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PubMed2005

The influence of methylene blue on the spontaneous contractity of the non-pregnant human myometrium and on the myometrial response to DEA/NO.

Nitric oxide (NO) is a potent inhibitor of spontaneous contractions of the human non-pregnant myometrium; however, the precise mechanism by which NO causes the myometrial smooth muscles to relax remains unclear. The aim of this study was to determine the influence of methylene blue (MB) on myometrial contractions and the response of the myometrium to DEA/NO in vitro. Concentration-response curves to DEA/NO were constructed in the absence and presence of MB (5x10(-6), 10(-4) and 10(-2) mol/l) and 5x10(-3) mol/l cystamine. Cystamine did not counteract the DEA/NO-induced relaxation of the myometrial strips. MB itself, excluding the lowest concentration, caused noticeable changes in spontaneous activity. The changes involved a concentration-dependent increase in the frequency of contractions, and a decrease in their amplitude. In conclusion, our results confirm that NO relaxes the human myometrium via a cGMP-independent mechanism. The results obtained in the presence of MB may be misleading because of its complex influence on myometrial contractile activity.

Adult↗
PubMed1975

The effect of sulfhydryl reagents on the heart rate and coronary flow of the isolated perfused guinea-pig heart.

The action of a number of compounds able to react with thiols was tested on guinea-pig hearts perfused at constant pressure. The SH reagents used were NaNO2, oxidized glutathione, cystamine, diamide, 1,5-difluoro-2,4-dinitrobenzene, nitroglycerol, sodium nitroferricyanide and HgCl2. 6,6'-Dithiodinicotinic acid, an SH reagent that does not penetrate the cell, produced no effect. All the other SH reagents produced an increase in coronary flow. All except oxidized glutathione and nitroglycerol increased the heart rate. The increase in heart rate and oxygen consumption could be completely blocked by dichloroisoproterenol; the increase in coronary flow was not affected. Difluorodinitrobenzene, diamide, cystamine and NaNO2 significantly decreased the acid-soluble thiol content of the heart. For these compounds, there was a significant correlation between the decrease in coronary flow and the decrease in thiols. We conclude that in the isolated heart, most SH reagents, if used at the appropriate concentration, will increase the heart rate, probably by relaasing catecholamines. They will also decrease the coronary resistance, probably by a direct effect on the coronary vessels.

Animals↗
PubMed1986

Equilibrium dialysis studies of the binding of radioprotector compounds to DNA.

Radioprotection by WR-2721, S-2-(3-aminopropylamino)ethyl phosphorothioate, is thought to involve its corresponding thiol (WR-1065) or symmetrical disulfide (WR-33278). It has been suggested that these metabolites concentrate close to the DNA target molecule; to test this hypothesis we have measured their in vitro binding to DNA. The binding of WR-33278 (0.05-0.4 mM) to calf thymus DNA (6 mM, with respect to DNA phosphate) was determined at 50, 100, and 150 mM KCl in 1 mM Tris, pH 7, by equilibrium dialysis. The binding of WR-1065 (0.5-8mM) was determined at 25, 50, and 100 mM KCl, under similar conditions, but with 2 mM EDTA and 3 mM dithiothreitol (DTT) added to the dialysis buffer to prevent thiol oxidation. Drug levels were quantitated by HPLC after fluorescent labeling with monobromobimane; disulfide samples were reduced with DTT prior to analysis. Dissociation constants (Kd = [Free Drug] [DNA site]/ [bound drug] ) under these conditions were found to vary with ionic strength, being in the range of 0.02 +/- 0.01 to 0.18 +/- 0.06 mM for WR-33278 and 0.43 +/- 0.24 to 3.5 +2/- 1.5 mM for WR-1065. WR-2721, glutathione, cysteine, and DTT showed no detectable binding to DNA in 25 mM KCl. However, cysteamine and cystamine did bind to DNA, with unbound drug to bound drug ratios of 8 +/- 2 and 0.6 +/- 0.1, respectively, at total drug concentrations of 1 mM. Cystamine and WR-1065 bound to DNA with comparable affinity under similar conditions. These results indicate that binding of WR-33278 and WR-1065 by DNA phosphate are probably significant in the mechanism of radioprotection by WR-2721.

