Home About us Contact
|
Home About us Contact | ||
|
|
||
mM EDTA (mm + edta)
Selected AbstractsDetermination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescenceELECTROPHORESIS, Issue 18-19 2004Lijun Yu Abstract High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (<10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (>1 200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2×104 cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings. [source] Mechanism of inhibition of purified leaping mullet (liza saliens) NADPH-cytochrome P450 reductase by toxic metals: Aluminum and thalliumJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2007Azra Bozcaarmutlu Abstract Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 ,M and 3 ,M, respectively. The Lineweaver,Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The Ki values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 ,M, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:340,3347, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20200 [source] Thiazide Diuretics Affect Osteocalcin Production in Human Osteoblasts at the Transcription Level Without Affecting Vitamin D3 ReceptorsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2000D. Lajeunesse Abstract Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1,100 ,M) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]- induced OC release by these cells (maximal effect, ,40,50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (,47.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 ,M HCTZ). HCTZ (100 ,M) also stimulated calcium (Ca2+) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(,-amino ethyl ether)- N,N,N',N' -tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, ,22.5 ± 6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1,100 ,M) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate. (J Bone Miner Res 2000;15:894,901) [source] PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAEJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2000SANIYE YARAR ABSTRACT The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE-cellulose column chromatography techniques. Trehalase was precipitated between 35,50% ammonium sulfate saturation and approximately 5,8 fold purification was achieved. The yeast cAMP-dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE-cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, "cryptic trehalase" (tre-c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE-ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE-cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6,6.8 and 30C, respectively. The kinetic parameters, Km and Vmax, were estimated as 11.78 mM trehalose and 12.47 ,mole glucose/min-mg protein, respectively. [source] Use of Lysozyme, Nisin, and EDTA Combined Treatments for Maintaining Quality of Packed Ostrich PattiesJOURNAL OF FOOD SCIENCE, Issue 3 2010Marianna Mastromatteo ABSTRACT:, The antimicrobial effectiveness of lysozyme, nisin, and ethylene diamine tetraacetic acid (EDTA) combination treatments (Mix1: 250 ppm lysozyme, 250 ppm nisin, 5 mM EDTA; Mix2: 500 ppm lysozyme, 500 ppm nisin, 5 mM EDTA) on bacterial growth of ostrich patties packaged in air, vacuum, and 2 different modified atmospheres (MAP1: 80% O2, 20% CO2; MAP2: 5% O2, 30% CO2, 65% N2) was evaluated. Moreover, the lipid oxidation was evaluated as well as color and sensory characteristics. The growth of total viable counts and lactic acid bacteria were strongly inhibited by the antimicrobial treatments in all the running time (Inhibition Index >97%) whereas for Enterobacteriaceae,and Pseudomonas,spp. lower inhibition indices from 12% to about 28% were observed. The lipid oxidation was more pronounced in the control respect to the treated meat patties. Moreover, the mixture at low concentration of lysozyme and nisin showed the best antioxidative effect. High concentrations of lysozyme and nisin showed the greatest color loss. Also, off-odors for the untreated patties developed faster than the treated samples. Practical Application: Great interest is developing in food bio-preservation, because of the ever-increasing needs to protect consumers' health and to valorize the naturalness and safety of food products. [source] MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE -INDUCED DEFICIENCY OF METHYL DONORSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000R. Bianchi We used cycloleucine (CL) , which prevents methionine conversion to S-adenosyl-methionine (SAMe) by inhibiting ATP-L-methionine-adenosyl-transferase (MAT) , to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35,50%. SAMe p-toluensulphonate (SAMe-SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL-treated animals killed at day 18 after birth, starting from labelled substrates ([3H]-choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL-treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30,40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL. Among proteins, in sciatic nerves of CL-treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe-SD4 treatment counteracted, and in some cases normalized, these changes. In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe-SD4 administration. The financial support of Telethon-Italy (grant No. D 51) is gratefully acknowledged. [source] Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007Gauri Misra Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2,µl 10,mg,ml,1 protein in 50,mM Tris,HCl pH 8.0, 50,mM NaCl, 5,mM EDTA equilibrated against 500,µl reservoir solution consisting of 2.25,M ammonium formate and 0.1,M HEPES buffer pH 7.25. Diffraction data extending to 2.0,Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3,Å. The crystals are hexagonal in shape and belong to space group P6322. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (VM) of 2.1,Å3,Da,1 and a solvent content of about 36.9%. [source] | |||||||||||||