Addition Of Beta-mercaptoethanol Research Articles

Влияние простатических пептидов на трансмембранный перенос ломефлоксацина.

Introduction. The use of peptides that penetrate the cell without the participation of membrane proteins and are capable of intracellular transport of low molecular weight substances associated with them opens up a new stage in the development of targeted drug delivery to therapeutic targets. Aim. Evaluation of the effect of prostatic peptides of Vitaprost® (prostate extract) on the transmembrane transport of the antimicrobial drug lomefloxacin. Material and methods. In the first series, the effects of prostatic peptides on the ability of lomefloxacin to pass through artificial membranes by passive diffusion (PAMPA method – parallel artificial membrane permeability assay) were studied. In the second series, the effects of prostatic peptides on the cell permeability of lomefloxacin were studied in the Caco-2 model. Results. Comparative study of effective membrane permeability (logPe). for lomefloxacin in the absence (control group) and presence of prostatic peptides (a mixture of prostate extract at a concentration of 10 mg/ml) showed that the combined use of lomefloxacin and prostatic peptides leads to a significant increase in the logPe value, an increase of 29% on average. The specific nature of the identified interaction was confirmed: the addition of beta-mercaptoethanol (BME) to the donor compartment did not affect the permeability of pure lomefloxacin, but suppressed the cargo effect of prostatic peptides. The permeability coefficient (Papp) calculated on the Caco-2 model for lomefloxacin in combination with peptides was 13.04±1.80×10-6 cm/s, while for lomefloxacin it was 10.31±0.76×10-6 cm/s. Conclusion. Comparison of the obtained data of two series of experiments indicates the presence of a direct cargo effect of prostatic peptides on the transmembrane transport of lomefloxacin in vitro.

Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes.

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1mM) and high (0.7mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n=293, BME 0.1 n=270, BME 0.7 n=285) were stained with Hoechst 33342 and examined for nuclear stage after 26hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n=83, BME 0.1 n=65, BME 0.7 n=80). Cleavage rates were determined after 60hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1mM and 55.1% BME 0.7mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.

High efficiency transport of quantum dots into plant roots with the aid of silwet L-77

Quantum dots (QDs) are a novel type of small, photostable and bright fluorophores that have been successfully applied to mammalian and human live cell imaging. In this study, highly dispersive water-soluble mercaptoacetic acid (MAA)-coated CdSe/ZnS QDs were synthesized, which were suitable for investigation as fluorescent probe labels. The treatment of maize seedling roots with QDs showed that the surfactant silwet L-77 aided the efficient transport of QDs into maize roots. Under a concentration ranging from 0.128 to 1.28 microM, QDs caused very low cytotoxicity on maize seed germination and root growth. The addition of mercuric chloride to the Hoagland solution resulted in a decrease of QD content in root tissues, and this decrease was reversed upon the addition of beta-mercaptoethanol, which suggests that mercury-sensitive processes play a significant role in regulating QD flow in the maize root system. We speculate that the apoplastic pathway can contribute substantially to the total quantity of QDs reaching the stele. Therefore, based on this transport approach, MAA-coated QDs can be utilized for live imaging in plant systems to verify known physiological processes.

Implications of β-Mercaptoethanol in Relation to Folate Stability and to Determination of Folate Degradation Kinetics during Processing: A Case Study on [6S]-5-Methyltetrahydrofolic Acid

The effect of beta-mercaptoethanol (0-2%) addition to thermally and/or pressure-treated samples on [6S]-5-methyltetrahydrofolate was studied. Degradation of [6S]-5-methyltetrahydrofolate during both thermal and pressure treatments was mainly caused by oxidation, and the oxidized folates could be partly/completely reduced by beta-mercaptoethanol. The addition of beta-mercaptoethanol (2%) to the thermally and pressure-treated samples overestimated the "actual" stability of [6S]-5-methyltetrahydrofolate and misled the obtained kinetic information.

Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.

Characterization of fibroblast cytoplasmic proteins that bind to the 3' UTR of human catalase mRNA.

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3' untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3' UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of beta-mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.

Inhibition and acceleration of Na+/Ca2+/K+ exchange fluxes by Ag+ in bovine retinal rod outer segments.

