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Media Containing (media + containing)
Selected AbstractsCellular and molecular basis of cadmium-induced deformities in zebrafish embryosENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2000Shuk Han Cheng Abstract Cadmium is known to cause developmental defects in a varietyof vertebrate species, but relatively little is known about the underlying molecular mechanisms. In this study, we used zebrafish (Danio rerio) embryos as a model system to investigate cadmium-induced toxicities. Fertilized embryos collected at 5-h after fertilization were incubated for 18 h in culture media containing 1 to 1, 000 ,M CdCl2. The median embryolethal concentration (LC50) was 168 ,M, whereas the median effect concentration (EC50) for total adverse effect (mortality and developmental defects) was 138 ,M. Six major types of deformities were observed: head and eye hypoplasia, hypopigmentation, cardiac edema, yolk sac abnormalities, altered axial curvature, and tail malformations. The frequency of malformations increased with cadmium concentration. Somites of embryos with altered axial curvature were investigated using the antimyosin antibody MF-20. This study demonstrated, to our knowledge for the first time, reduced myotome formation in cadmium-induced spinal deformity. Embryos with head and eye hypoplasia were studied using the anti-neural tissue antibody zns-2, and a poorly developed central nervous system was revealed. Head and eye hypoplasia were associated with lack of expression of the sonic hedgehog gene, which controls the patterning of the neural tube and somites. Genes involved in tail formations, such as evenskipped 1 and no tail, were ectopically expressed in embryos with tail malformations. Our data support the hypothesis that fish embryonic malformations induced by cadmium might be mediated through ectopic expression of developmental regulatory genes. [source] The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009A.A.T. De Carvalho Abstract Aims:, To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. Methods and Results:, Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l,1 of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l,1, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. Conclusions:,Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. Significance and Impact of the Study:, The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria. [source] Homeostasis of neuroactive amino acids in cultured cerebellar and neocortical neurons is influenced by environmental cuesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Helle Waagepetersen Abstract Neuronal function is highly influenced by the extracellular environment. To study the effect of the milieu on neurons from cerebellum and neocortex, cells from these brain areas were cultured under different conditions. Two sets of cultures, one neocortical and one cerebellar neurons, were maintained in media containing [U- 13C]glucose for 8 days at initial concentrations of 12 and 28 mM glucose, respectively. Other sets of cultures (8 days in vitro) maintained in a medium containing initially 12 mM glucose were incubated subsequently for 4 hr either by addition of [U- 13C]glucose to the culture medium (final concentration 3 mM) or by changing to fresh medium containing [U- 13C]glucose (3 mM) but without glutamine and fetal calf serum. 13C Nuclear magnetic resonance (NMR) spectra revealed extensive ,-aminobutyric acid (GABA) synthesis in both cultured neocortical and cerebellar neurons after maintenance in medium containing [U- 13C]glucose for 8 days, whereas no aspartate labeling was observed in these spectra. Mass spectrometry analysis, however, revealed high labeling intensity of aspartate, which was equal in the two types of neurons. Addition of [U- 13C]glucose (4 hr) on Day 8 in culture led to a similar extent of labeling of GABA in neocortical and in cerebellar cultures, but the cellular content of GABA was considerably higher in the neocortical neurons. The cellular content of alanine was similar regardless of culture type. Comparing the amount of labeling, however, cerebellar neurons exhibited a higher capacity for alanine synthesis. This is compatible with the fact that cerebellar neurons could ameliorate a low alanine content after culturing in low glucose (12 mM) by a 4-hr incubation in medium containing 3 mM glucose. A low glucose concentration during the culture period and a subsequent medium change were associated with decreases in glutathione and taurine contents. Moreover, glutamate and GABA contents were reduced in cerebellar cultures under either of these conditions. In neocortical neurons, the GABA content was decreased by simultaneous exposure to low glucose and change of medium. These conditions also led to an increase in the aspartate content in both types of cultures, although most pronounced in the neocortical neurons. Further experiments are needed to elucidate these phenomena that underline the impact of extracellular environment on amino acid homeostasis. © 2004 Wiley-Liss, Inc. [source] Synergistic enhancement of collagenous protein synthesis by human gingival fibroblasts exposed to nifedipine and interleukin-1-beta in vitroJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 1 2000R. B. Johnson Abstract: Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; however, many clinicians suggest that poor oral hygiene may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis by gingival fibroblasts is synergistically enhanced when they are exposed to both nifedipine (N) and the pro-inflammatory cytokine, interleukin-1-beta, a cytokine expressed in inflamed gingiva. Human gingival fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group 1 was exposed to media containing 0, 5, 50, or 500 pg/ml IL-1-beta, or 10,7 M N for 7 days; Group 2 was exposed to those concentrations of IL-1-beta +10,7 M N. [3H]-proline was added to the medium for the final 24 h. