Home About us Contact
|
Home About us Contact | ||
|
|
||
Medium Containing (medium + containing)
Terms modified by Medium Containing Selected AbstractsRole of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulationCYTOSKELETON, Issue 2 2006Zoltįn Krasznai Abstract A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing ,1 ,M Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3,,4, -dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 ,M, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50 = 2.44 ,M). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role. Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source] Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation methodENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2009Samir C. Debnath Abstract Reproducible protocol for regeneration of complete plantlets from ,Bounty' strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid-gelled medium containing 4 ,M thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2,,M TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1,,M in the bioreactor system but inhibited shoot elongation. TDZ-induced shoots were elongated and rooted in vitro on gelled medium containing 2,,M zeatin. Such bioreactor-derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin-containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production. [source] Role of ethylenediaminetetraacetic acid on lead uptake and translocation by tumbleweed (Salsola kali L.)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2007Guadalupe de la Rosa Abstract Tumbleweed plants (Salsola kali L.) grown in agar and liquid media demonstrated a high capacity to accumulate Pb in their different parts without affecting biomass. Whereas shoot elongation and biomass were not significantly affected by high tissue concentrations of Pb, root growth was significantly affected relative to controls. Roots, stems, and leaves demonstrated Pb concentrations of 31,000, 5,500, and 2,100 mg/kg dry weight, respectively, when plants were grown in the agar medium containing 80 mg Pb/L. Application of ethylenediaminetetraacetic acid (EDTA) to Pb-contaminated media dramatically reduced the total acquisition of Pb from both types of media. However, EDTA significantly increased the translocation of Pb from roots to the aerial parts, as evidenced by a multifold increase (23- and 155-fold for agar and liquid media, respectively) in the translocation concentration factor. The concentration of the antioxidant thiol compounds significantly increased (p < 0.05) in plants grown with uncomplexed Pb treatments relative to control plants. Scanning-electron microscopy and electron dispersive x-ray spectroscopic evaluation of leaf samples demonstrated an interesting pattern of Pb translocation in the presence or absence of EDTA. Large Pb crystals were found across the leaf tissues (palisade, spongy parenchyma, and conducting tissues) in the absence of EDTA. Lead nanoparticles also were seen when plants were grown in Pb-EDTA solution. Ultramicroscopic features of tumbleweed provide clear evidence for the unrestricted conduction of Pb from the root to the aerial parts, and this property makes the plant a good candidate for phytoremediation. [source] Neocortical Potassium Currents Are Enhanced by the Antiepileptic Drug LamotrigineEPILEPSIA, Issue 7 2002Cristina Zona Summary: ,Purpose: We used field-potential recordings in slices of rat cerebral cortex along with whole-cell patch recordings from rat neocortical cells in culture to test the hypothesis that the antiepileptic drug (AED) lamotrigine (LTG) modulates K+ -mediated, hyperpolarizing currents. Methods: Extracellular field-potential recordings were performed in neocortical slices obtained from Wistar rats aged 25,50 days. Rat neocortical neurons in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated K+ currents. Results: In the in vitro slice preparation, LTG (100,400 ,M) reduced and/or abolished epileptiform discharges induced by 4-aminopyridine (4AP, 100 ,M; n = 10), at doses that were significantly higher than those required to affect epileptiform activity recorded in Mg2+ -free medium (n = 8). We also discovered that in cultured cortical cells, LTG (100,500 ,M; n = 13) increased a transient, 4AP-sensitive, outward current elicited by depolarizing commands in medium containing voltage-gated Ca2+ and Na+ channel antagonists. Moreover, we did not observe any change in a late, tetraethylammonium-sensitive outward current. Conclusions: Our data indicate that LTG, in addition to the well-known reduction of voltage-gated Na+ currents, potentiates 4AP-sensitive, K+ -mediated hyperpolarizing conductances in cortical neurons. This mechanism of action contributes to the anticonvulsant effects exerted by LTG in experimental models of epileptiform discharge, and presumably in clinical practice. [source] The Synthesis of the Dimethyl Ester of Quino[4,4a,5,6- efg]-Annulated 7-Demethyl-8-deethylmesoporphyrin and Three of Its Isomers with Unprecedented peri -Condensed