proven will have a significant impact on our understanding of the epidemiology of AHS. No AHSVs could be detected in the other 15 species of Culicoides assayed, which suggests that some of the southern African Culicoides species are refractory to AHSV infection. However, further work with larger numbers of each species will be necessary to confirm this observation. [source] A mini-bag technique for evaluation of fungicide effects on Trichoderma spp in mushroom compostPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 4 2004Salem O Abosriwil Abstract An in vivo technique was developed to observe colonisation of mushroom compost by Trichoderma spp. Isolates of T harzianum (Th2), T harzianum (Th1), T koningii (Tk) and T viride (Tv) were artificially introduced into compost using a mini-bag system. Wheat grains, colonised by Trichoderma spp, were placed centrally on a layer of compost at the bottom of 1-litre polythene bags which were then filled with 350 g of spawned or un-spawned compost, and partially sealed. After 14 and 21 days incubation at 27 °C, the bags were assessed for recovery of Trichoderma from middle and top zones using a needle stab re-isolation technique and a visual colonisation scoring system. Visible green mould contamination, similar to that observed in practice, developed within 21 days. The visual colonisation scoring was reliably related to the re-isolation success. In this evaluation, Trichoderma spp showed considerable differences in their relative abilities to colonise spawned and un-spawned compost, with Th2 isolates being consistently superior to the other isolates of Th1, Tk and Tv in colonising spawned compost. This technique was employed to evaluate the effects of fungicides on the colonisation of mushroom compost by three Trichoderma spp: Th2, Th1 and Tk, using 1-litre and 5-litre mini-bag systems. Aqueous suspensions of benomyl, carbendazim, thiabendazole, prochloraz and prochloraz + carbendazim incorporated into the compost at 50 mg litre,1, or applied to spawn at 50 mg kg,1, reduced the colonisation by Trichoderma spp. Prochloraz and prochloraz + carbendazim were superior to benomyl, carbendazim or thiabendazole in reducing compost colonisation by Th2, Th1 and Tk, with Th2 being the most persistent type, capable of colonising treated compost in the presence of all five fungicides. The prochloraz + carbendazim mixture, not normally used in mushroom production, was equal to or better than prochloraz alone. The incidence of green mould colonisation by Th2 was as extensive in the 5-litre compost bags as in the 1-litre bags, but colonisation by Th1 and Tk was more apparent in the 5-litre bags. The in vivo mini-bag evaluations using wheat grain Trichoderma inoculum and needle stab re-isolation procedures proved an efficient method for studying colonisation and screening for effectiveness of fungicides applied to mushroom compost or spawn. Copyright © 2003 Society of Chemical Industry [source] Apoptotic activity of doxazosin on prostate stroma in vitro is mediated through an autocrine expression of TGF-,1THE PROSTATE, Issue 3 2001Kenneth Y. Ilio Abstract Background Doxazosin, an alpha-adrenergic antagonist, has been shown to induce apoptosis in prostatic stromal cells. The mechanism of this apoptotic action by Doxazosin remains undefined. The present study was carried out to demonstrate that the effect of Doxazosin on apoptosis of prostate stromal cells is mediated through an autocrine action of TGF-,1. Methods Primary cultures of human prostate cells were treated with varying concentrations of Doxazosin (0, 0.1, 1, 10, and 100 ,M) for a period up to 3 days. At the end of the 3-day culture, cell numbers were counted. Apoptosis was assessed by a colorimetric terminal deoxyribonucleotide transferase labeling technique. TGF-,1 was determined by enzyme-linked immunosorbent assay (ELISA). Results Compared to control cultures, cell numbers were significantly decreased as much as 68.4% in cultures treated with 10 ,M of Doxazosin after 3 days incubation, while apoptosis increased by 64.7% in cultures treated with the same concentration of Doxazosin after 24 h. This decrease in cell number was reversed when antibody to TGF-,1 was added to these cultures. Addition of TGF-,1 (0, 1.0, and 10 ng/mL) to the cultures also decreased the cell numbers. Quantitation of TGF-,1 in lysates of cells by ELISA revealed that the cells treated with Doxazosin (10 ,M) produced as much as 62.5% more TGF-,1 than in that of untreated cells. Conclusions These results demonstrate that the apoptotic effect of Doxazosin on human prostatic stromal cells is mediated through an autocrine production of TGF-,1. Prostate 48:131,135, 2001. © 2001 Wiley-Liss, Inc. [source] Effects of sublethal levels of tributyltin chloride on a new toxicity test organism, Liza saliens (osteichthyes, mugilidae): a histological studyAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 6 2006P. D'Agati Abstract The histopathological effects of 10,7 and 10,9M tributyltin(IV)chloride,TBTCl, solutions on different Liza saliens organs have been studied by light microscope. The fish were sacrificed after 3,4 h incubation in 10,7M TBTCl solution or after 15 days incubation in 10,9M solution. The observed histopathological changes were dose- and time-dependent. The 10,7M TBTCl concentration resulted in major damage to the gill epithelium, indicating that TBTCl primarily interfered with the respiration, osmoregulation, acid balance and nitrogenous waste excretion processes. After incubation in 10,9M TBTCl solution the fish lived 20 or more days, but many of the organs were altered. Thymus atrophy, reduced spleen and altered head kidney were observed. These histological results indicated that TBTCl interfered with organ immunodefense and altered main metabolic pathways in Liza saliens. The presence of melano-macrophage centers, only in TBT-treated liver and spleen, can be considered a tool to facilitate, with other biomarkers, the detection of alterations by toxicants. Regarding the pancreas activity in 10,7M solutions, it has been noted that, in the exocrine cells, very few zymogen granules were still present and the Langerhans islets were more altered. In 10,9M solution the exocrine pancreatic cells had no granules and the islet cells presented degenerative alterations. In addition, TBTCl, which altered the pancreas and gonad morphology, could again be considered an endocrine disrupter even if biochemical data are still necessary. Finally, the Liza saliens juveniles could be considered an interesting biological model for experiments with contaminants, due to their ease of adaptation to experimental conditions and food chain position. Copyright © 2005 John Wiley & Sons, Ltd. [source] Cyst-based toxicity tests XII,Development of a short chronic sediment toxicity test with the ostracod crustacean Heterocypris incongruens: Selection of test parametersENVIRONMENTAL TOXICOLOGY, Issue 6 2002Belgis Chial Abstract Experiments were carried out with neonates of the freshwater ostracod Heterocypris incongruens hatched from cysts in order to develop a new culture/maintenance-free solid-phase microbiotest for the toxicity assessment of contaminated sediments. Based on preliminary investigations, a number of test parameters were investigated for a short-chronic assay: hatching time, size of the cups of the multiwell test plates, feeding of the test organisms prior to the test, amount of supplemental algal food, volume of sediment, and duration of the test. On the basis of the findings, a test protocol was formulated for a 6-day assay in 12-cup multiwell plates with 10 organisms per cup and 3 replicates. The test organisms were collected 52 h after the start of the incubation of the cysts in standard freshwater at 25°C under continuous illumination after a 4-h prefeeding with 1.3 mg/mL Spirulina. The test biota in the cups were exposed to 300 ,L of test sediment in 2 mL of standard freshwater with 3 × 107 live algal cells (Raphidocelis subcapitata) as food supplement. Calibrated sand was used as a reference sediment. Mortality and growth of the ostracods were determined after 6 days' incubation at 25°C in darkness. The selected test parameters for the new microbiotest were found adequate for toxicity determination of natural sediments compared with the 10-day contact test with the amphipod Hyalella azteca. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 520,527, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10085 [source] | ||||||||||||||||