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (d.p.m./103 cells) were compared by factorial ANOVA and Scheffè comparisons. Collagenous protein synthesis was significantly reduced by 5 pg/ml IL-1-beta +10,7 M N and enhanced by 500 pg/ml IL-1-beta +10,7 M N as compared to N or IL-1-beta alone. Thus, patients may be more susceptible to gingival overgrowth coincident to nifedipine therapy as a result of the synergistic enhancement of connective tissue synthesis by these agents. [source] Localization of insulin-like growth factor binding protein-2 in chondrocytes of bovine articular cartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2003Teresa I. Morales Purpose: Previous work indicated that transforming growth factor (TGF-,) treatment of bovine articular cartilage resulted in an accumulation of insulin-like growth factor binding protein-2 (IGF-BP-2). The purpose of the work presented in this paper was to define the localization of the IGF-BP-2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF-,. Method: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF-,1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti-IGF-BP-2 performed. Results: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF-BP-2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF-, treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue. Conclusions: It is concluded that IGF-BP-2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF-, increases the number of cells with positive immunostaining. These data help to support the postulate that TGF-, exerts at least some of its actions in articular cartilage via cross-talk mechanisms involving the IGF-BP-2 system. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003H. R. Haase Background:, Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage. Objectives:, We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Methods:, The cell populations were assessed for mineralization potential after long-term culture in media containing ,-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results:, As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion:, The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers. [source] Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cellsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2003Vanessa M. D'Souza Abstract The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased Vmax, while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway. © 2003 Wiley-Liss Inc. and the American Pharmaeceutical Association J Pharm Sci 92:594,603, 2003 [source] Ethanol Increases Fetal Human Neurosphere Size and Alters Adhesion Molecule Gene ExpressionALCOHOLISM, Issue 2 2008Sharada D. Vangipuram Background:, Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). Methods:, Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. Results:, Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of , and , Laminins 1, , Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan ,. No change in the expression of , Actin was observed while the expression of , Integrin 2 was significantly suppressed. Conclusions:, ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD. [source] Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteinsMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2007Neeraj Sinha Abstract In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different 13C labeling schemes with the purpose of overcoming the interfering effects of 13C13C dipole,dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% 13C in all sites and metabolically labeled from bacterial growth on media containing [2- 13C]-glycerol or [1,3- 13C]-glycerol enables direct 13C detection in solid-state NMR experiments without the need for homonuclear 13C13C dipole,dipole decoupling. The 13C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent 15N-detected spectra. The isotopic labeling pattern was analyzed for [2- 13C]-glycerol and [1,3- 13C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored 13C-labeled samples, in contrast to their failure with conventional 100% uniformly 13C-labeled samples. Copyright © 2007 John Wiley & Sons, Ltd. [source] High salt-treatment-induced Na+ extrusion and low salt-treatment-induced Na+ accumulation in suspension-cultured cells of the mangrove plant, Bruguiera sexangulaPLANT CELL & ENVIRONMENT, Issue 10 2001M. Kura-Hotta Abstract A suspension-cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non-adapted cells were transferred to media containing 0,200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na+, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na+ and Cl, concentrations. In the latter treatment, the intracellular concentration of Na+ returned to the original low level within 2 weeks. It took a longer time for Cl, to return to its original level. As a result, the Na+ and Cl, concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na+/H+ antiporter, suppressed the recovery of Na+ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate-dependent H+ -ATPase and the Na+/H+ antiporter in the plasma membrane fraction. [source] Effective biosynthesis of poly(3-hydoxy- butyrate- co -4-hydroxybutyrate) with high 4-hydroxybutyrate fractions by Wautersia eutropha in the presence of ,-amino acidsPOLYMER INTERNATIONAL, Issue 1 2008Hiroshi Kimura Abstract BACKGROUND: Biopolymers produced by microbes are in demand as their biodegradable and biocompatible properties make them suitable for disposable products and for potential use as biomaterials for medical applications. The effective