Quinoline Porphyrin Structures.EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 19 2004Molecules with Outstanding Properties as Sensitizers for Photodynamic Therapy in the Far-Red Region of the Visible Spectrum Abstract The mesoporphyrin dimethyl ester nickel complex has been formylated via the Vilsmeier method. The four possible mono meso-formyl derivatives were isolated and characterized. Wadsworth,Emmons coupling with the anion of (diethylphosphono)acetonitrile converted these aldehydes into the four novel meso acrylonitriles. Brief treatment of these acrylonitrile systems in hot trichloroacetic acid resulted in the formation of four achiral porphyrin derivatives with unprecedented nickel complexes of quino-fused porphyrins. Subsequent removal of the nickel gave four quino-porphyrin free bases: quino[4,4a,5,6- efg]-annulated 7-demethyl-8-deethylmesoporphyrin dimethyl ester 6a, 2,-(methoxycarbonyl)quino[4,4a,5,6- jkl]-annulated 12-demethyl-13-de[2,-(methoxycarbonyl)ethyl]mesoporphyrin dimethyl ester 6b, 2,-(methoxycarbonyl)quino[4,4a,5,6- qrs]-annulated 18-demethyl-17-de(2,-methoxycarbonylethyl)mesoporphyrin dimethyl ester 6c and quino[4,5,6,7- abt]-annulated 2-demethyl-3-deethylmesoporphyrin dimethyl ester 6d. The structures of these systems were unambiguously determined via mass spectroscopy and a plethora of NMR techniques. In the same way, etioporphyrin and octaethylporphyrin were converted into the corresponding peri -condensed quinoporphyrins as products, which shows that the formation of novel pericondensed quino-porphyrins is a general reaction in the porphyrin series and will have a wide scope in this field. Also, a plausible reaction mechanism for the formation of the quinoporphyrin systems was derived. As a first test for the use of these systems as sensitizers in far-red phototherapy, the quantum yield of singlet oxygen generation by 6a in toluene was studied. This quantum yield is 0.77, which is even higher than the singlet oxygen generation by sensitized meso-tetraphenylporphyrin. Secondly, when Chinese Hamster ovary (CHO) cells were incubated in medium which contained up to 15 ,g/ml of 6a, the survival rate of the cells in the dark is complete within experimental error, showing that under these conditions, 6a is not toxic to CHO cells. When CHO cells incubated in medium containing 6a in concentrations of 1 ,g/ml and higher were treated with white light of intensity 30 mW/cm2 for 15 minutes, complete cell death was observed. Based on these facts, we expect that all four achiral systems will show very promising properties to form the basis of a photodynamic therapy in far-red light. The fact that these systems are achiral is an additional bonus for medical applications. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Bumetanide, the Specific Inhibitor of Na+ -K+ -2Cl, Cotransport, Inhibits 1,,25-Dihydroxyvitamin D3 -Induced Osteoclastogenesis in a Mouse co-culture SystemEXPERIMENTAL PHYSIOLOGY, Issue 5 2003Hyun-A Lee The Na+ -K+ -2Cl, cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-,B ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1,,25-dihydroxyvitamin D3 (1,,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2 -terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1,,25(OH)2D3 -induced osteoclastogenesis partly via the phosphorylation of JNK. [source] Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvaceaFEBS JOURNAL, Issue 2 2004Shicheng Chen We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ,,20,30% of peak levels throughout the subsequent stages of sporophore development. [source] Lithium-mediated suppression of morphogenesis and growth in Candida albicansFEMS YEAST RESEARCH, Issue 4 2008Layla F. Martins Abstract Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg2+. In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC50 of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg2+. These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway. [source] Effects of ouabain on contractions induced by manganese ions in Ca2+ -free, isotonic solutions with varying concentrations of K+ in guinea-pig taenia coliFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 3 2005Tetsuyuki Nasu Abstract The action of ouabain, a cell membrane Na+, K+ -ATPase blocker, on contractions induced by manganese ions (Mn2+) in Ca2+ -free, isotonic solutions with varying concentrations of K+ in the external medium were investigated in order to evaluate the underlying role of external Na+ in Mn2+ -induced contractions in isolated taenia coli of the guinea-pig. Mn2+ at 5 mm induced greater contractions as external isotonic K+ concentrations progressively increased from 10 to 100 mm. Ouabain (2 × 10,4 m) completely inhibited tension development stimulated by 5 mm Mn2+ in isotonic, 30 mm K+ (96 mm Na+) medium. Whereas, the tension inhibitory effects of ouabain became progressively weaker as isotonic, external K+ concentrations increased to 60 mm, which successively decreased external Na+ concentrations. Eventually, ouabain failed