Amifostine↗
PubMed1982

Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin.

Guanylate cyclase from 105,000 X g particulate fractions of rat liver homogenates (20 pmoles of cyclic GMP formed/min/mg protein) was solubilized in the absence of detergents by incubating fractions 12 min at 37 degrees with 5 ug/ml trypsin. Optimal solubilization was dependent upon trypsin and particulate preparation concentrations. Virtually no activation of particulate guanylate cyclase was observed at any time point or trypsin concentration tested. Guanylate cyclase solubilized with trypsin was purified about 500-fold (9.4 nmoles/min/mg protein) using ammonium sulfate precipitation, GTP-affinity chromatography, and preparative polyacrylamide gel electrophoresis. Activity eluted as a single peak on Sepadex G-200 (Stokes radius = 40 A) and migrated as a single peak on sucrose density gradients (S20,w = 4.6). Thus, the tryptic fragment was estimated to be about 80,000 daltons (Mr) with a frictional ratio (f/fo) of 1.4. These partially purified preparations exhibited linear double reciprocal plots with Mn-GTP and Hill coefficients of 1.0. This is in contrast to the crude membrane-associated enzyme which had a Hill co-efficient of 1.5. Membrane-bound and trypsin-solubilized guanylate cyclase were activated 3- and 4-fold with nitric oxide and were inhibited with 1mM cystamine. Cystamine inhibition could be partially reversed with 7.5 mM dithiothreitol. These studies indicate that particulate guanylate cyclase solubilized by limited proteolysis is amenable to purification by "classical" chromatographic techniques. The partially purified fragment contains the catalytic site, the site for nitric oxide activation, and at least one sulfhydryl group required for activity.

Animals↗
PubMed1984

[Effect of beta-endorphin on G-cells in rats with experimental duodenal ulcer].

Immunohistochemistry was used to study the changes in the number of G cells in the antral part of the stomach of rats (40 animals) with cystamine-induced duodenal ulcer treated with beta-endorphine. In the stomach of rats with cystamine-induced ulcer the number of G cells was discovered to be significantly increased, which was removed by an opioid peptide. Naloxone did not block the action of beta-endorphine. Thus, beta-endorphine changes the number of G cells, the drug action being not associated with opiate receptors.

Animals↗
PubMedDate not supplied

PQQ as redox shuttle for quinoprotein glucose dehydrogenase.

The role of pyrroloquinoline quinone (PQQ) as a redox shuttle between an electrode and the active site of soluble quinoprotein glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been investigated using both electrochemical and spectrophotometric methods. Reversible redox behavior of PQQ was observed at cystamine-modified gold electrodes. sGDH is able to reduce free PQQ, i.e. PQQ that is not bound to the enzyme and therefore could act as a mediator between the enzyme and the cystamine-modified electrode. The second order rate constants for the reduction of PQQ by sGDH are 6 x 10(3) M(-1) S(-1) and 64 M(-1) S(-1) in the absence and in the presence of calcium ions, respectively. Similarly, the interaction with a second redox protein is realized via the PQQ shuttle. Using DC voltammetry, the reduction rate of cytochrome c (cyt c) by PQQH2 was determined to be on the order of 10(4) M(-1) S(-1)

Acinetobacter↗
PubMed1998

Role of sperm head syndecan at fertilization in fish.

Fish sperm head plasma membranes have been demonstrated to contain syndecan (transmembrane heparan sulfate proteoglycan) immunofluorohistochemically and using immunoblot analysis and transglutaminase (TGase) by histochemistry. In order to examine the involvement of syndecan in fertilization, mature eggs were inseminated by direct mixing with untreated sperm or with sperm pretreated with an antiheparan sulfate (HS) antibody monoclonal (mAb), bovine serum albumin, human transferrin, or a TGase inhibitor (monodansylcadaverine, cystamine, and iodoacetamide). The fertilization rates of eggs inseminated with untreated and albumin-pretreated sperm were approximately 99.3% and 94.7%, respectively. Those of eggs pretreated with the anti-HS antibody and transferrin were 0% and 5.4%, respectively, whereas use of sperm pretreated with TGase inhibitors resulted in fertilization rates of approximately 13.2-17.8%. These results indicate that sperm head syndecan play an important role in fish sperm-egg contact and/or binding and that TGase inhibitors may reduce the fertilization rate by inhibiting sperm motility.