microbial production of copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate(4HB) with high molar fractions of 4HB unit by a wild-type Wautersia eutropha H16 was investigated in culture media containing 4-hydroxybutyric acid (4HBA) and different carbon substrates in the presence of various ,-amino acids. RESULTS: The addition of carbon sources such as glucose, fructose and acetic acid to the culture medium containing 4HBA in the presence of ,-amino acids resulted in the production of random poly(3HB- co -4HB) with compositions of up to 77 mol% 4HB unit, but the yields of copolyesters with 60,77 mol% 4HB units were less than 15 wt% of dried cell weights. In contrast, when carbon sources such as propionic acid and butyric acid were used as the co-substrates of 4HBA in the presence of ,-amino acids, poly(3HB- co -4HB) copolyesters with compositions of 72,86 mol% 4HB were produced at maximally 47.2 wt% of dried cell weight (11.3 g L,1) and the molar conversion yield of 4HBA to 4HB fraction in copolyesters was as high as 31.4 mol%. Further, poly(3HB- co -4HB) copolyesters with compositions of 93,96 mol% 4HB were isolated at up to 35.2 wt% of dried cell weights by fractionation of the above copolymers with chloroform/n-hexane. CONCLUSION: The productivity of copolyesters with over 80 mol% 4HB fractions was as high as 0.146 g L,1 h,1 (3.51 g L,1 for 24 h) by flask batch cultivation. Copyright © 2007 Society of Chemical Industry [source] Secondary metabolite production by the fungal pathogen Eutypa lata: Analysis of extracts from grapevine cultures and detection of those metabolites in plantaAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 2 2006RICHARD LARDNER Eutypa dieback of grapevines is caused by the fungal pathogen Eutypa lata and reduces vineyard longevity worldwide. Early detection could reduce losses due to this disease, so our aim was to identify acetylenic phenol metabolites of E. lata that could prove suitable as chemical markers in an early diagnostic test for the pathogen. Accordingly, secondary metabolite production by 30 isolates of E. lata grown on media derived from canes of three grapevine cultivars was analysed using HPLC. Six metabolites, namely eutypinol, methyl eutypinol, eulatachromene, eutypine, 2- iso -propenyl-5-formylbenzofuran and eulatinol, were detected in culture filtrates. Most abundant were eutypinol and methyl eutypinol, produced by 97 and 83% of isolates, respectively. There was no apparent correlation between secondary metabolite production on media containing milled canes from the three cultivars of grapevine, and the field tolerance of these same cultivars to Eutypa dieback. When various other fungi commonly isolated from grapevine trunks in Australia were grown on milled cane, no secondary metabolites characteristic of E. lata were detected, suggesting such compounds are specific to E. lata. To examine the detection of secondary metabolites in planta, micropropagated grapevine plantlets were treated with purified or crude culture filtrates from nine isolates of E. lata grown on malt yeast broth. Various secondary metabolites were identified in treated plantlets, however, no single compound was detected consistently. Eutypinol was detected in micropropagated grapevine plantlets inoculated with mycelium of E. lata, however, no metabolites were detected in the sap of vines which had been artificially inoculated with the pathogen. [source] Zero valent iron as an electron-donor for methanogenesis and sulfate reduction in anaerobic sludgeBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Srilakshmi Karri Abstract Zero valent iron (ZVI) is a reactive media commonly utilized in permeable reactive barriers (PRBs). Sulfate reducing bacteria are being considered for the immobilization of heavy metals in PRBs. The purpose of this study was to evaluate the potential of ZVI as an electron donor for sulfate reduction in natural mixed anaerobic cultures. The ability of methanogens to utilize ZVI as an electron-donor was also explored since these microorganisms often compete with sulfate reducers for common substrates. Four grades of ZVI of different particle sizes (1.120, 0.149, 0.044, and 0.010 mm diameter) were compared as electron donor in batch bioassays inoculated with anaerobic bioreactor sludge. Methanogenesis was evaluated in mineral media lacking sulfate. Sulfate reduction was evaluated in mineral media containing sulfate and the specific methanogenic inhibitor, 2-bromoethane sulfonate. ZVI contributed to significant increases in methane production and sulfate reductioncompared to endogenous substrate controls. The rates of methane formation or sulfate reduction were positively correlated with the surface area of ZVI. The highest rates of 0.310 mmol CH4 formed/mol Fe0·day and 0.804 mmol SO reduced/ mol Fe0·day were obtained with the finest grade of ZVI (0.01 mm). The results demonstrate that ZVI is readily utilized as a slow-release electron donor for methanogenesis and sulfate reduction in anaerobic sludge; and therefore, has a promising potential in bioremediation applications. © 2005 Wiley Periodicals, Inc. [source] Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C, activationCELL PROLIFERATION, Issue 6 2001P. W. Sylvester Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0,250 µm) of ,-, ,-, or ,-tocopherol or ,-, ,-, or ,-tocotrienol. Treatment with growth inhibitory doses of ,-tocopherol (100 µm), ,-tocotrienol (50 µm), or ,- or ,-tocotrienol (10 µm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC,. However, these treatments were found to inhibit EGF-induced PKC, activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 µm,- or ,-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC, activation. [source] | ||||||||||||||||