to affect contractions stimulated by Mn2+ in isotonic, 126 mm K+, Na+ -deficient medium. Ouabain caused progressively greater increase in cellular Na+ concentrations as the Na+ concentrations increased in the isotonic, K+ medium. While, pyruvate, which penetrates cell independently of external Na+, reversed the inhibition of tension by ouabain in isotonic, 30 mm K+, Na+ -sufficient (96 mm) medium containing 5 mm Mn2+. These results suggested that Mn2+ induced the contraction, which was maintained by glucose transport depending on external Na+, in the case of Na+ -sufficient medium in K+ -depolarized taenia coli. However, it induced the contraction independent of external Na+, in the case of Na+ -deficient, K+ medium. Ouabain might exhibit greater inhibition of the contraction induced by Mn2+ as the decrease in the Na+ gradient across the cell membranes continues. [source] Astrocyte-derived factors modulate the inhibitory effect of ethanol on dendritic developmentGLIA, Issue 4 2002Penelope A. Yanni Abstract Numerous studies in vivo and in vitro have demonstrated that ethanol disrupts neuromorphogenesis. However, it has not been determined what role, if any, is played by non-neuronal cells in mediating this effect. We recently reported that ethanol inhibits dendritic development in low-density cultures of fetal rat hippocampal pyramidal neurons (Yanni and Lindsley, 2000: Dev Brain Res 120:233,243). In this culture system, cortical astrocytes precondition neuronal culture media for 2 days before the addition of neurons, which then develop on a separate substrate in coculture with the astrocytes. To determine whether astrocyte response to ethanol mediates the effects of ethanol on neurons, the present study compared dendritic development of neurons after 6 days in medium containing 400 mg/dl ethanol in coculture with live astrocytes and in conditioned medium from astrocytes that were never exposed to ethanol. The same experiment was also performed with and without ethanol present during astrocyte preconditioning of the medium. The effects of ethanol differed depending on when it was added to the cultures relative to addition of newly dissociated neurons. However, the effects of ethanol were not related to whether neurons were cocultured with live astrocytes. When astrocytes preconditioned the medium normally, ethanol added at plating inhibited dendritic development of neurons regardless of whether they were maintained in coculture with live astrocytes or in conditioned medium. In surprising contrast, the presence of ethanol during astrocyte preconditioning of the media had a growth promoting effect on subsequent dendrite development despite the continued presence of ethanol in the medium. Thus, astrocytes release soluble factors in response to ethanol that can protect neurons from the inhibitory effects of ethanol on dendritic growth, but the timing of neuronal exposure to these factors, or their concentration, may influence their activity. GLIA 38:292,302, 2002. © 2002 Wiley-Liss, Inc. [source] Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epitheliumGLIA, Issue 1 2001Monica M. Jablonski Abstract In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments. GLIA 35:14,25, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of prolonged hydroxytamoxifen treatment of MCF-7 cells on mitogen activated kinase cascadeINTERNATIONAL JOURNAL OF CANCER, Issue 5 2002Fanjaniriana Rabenoelina Abstract Resistance to the antiestrogen tamoxifen is the main stumbling block for the success of breast cancer therapy. We focused our study on cellular alterations induced by a prolonged treatment with the active tamoxifen metabolite hydroxytamoxifen (OHT). We show that a prolonged OHT treatment (for up to 7 days) led to a progressive increase in the level of phosphorylated p44/42 mitogen activated kinase (MAP kinase) induced by 10,7 M TPA stimulation, without any significant change in the protein level. This effect was also observed in MCF-7 cells grown first in medium containing dextran-coated charcoal-treated FCS (DCC medium) for 20 days prior to OHT treatment, indicating a specific effect of the antiestrogen and not an effect of estrogen deprivation. It was prevented by cotreatment with estradiol and not observed in the estrogen receptor negative HeLa cell line, suggesting that it was mediated by the estrogen receptor. TPA induced phosphorylation of MEK1/2 was also raised by OHT treatment, without any change in their protein level or Raf-1 and H-Ras levels. When the MCF-7R OHT resistant cell line was grown in antiestrogen containing medium, the level of phosphorylated p44/42 MAP kinase was also high but reversed when the antiestrogen was removed. The 2 other MAP kinase, JNK and P38 pathways were not affected in the same way by OHT treatment. In conclusion, our data reveal that a prolonged OHT treatment, by increasing p44/42 MAPK activity, affects a key step in the growth control of MCF-7 cells, although not sufficiently to overcome the growth inhibitory effect of the drug. © 