The effect of Ag+ on Ca2+ fluxes mediated by the retinal rod Na+/Ca2+/K+ exchanger was investigated in intact bovine rod outer segments (ROS). Intracellular Na+ concentration ([Na+]in)-dependent Ca2+ influx and extracellular Na+ concentration ([Na+]out)-dependent Ca2+ efflux were monitored by changes in cytosolic free Ca2+ measured with the fluorescent Ca(2+)-indicating dye fluo 3. Ag+ was the most effective inhibitor of Na+/Ca2+/K+ exchange fluxes described to date, with half-maximal inhibition observed at 2-8 microM Ag+. Inhibition by Ag+ could be reversed by addition of beta-mercaptoethanol but not by addition of cysteine. Reversal by beta-mercaptoethanol resulted in a marked acceleration of [Na+]out-dependent lowering of cytosolic free Ca2+ but not of [Na+]in-dependent Ca2+ influx. We suggest that Ag+ inhibits and accelerates Na+/Ca2+/K+ exchange fluxes by binding to cysteine residues on the cytosolic surface of the exchanger protein.

Studies on the inhibitory action of leminoprazole against rabbit gastric H+,K(+)-ATPase

Inhibitory action of leminoprazole ((+/-)-2-[[2-(isobutylmethylamino)benzyl]sulfinyl]-1H-benzimidazol e, NC-1300-O-3, LEM) against the H+,K(+)-ATPase activity in rabbit gastric vesicles was investigated. LEM inhibited the H+,K(+)-ATPase activity in leaky vesicles in a concentration- and time-dependent manner. When preincubated with gastric vesicles (20 micrograms protein/ml) for 30 min at 37 degrees C in medium (pH 6.1 or 7.4), the IC50 values were 5.3 microM and 19 microM, respectively. The inhibitory action of LEM was not competitive with respect to K+ and was not reversed by dilution, suggesting that the inhibitory action is irreversible. Inhibition of the enzyme activity by LEM was not found when beta-mercaptoethanol (0.1 mM) was premixed with enzyme before addition of LEM, and it was partially recovered by addition of beta-mercaptoethanol or dithiothreitol (50 mM) after LEM treatment. These results suggest that LEM reacts with essential SH groups of H+,K(+)-ATPase and inactivates the enzyme by forming a covalent disulfide bond. The inhibitory activity of LEM was more potent at pH 6.1 than at pH 7.4, and the rate of the reaction of LEM with GSH was enhanced by lowering the pH of the medium. The inhibition of proton transport by LEM (30 microM) was found after the intact vesicles were fully acidified. LEM also strongly inhibited the valinomycin-stimulated H+,K(+)-ATPase activity. Therefore, it is considered that LEM inhibits H+,K(+)-ATPase activity by an unknown activated reaction under the acidic condition. Alternatively, the possibility was also suggested that an acidic condition is not always necessary for the inhibition of H+,K(+)-ATPase activity by LEM, since LEM, at higher concentration, inhibited the initial rate of acidification and inhibited nigericin-stimulated H+,K(+)-ATPase activity in intact vesicles.

Partial purification of a bacterial lectinlike substance from Eikenella corrodens.

A bacterial lectinlike substance, which is considered to participate in the adherence of Eikenella corrodens to various host cells, was purified from E. corrodens cells. The substance was extracted in 1% Triton X-100 with sonication from the cell envelope of E. corrodens 1073 and partially purified by galactosamine affinity chromatography and gel filtration chromatography based on its hemagglutination (HA) activity. The lectinlike substance was purified about 256-fold as evaluated by its specific HA activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified lectinlike substance (PPL) produced a single protein band of large molecular weight when it was applied to the gel without the addition of beta-mercaptoethanol and heating. Chemical analysis showed that PPL contained 14.4 micrograms of hexose per 100 micrograms of protein and that it did not contain muramic acid, glucosamine, or 2,6-diaminopimelic acid, which are characteristic of peptidoglycans. The HA activity of PPL was inhibited by EDTA but restored by adding Ca2+. The HA activity was remarkably inhibited by sugars containing N-acetyl-D-galactosamine and D-galactose. These results indicate that the lectinlike substance on the E. corrodens cells is an essential factor for the adherence to host cells.

Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent.

Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions. Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo provide protection from inactivation. The protection obtained with ATP is not dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate. The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.

Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the effect of heat on the dissociation, denaturation, and aggregation of sesame alpha-globulin.

alpha-globulin, the major fraction of sesame seed (Sesamum indicum L.) proteins, coagulates on heating. Dissociation, denaturation, and aggregation of the protein upon heating were studied by gel filtration, polyacrylamide gel electrophoresis, sedimentation velocity, pktapp of tyrosyl groups, and fluorescence measurements. Addition of beta-mercaptoethanol does not reduce the extent of heat coagulation. The reassociation of heat denatured subunits through hydrophobic interaction results in the formation of insoluble precipitate.

Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma.

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.

Endonuclease II of Escherichia coli: degradation of gammairradiated DNA.

Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.