Animals↗
PubMed1993

Modulation of glutathione S-transferase activity by a thiol/disulfide exchange reaction and involvement of thioltransferase.

Low concentrations of cystamine and cystine inactivated human placenta glutathione S-transferase (GST-pi) in cytosolic fraction very effectively, as did the purified enzyme, through the thiol/disulfide exchange reaction. Mixed disulfide formation of GST-pi in cytosol was prevented by thioltransferase existing in cytosol with a low concentration of GSH. This protection of GST-pi activity was more effective with the participation of glutathione reductase. The incorporation of half-[14C]cystine into a GST-pi molecule according to the inactivation was provided by autoradiography. Purified human placenta thioltransferase (1900-fold from cytosol) could release the incorporated half-[14C]cystine from a GST-pi molecule with restoration of enzyme activity. Thioredoxin in placenta cytosol could not protect the GST-pi activity from inactivation at all.

Chromatography, Affinity↗
PubMed1993

An amplified assay for thiols based on reactivation of papain.

A sensitive spectrophotometric assay has been developed for thiol (sulfhydryl) groups using an inactive disulfide derivative of papain (papain-S-SCH3). The thiol-disulfide interchange reaction of a thiol with papain-S-SCH3 results in the stoichiometric formation of active papain (papain-SH). The reactivated papain catalyzes the hydrolysis of a chromogenic substrate, resulting in an amplified spectrophotometric signal proportional to the initial amount of thiol. A variety of thiols, e.g., cysteine, glutathione, penicillamine, cysteine methyl ester, and cysteamine, yield similar linear plots for the activity of papain vs the initial amount of thiol. An unknown concentration of a thiol is measured using a standard plot for the activity of papain vs the amount of thiol, obtained for the same thiol or for a similar thiol. Thiol groups on proteins and thiol groups of high values of pKa (2-mercaptoethanol, 3-mercaptopropanoic acid) can also be assayed using papain-S-SCH3 in the presence of excess cystamine. The assay is about 100-fold more sensitive than that using Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)]. A 0.4 microM solution of cysteine produces an absorbance change of 0.55 at 410 nm after 30 min in the assay, compared to a predicted change in absorbance of 0.0054 using Ellman's assay.

Aniline Compounds↗
PubMed1993

Reversible modification of cysteine residues of NADPH-cytochrome P-450 reductase.

A reversible chemical modification of SH-groups of NADPH-cytochrome P-450 reductase is the subject of the present study. The enzyme was modified using first biradical RS-SR (R being the imidazolidine derivative) and a new affinity reductase inhibitor beta-cystamine adenosine diphosphate (ANSSN). These reagents were shown to be covalently bound to reductase SH-groups via the reaction of thiol-disulfide exchange resulting in the loss of reducing activity for cytochrome c. NADP+ protected reductase from inactivation and decreased the extent of the modification by RS-SR. The modification of reductase was reversible: the modified enzyme was partially reactivated with glutathione and dithiothreitol. The method proposed can be used to study both the reductase structure and the reversible inhibition of microsomal monooxygenase systems.

Adenosine Diphosphate↗
PubMed1999

The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.

Acetylcysteine↗
PubMed1993

Difference in glutathione S-transferase response to oxidative stress between porcine and bovine lens.

Porcine and bovine lens GSTs were compared in the stability against various oxidative stress which is a major factor of cataract formation in order to clarify the role of lens glutathione S-transferase (GST) and its relation to cataractogenesis. Class pi porcine lens GST was inactivated reversibly by biological disulfides, cystine and cystamine, and also inactivated by active oxygen species such as O2- generated through xanthine-xanthine oxidase system and H2O2. On the other hand, class mu bovine lens GST was insensitive to such applied oxidative stress. Furthermore, 1,2-naphthoquinone, which is a metabolite of naphthalene and an actual inducer of naphthalene cataract, strongly inactivated porcine lens GST though it did not affect bovine enzyme. Thus, porcine and bovine lens GSTs had different sensitivity to various oxidative stress which could induce cataract formation. The results suggest that the differential expression of GST isozymes among animals may explain the variation in the cataract formation caused by oxidative stress.