2002 Wiley-Liss, Inc. [source] Effect of urothelium on bladder contractility in diabetic ratsINTERNATIONAL JOURNAL OF UROLOGY, Issue 7 2005MURAT KO Abstract Aim: It is known that physiopathological changes in diabetes affect the function of the bladder. In this study, we aimed to demonstrate the possible effects of diabetes on the urothelium during this physiopathological process. Methods: Diabetes was induced in rats by tail vein injection of 35 mg/kg streptozotocin. Eight weeks later, intact and denuded bladder strips were prepared from these rats. Electrical field stimulation (EFS; 0.5,32 Hz), carbachol (10,8,10,3 mol/L; cumulative dosage-response curves) and KCl (120 mmol/L) were used for the evaluation of the contractile responses. All responses were expressed as mg tension developed per mg of bladder tissue. Weights of rats and of their bladders, blood glucose levels, and frequency- and concentration,response curves were compared using anova, the paired t -test and the independent t -test. Differences were considered significant at P < 0.05. Results: Although no differences related to the weight of bladders of the control and diabetic groups were observed, there were differences in blood glucose levels and body weights between the two groups. Similarly, although there were no differences between the data obtained with EFS and KCl from tissues with intact and denuded strips in the control group, carbachol responses significantly differed between intact and denuded strips in the non-diabetic group. These differences were not observed in the diabetic group. In the control groups, in the presence of additional strips with intact urothelium placed in the medium containing denuded tissue, the differences in contractile responses between the intact control strip and the denuded strip disappeared. Conclusions: Diabetes possibly changes the interaction between the relaxant factors that are released from urothelium and muscarinic stimulation, but these interactions are not completely understood yet. Consequently, the response of the bladder to contractile stimulants is also affected. Further studies are required to reveal the mechanism by which diabetes influences the urothelium. [source] Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroidesJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2010P.I. Nikel Abstract Aims:, Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen-restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results:, Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4-fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro-oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l,1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l,1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl-coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two-stage bioreactor cultures were conducted in a minimal medium containing 100 ,g l,1 calcium d -pantothenate to evaluate oxic acetyl-CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l,1 with a volumetric productivity of 0·34 ± 0·02 g l,1 h,1. Conclusions:,Escherichia coli responded to adhE over-expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl-CoA played a key role in micro-oxic ethanol synthesis and growth. Significance and Impact of the Study:, Insight into the micro-oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis. [source] The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentationsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2007J.C. Bohlscheid Abstract Aim:, To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. Methods and Results:, Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l,1) and biotin (0, 1 or 10 ,g l,1) as variables. All media contained 240 g l,1 glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 ,g l,1 had no effect. Nitrogen × biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H2S). Maximum H2S production occurred in the medium containing 60 mg l,1 YAN and 1 ,g l,1 biotin. Conclusions:, Nitrogen × biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. Significance and Impact of the Study:, This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations. [source] Flocculation onset in Saccharomyces cerevisiae: the role of nutrientsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2005S. Sampermans Abstract Aims:, To examine the role of the nutrients on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. Methods and Results:, Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. For cells grown in chemically defined medium (yeast nitrogen base with glucose) or in rich medium (containing yeast extract, peptone and fermentable sugars: fructose or maltose), the onset of flocculation occurred after the end of exponential respiro-fermentative phase of growth being coincident with the attainment of the lower level of carbon source in the culture medium. Cells, in exponential respiro-fermentative phase of growth, transferred to a glucose-containing medium without nitrogen source, developed a flocculent phenotype, while these carbon source starved cells, in the presence of all other nutrients that support growth, did not flocculate. In addition, cells in exponential phase of growth, under catabolite repression, when transferred to a medium containing 0·2% (w/v) of fermentable sugar (fructose or maltose) or 2% (v/v) ethanol, showed a rapid triggering of flocculation, while when incubated in 2% (v/v) glycerol did not develop a flocculent phenotype. Conclusions:, The onset of flocculation occurs when a low sugar and/or nitrogen concentration is reached in culture media. The triggering of flocculation is an energetic dependent process influenced by the carbon source metabolism. The presence of external nitrogen source is not necessary for developing a flocculent phenotype. Significance and Impact of the Study:, This work contributes to the elucidation of the role of nutrients on the onset of flocculation in NewFlo phenotype yeast strains. This information might be useful to the brewing industry, in the control of yeast flocculation, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality. [source] Hydrocarbon accumulation by picocyanobacteria from the Arabian GulfJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2001R.H. Al-Hasan Aims:,The objective of this work was to study picocyanobacteria in the Arabian Gulf water in relation to oil pollution. Methods and Results:,Epifluorescent microscopic counting showed that offshore water samples along the Kuwaiti coast of the Arabian Gulf were rich in picocyanobacteria which ranged in numbers between about 1 × 105 and 6 × 105 ml,1. Most dominant was the genus Synechococcus; less dominant genera were Synechocystis, Pleurocapsa and Dermocarpella. All isolates grew well in an inorganic medium containing up to 0·1% crude oil (w/v) and could survive in the presence of up to 1% crude oil. Hydrocarbon analysis by gas liquid chromatography (GLC) showed that representative strains of the four genera had the potential for the accumulation of hydrocarbons (the aliphatic n -hexadecane, aromatic phenanthrene and crude oil hydrocarbons) from aqueous media. Electron microscopy showed that the cells of these strains appeared to store hydrocarbons in their inter thylakoid spaces. Analysis by GLC of constituent fatty acids of total lipids and individual lipid classes from representative picoplankton strains grown in the absence and presence of hydrocarbons showed, however, that the fatty acid patterns were not markedly affected by the hydrocabon substrates, meaning that the test strains could not oxidize the accumulated hydrocarbons. Conclusions:,The Arabian Gulf is among the water bodies of the world richest in picocyanobacteria. These micro-organisms accumulate hydrocarbons from the water body, but do not biodegrade these compounds. It is assumed that hydrocarbon-utilizing bacteria that were always found associated with all picocyanobacteria in nature may carry out the biodegradation of these compounds. Significance and Importance of the Study:,The results shed light on the potential role of picocyanobacteria in controlling marine oil pollution. [source] Biodegradation of poly(butylene adipate- co -butylene terephthalate)/layered-silicate nanocompositesJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2007Yoshihiro Someya Abstract The biodegradability of poly(butylene adipate- co -butylene terephthalate) (PBAT) and PBAT/starch composites with layered silicates prepared by melt intercalation was evaluated with aerobic biodegradability tests in soil and in an aqueous medium containing activated sludge. Nonmodified montmorillonite (MMT) and octadecylamine-modified montmorillonite (ODA-M), known to give a microcomposite and an intercalated nanocomposite for PBAT, respectively, were used as layered silicates. After they were buried in the soil for 8 months, the PBAT/MMT microcomposite exhibited a higher weight loss than the control PBAT, whereas the PBAT/ODA-M nanocomposite showed a lower weight loss instead. Also, the biodegradability test in the aqueous medium, by determining the biochemical oxygen demand, showed that the addition of MMT and/or starch to PBAT promoted biodegradation, whereas the addition of ODA-M did not. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007 [source] Overexpression of RGPR-p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: Involvement of TTGGC motifJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Natsumi Sawada Abstract A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the ,710/+18 LUC construct (wild-type) or ,710/+18 LUC construct (mutant) with deletion of ,523/,435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the ,710/+18 LUC construct vector or the ,710/+18 LUC construct with deletion of ,523/,435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1,34) (PTH; 10,7 M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of ,523/,435 sequence of regucalcin promoter. This was also seen using the ,710/+18 LUC construct with deletion of ,523/,503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10,7 M), Bay K 8644 (10,6 M), phorbol 12-myristate 13-acetate (PMA; 10,6 M), or N6, 2,-dibutyryl cyclic adenosine 3,, 5,-monophosphate (DcAMP; 10,4 M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10,6 M), staurosporine (10,9 M), PD 98059 (10,8 M), wortmannin (10,8 M), genistein (10,6 M), vanadate (10,6 M), or okadaic acid (10,6 M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. J. Cell. Biochem. 