Animals↗
PubMed1975

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts.

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.

Chloroplasts↗
PubMed1990

Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals.

The importance of mitochondrial ATP formation and extracellular acidosis was evaluated in hepatocyte suspensions after different toxic treatments. Acidotic pH was protective against cell killing from all toxic treatments examined except for pronase, a toxic protease. Fructose, a substrate for glycolytic ATP formation, provided good protection against toxicity from cyanide, oligomycin, t-butyl hydroperoxide, menadione and cystamine. Protection by fructose against CCCP, gramicidin and Br-A23187 required oligomycin. This indicated that these ionophores were causing cytotoxicity by uncoupling oxidative phosphorylation. Fructose provided little protection against pronase and HgCl2, the latter compound being a potent inhibitor of glycolysis. In conclusion, disruption of mitochondrial ATP formation was a common event contributing to the toxicity of chemical oxidants and ionophores. Acidotic pH was generally protective under these conditions of impaired ATP generation.

Animals↗
PubMed1991

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis.

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.

Animals↗
PubMed1993

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation.

Evidence that rat liver microsomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver microsomes has been obtained. Activation of the microsomal glutathione transferase in microsomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated microsomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the microsomal glutathione transferase (Glutaryl-L-Cys-Gly and alpha-L-Glu-L-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (alpha-D-Glu-L-Cys-Gly, gamma-D-Glu-L-Cys-Gly, beta-L-Asp-L-Cys-Gly, alpha-L-Asp-L-Cys-Gly and alpha-D-Asp-L-Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium-dependent glutathione peroxidase (mercaptosuccinate at 50 microM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver microsomes against lipid peroxidation. Purified microsomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in microsomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 microM NADPH and 10 microM Fe-EDTA (2:1). This model system was used to investigate whether microsomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional microsomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100 microM bromosulphophtalein) of the enzyme. Apparently, rat liver microsomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.

Amino Acid Sequence↗
PubMed1991

Transglutaminase is involved in the fusion of mouse alveolar macrophages induced by 1 alpha, 25-dihydroxyvitamin D3.

We have reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] induces fusion of mouse alveolar macrophages directly by a mechanism involving spermidine-dependent protein synthesis (Tanaka, H. et. al., 1989, Exp. Cell Res. 180, 72-83). The macrophage fusion induced by 1 alpha,25(OH)2D3 occurred in a calcium-dependent manner (Jin, C.H. et al., 1988, J. Cell. Physiol. 137, 110-116). In the present study, we examined the possibility that transglutaminase, a calcium-dependent enzyme, is involved in the fusion of macrophages induced by 1 alpha,25(OH)2D3. The activity of transglutaminase increased greatly 12 h after 1 alpha,25(OH)2D3 was ended and reached a maximum at 48 h. Western blot analysis of the cell lysate using an anti-transglutaminase antibody showed that 1 alpha,25(OH)2D3 induced a 77-kDa protein corresponding to transglutaminase. When spermidine synthesis was inhibited by adding methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, the increase in the transglutaminase synthesis by 1 alpha,25(OH)2D3 was markedly inhibited with concomitant inhibition of fusion. Adding more spermidine restored both the synthesis of transglutaminase and the fusion. The treatment of macrophages with cystamine, an inhibitor of transglutaminase, inhibited the fusion in parallel with the suppression of transglutaminase activity, both induced by 1 alpha,25(OH)2D3. These results clearly indicate that 1 alpha,25(OH)2D3 induces transglutaminase by a spermidine-dependent mechanism and that this enzyme is involved in a biological reaction(s) essential for inducing macrophage fusion.

Animals↗
PubMed1986

The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system.

Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+-activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+-dependent, non-lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.

Adenosine Triphosphate↗