99: 589,597, 2006. © 2006 Wiley-Liss, Inc. [source] ,-cryptoxanthin stimulates cell differentiation and mineralization in osteoblastic MC3T3-E1 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Satoshi Uchiyama Abstract The effect of ,-cryptoxanthin, a kind of carotenoid, on cell differentiation and mineralization in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in a minimum essential medium containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or ,-cryptoxanthin (10,8 to 10,6 M) without FBS. Cells were cultured for 3 to 21 days. Gene expression in osteoblastic cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with ,-cryptoxanthin (10,7 or 10,6 M) for 3 days caused a significant increase in Runx2 type 1, Runx2 type 2, ,1 (I) collagen, and alkaline phosphatase mRNA levels in osteoblastic cells. These increases were completely blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-,- D -ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. Meanwhile, vitamin A (10,6 M) did not have a significant effect on Runx2 type 1 mRNA expression in the cells. The effect of ,-cryptoxanthin (10,6 M) in stimulating Runx2 type 1 and ,1 (I) collagen mRNA levels, protein content, and alkaline phosphatase activity in the cells was also seen in the presence of vitamin A (10,6 M), suggesting that the mode of ,-cryptoxanthin action differs from that of vitamin A. Prolonged culture with ,-cryptoxanthin (10,6 M) for 3 to 21 days caused a significant increase in cell number, deoxyribonucleic acid (DNA) content, protein content, and alkaline phosphatase activity in osteoblastic cells, suggesting that ,-cryptoxanthin stimulates cell proliferation and differentiation. Moreover, culture with ,-cryptoxanthin (10,7 or 10,6 M) for 5 to 21 days caused a remarkable increase in mineralization. This study demonstrates that ,-cryptoxanthin has a stimulatory effect on cell differentiation and mineralization due to enhancing gene expression of proteins, which involve in bone formation in osteoblastic MC3T3-E1 cells. © 2005 Wiley-Liss, Inc. [source] Fermentative production of L(+)-lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2008Young-Jung Wee Abstract BACKGROUND: Attempts were made to determine the lactic acid production efficiency of novel isolate, Enterococcus faecalis RKY1 using four different starches (corn, tapioca, potato, and wheat starch) with different concentrations (50, 75, 100, and 125 g L,1) and corn steep liquor as an inexpensive nitrogen source. RESULTS: The yield of lactic acid from each starch was higher than 95% based on initial starch concentrations. High lactic acid concentration (129.9 g L,1) and yield (1.04 g-lactic acid g,1 -starch) were achieved faster (84 h) from 125 g L,1 of corn starch. Among the starches used, tapioca starch fermentation usually completed in a shorter incubation period. The final dry cell weight was highest (7.0 g L,1) for the medium containing 75 g L,1 of corn starch, which resulted in maximum volumetric productivity of lactic acid (3.6 g L,1 h,1). The addition of 30 g L,1 corn steep liquor supplemented with a minimal amount of yeast extract supported both cell growth and lactic acid fermentation. CONCLUSION:Enterococcus faecalis RKY1 was found to be capable of growing well on inexpensive nutrients and producing maximum lactic acid from starches and corn steep liquor as lower-cost raw materials than conventionally-used refined sugars such as glucose, and yeast extract as an organic nitrogen source in laboratory-scale studies. These fermentation characteristics are prerequisites for the industrial scale production of lactic acid. Copyright © 2008 Society of Chemical Industry [source] Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomesJOURNAL OF FISH DISEASES, Issue 8 2005M-J Kim Abstract Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING. [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] Sulphide-induced energy deficiency in colonic cells is prevented by glucose but not by butyrateALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2002S. J. Hulin Background: In ulcerative colitis, hydrogen sulphide is postulated to impair colonocyte butyrate metabolism, leading to cellular energy deficiency and dysfunction. Aims: To determine the effects of sulphide exposure on butyrate metabolism and adenosine triphosphate levels of HT29 colonic epithelial cancer cells, and to establish whether energy deficiency can be prevented by increased butyrate concentrations or the presence of glucose. Methods: HT29 cells were maintained in medium containing 3 mM butyrate, 5 mM glucose, or both substrates. Oxidation rates were measured by 14CO2 release from 14C-labelled substrates. Cellular adenosine triphosphate was assayed using the luciferin/luciferase chemiluminescent method. The effects of sulphide (0,5 mM) on substrate oxidation and adenosine triphosphate levels and of increasing butyrate concentration (0,30 mM) with sulphide were observed. Results: HT29 cells showed similar energy substrate usage to primary colonocyte cultures. Sulphide exposure inhibited butyrate oxidation and led to a reduction in cellular adenosine triphosphate. This fall was prevented by co-incubation with glucose, but not by increasing concentrations of butyrate. Conclusions: HT29 cells utilize butyrate as an energy substrate and represent a useful in vitro model of the effects of sulphide on colonocytes. Sulphide inhibits butyrate oxidation and leads to demonstrable energy deficiency, prevented by the presence of glucose but not by increased butyrate concentrations. [source] Three gene products govern (p)ppGpp production by Streptococcus mutansMOLECULAR MICROBIOLOGY, Issue 6 2007José A. Lemos Summary The current dogma implicating RelA as the sole enzyme controlling (p)ppGpp production and degradation in Gram-positive bacteria does not apply to Streptococcus mutans. We have now identified and characterized two genes, designated as relP and relQ, encoding novel enzymes that are directly involved in (p)ppGpp synthesis. Additionally, relP is co-transcribed with a two-component signal transduction system (TCS). Analysis of the (p)ppGpp synthetic capacity of various mutants and the behaviour of strains lacking combinations of the synthetase enzymes have revealed a complex regulon and fundamental differences in the way S. mutans manages alarmone production compared with bacterial paradigms. The functionality of the RelP and RelQ enzymes was further confirmed by demonstrating that expression of relP and relQ restored growth of a (p)ppGpp0Escherichia coli strain in minimal medium, SMG and on medium containing 3-amino-1,2,4-triazole, and by demonstrating (p)ppGpp production in various complemented mutant strains of E. coli and S. mutans. Notably, RelQ, and RelP and the associated TCS, are harboured in some, but not all, pathogenic streptococci and related Gram-positive organisms, opening a new avenue to explore the variety of strategies employed by human and animal pathogens to survive in adverse conditions that are peculiar to environments in their hosts. [source] BH4 peptide derived from Bcl-xL and Bax-inhibitor peptide suppresses apoptotic mitochondrial changes in heat stressed bovine oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2009Paolete Soto Mitochondria play an important role in the integration and transmission of cell death signals mediated by the Bcl-2 family proteins. Experiments were conducted to determine whether the anti-apoptotic peptides BH4 domain of Bcl-xL (TAT-BH4) and Bax inhibitor peptide (BIP) suppresses heat stress (HS) injury in oocytes by reduction of apoptotic-like events. Cumulus,oocyte complexes (COCs) were matured at 39°C (control) or 41°C (HS) for 21 hr then placed in maturation medium containing 0 or 100 µM BIP in water and 0 or 1 µM TAT-BH4 in dimethyl sulfoxide (DMSO), or a combination of both peptides (BIP,+,BH4). Peptide effects on embryo development, DNA fragmentation, mitochondrial membrane potential (,,m), and mitochondrial DNA (mtDNA) copy number were measured. All groups were fertilized and cultured in vitro at 39°C for 8 days. Compared to control, HS-treated oocytes induced a decrease in embryo development (P,<,0.05), increase in proportion of TUNEL-positive chromatin in oocytes and blastocysts (P,<,0.05), and loss of oocyte ,,m (P,<,0.001). In the presence of BIP or BIP,+,BH4, development of HS-treated oocytes into blastocysts was increased (P,<,0.05). Conversely, COCs matured with TAT-BH4 at 41°C showed reduced embryonic development (P,<,0.05). Exposure of HS-treated to each or both peptides resulted in a reduction of TUNEL frequency in oocytes and blastocysts cells derived from these oocytes (P,<,0.05). The loss of ,,m in HS-treated oocytes was not restored by exposure to BIP,+,BH4 and there was no effect in mtDNA copy number. In conclusion, the present results show that HS-induced apoptosis in bovine oocytes involves Bax and BH4 domain-dependent pathways. Mol. Reprod. Dev. 76: 637,646, 2009. © 2008 Wiley-Liss, Inc. [source] Magnetic resonance imaging and biological properties of pancreatic islets labeled with iron oxide nanoparticlesNMR IN BIOMEDICINE, Issue 8 2009Hoe Suk Kim Abstract This study was undertaken to investigate the in vitro effect of islet labeling with iron oxide nanoparticles for MRI on islet viability, insulin secretion, and gene expression. Isolated rat islets were labeled with Resovist (25,200,µg Fe/mL, a clinically approved MRI contrast agent) in the presence or absence of poly- l -Lysine (PLL, 1.5,µg/mL) for 48,h. The iron content of labeled islets was found to increase in a dose-dependent manner. More than 90% of the islets were labeled with 100,µg Fe/mL. We confirmed the localizations of iron oxide nanoparticles within islet , -cells by insulin immunostaining. As the concentration of Resovist increased, T2 values as determined by T2 -weighted MRI on a 1.5,Tesla MR scanner decreased. Labeling of 100 islets in a medium containing 100,µg Fe/mL of Resovist in the absence of PLL provided sufficient contrast for islet visualization on T2 -weighted MRI. MTT assays showed that the viability of labeled islets was not different from that of unlabeled islets. No statistical difference was observed between labeled (2.91,±,0.36) and unlabeled islets (2.83,±,0.61) in terms of the ability to secrete insulin, as determined by the glucose stimulation index. We also evaluated the effect of iron oxide incorporation on the gene expressions in islet cells using RT-PCR (reverse transcriptase PCR). Insulin expression in labeled islets was significantly elevated (1.83,±,0.25 fold vs. unlabeled; p,=,0.005), but not the expression of somatostatin (1.39,±,0.18 fold vs. unlabeled; p,=,0.085) or glucagons (1.28,±,0.13 fold vs. unlabeled; p,=,0.09). Expression of an important transcription factor for insulin gene transcription, BETA2 (beta-cell E-box trans-activator), was increased in labeled islets (1.67,±,0.15 fold vs. unlabeled; p,=,0.029). The findings of this study indicate that Resovist provides a satisfactory means to image islets and has no deleterious effect on islet function or gene expression. Copyright © 2009 John Wiley & Sons, Ltd. [source] Cadmium partitioning and gene expression studies in Nicotiana tabacum and Nicotiana rusticaPHYSIOLOGIA PLANTARUM, Issue 3 2006Lucien Bovet To better understand the differences in cadmium (Cd) uptake, partitioning and gene regulation between Nicotiana tabacum and Nicotiana rustica, we compared these two species for root and leaf Cd concentrations after different Cd exposures, 109Cd root-to-shoot transport, Cd tolerance as well as differential gene expression in roots exposed or not to CdCl2 using reverse transcriptase,PCR (RT-PCR). When grown in 1 ,M CdCl2 for 7 days, N. rustica exhibited higher root and lower leaf Cd contents than N. tabacum. Data were confirmed by radiolabeling experiments, which further showed that some 109Cd accumulated in the distal part of lateral roots in N. rustica. Visual inspection of leaves suggested that N. rustica was somewhat more tolerant to high Cd exposure (50 ,M CdCl2) compared with N. tabacum. At such a high Cd concentration, Cd toxic effects on N. tabacum leaves were apparently not directly related to the homeostasis of Fe and Mn. However, the Zn levels were different in N. rustica compared with N. tabacum in absence and presence of Cd treatments. Root growth experiments revealed that N. tabacum, but not N. rustica, root length was reduced in bactoagar medium containing 20 ,M CdCl2. Complementary DNA microarrays were used as a screening approach to demonstrate by RT-PCR that some gene products were differentially regulated by Cd in N. rustica and in N. tabacum. In addition, "NtIRT1,"NtMTP1a, "NtHMA3" and "NtNAS3" were inducible by Cd in N. tabacum. Interestingly "NtIRT1" and NtMTP1a were differently expressed between the two species. Our results suggest different pathways for Cd sequestration and transport between these two species. [source] In vitro selection and plant regeneration of copper-tolerant plants from leaf explants of Nicotiana tabacum L. cv. ,Xanthi'PLANT BREEDING, Issue 4 2007G. R. Rout Abstract Copper tolerance of Nicotiana tabacum L. var. Xanthi in vitro was achieved through plant regeneration from leaf explants on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/l BA, 0.1,0.25 mg/l IAA and 60 ,m Cu. Tolerant organogenic calli showed more vigorous growth in medium containing 60 ,m Cu than the non-tolerant calli. Standard growth parameters such as fresh and dry weight of organogenic callus, growth tolerance index (GTI), enzyme activity (peroxidase and catalase) and copper accumulation were used as indicators of copper tolerance. The activities of peroxidase and catalase as well as estimation of protein, total amino acid and chlorophyll were greater in tolerant calli than non-tolerant ones. The GTI in the 4 weeks after the beginning of treatments yielded significant differences among the tolerant and non-tolerant organogenic callus cultures. The accumulation of copper in the tolerant calli increased significantly with an increase in copper concentration in the medium. Shoot bud regeneration was achieved in both tolerant and non-tolerant organogenic calli on MS medium containing 0.5 mg/l BA and 0.1 mg/l IAA. The tolerant regenerated shoots were rooted on half-strength basal MS medium with 60 ,m Cu for selection of tolerant clones. This study may help in the selection and characterization of Cu-tolerant lines of N. tabacum cv. ,Xanthi' for building conservation strategies and also for phytoremediation programmes. [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] | ||||||||||||||||||||||||||||||||||||||||