Alpha,beta-unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione

Monoclonal antibodies have been obtained by fusing mouse myeloma cells with spleen cells of mice immunized with crude thyroid membranes. Among the antibodies reactive with different thyroid antigenic components, three were found to specifically react with TSH receptor molecules. These antibodies displayed characteristic staining patterns on frozen sections of thyroid tissue from patients with various thyroid diseases upon identification of antibody binding by indirect peroxidase staining. No specific reactivity was detected with tissue from other human organs, such as pancreas, liver, fat, and muscle. The results demonstrate that the immunoperoxidase technique and the specificity of the monoclonal antibodies produced permitted the identification of cellular constituents that might be important antigens in autoimmune thyroid disease.     

Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography

The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.     

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.     

Spectrofluorometric determination of substituted tetrahydrocarbazoles by a methylene blue sensitized photolytic reaction

A spectrofluorometric method was developed for the determination of 6-chloro-9-[2-(2-methyl-5-pyridyl)ethyl]-1,2,3,4-tetrahydrocarbazole-2-methanol hydrochloride and its carboxylic acid analog in blood and urine. It involves extraction of both compounds at neutral pH, either from blood into ethyl acetate (the residue of which is dissolved in either) or from urine directly into ether. Both the alcohol and the acid are separated from each other by selective extraction into acid or base, respectively, and then reextracted into either from the respective aqueous medium by appropriate pH adjustment. The residues of the ether extracts containing the compounds are dissolved separately in 0.25 N NH4OH. Methylene blue is added to all samples, which are then exposed to UV energy for 15 min to produce the fluorophores. The fluorescence of the solutions is read at 370 nm, with excitation at 340 nm. The linear range of quantitation of both compounds is 0.02-10 mug/each/ml of final solution. The method was applied to the determination of blood levels and urinary excretion of the alcohol and its acid metabolite in a dog.     

Protoporphyrin IX accumulation in cells treated with 5-aminolevulinic acid: dependence on cell density, cell size and cell cycle

NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.     

The ratio of reduced glutathione/oxidized glutathione is maintained in the liver during short-term hepatic hypoxia

Controversy persists as to whether reperfusion-induced injuries actually occur in the hepatocyte. The liver is the major source of glutathione, a scavenger of hydrogen peroxide. The aim of this study was to evaluate the sensitivity of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH:GSSG] as an index of hepatic metabolic stress. A total of 121 rats were studied. The superior mesenteric vein (SMV) was occluded for 30 min, and this was followed by 0, 10, or 120 min of reperfusion. Total glutathione and GSSG levels in the liver, bile, and plasma were quantified, using glutathione reductase-coupled enzymatic assays. Results indicated that the hepatic GSH/GSSG ratio was maintained after an occlusion of the SMV, despite a decrease in adenosine triphosphate (ATP) level and energy charge potential. However, plasma levels of total glutathione and GSSG in the inferior vena cava increased after SMV occlusion and continued to increase after reperfusion. Biliary GSSG efflux decreased during 30-min occlusion of the SMV, and remained low even after reperfusion. The liver maintains homeostasis despite a decrease in biliary GSSG efflux, probably by secreting excess GSSG into the hepatic vein when the SMV is occluded. We conclude that the total amount of glutathione and GSSG in the plasma is directly correlated with oxidative stress in the liver.     

Simultaneous high-performance capillary electrophoresis analysis of the reduced and oxidised forms of ascorbate and glutathione

We describe here a procedure for the simultaneous analysis of the oxidised and reduced forms of the major cellular hydrophillic antioxidants, ascorbic acid (vitamin C) and glutathione (gamma-L-glutamyl-L-cysteinylglycine), by high-performance capillary electrophoresis. Separations are performed in uncoated fused-silica capillaries using 200 mmol/l borate pH 9.0, containing 20% (v/v) acetonitrile as the background electrolyte with fixed-wavelength UV absorbance detection at 185 nm. The influence of pH, organic solvent and other additives on the resolution of these compounds is described and we show that the optimised protocol is capable of simultaneously resolving other thiol components including, N-acetylcysteine and methyl-S-glutathione. The method is suitable for the analysis of these antioxidants in Arabidopsis and Nicotiana leaf tissue and is compatible with the use of the high ionic strength, acidic extraction solvents which are necessary to quench the redox equilibria of these labile components.     

Kinetic study of the reaction of glutathione peroxidase with peroxynitrite

Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 +/- 0.8) x 10(6) M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 degreesC. The rate constant for oxidized GPx is about 10 times lower, (0.7 +/- 0.2) x 10(6) M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.     

Characterization of the enzymatic and nonenzymatic reaction of 13-oxooctadecadienoic acid with glutathione

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.     

Secretion of plasminogen activator by cultured rat endometrial stromal cells from uteri differentially sensitized for the decidual cell reaction

OBJECTIVE: We investigated whether patient-centered communication skills can be taught to residents in Internal Medicine by using a time-limited behaviorally oriented intervention. METHOD: Residents working at the Department of Internal Medicine were randomly assigned to an intervention group (IG; N = 19) or a control group (CG; N = 23). In addition to 6 hours of standard medical education per week, the IG received specific communication training of 22.5 hours duration within a 6-month period. Initially and 10 months later, participants performed interviews with simulated patients. Interviews were rated by blinded raters who used the Maastricht History and Advice Checklist-Revised. RESULTS: Compared with the CG, the IG improved substantially in many specific communication skills. Both groups improved in the "amount of medical information identified" and in the ability to "communicate about feasibility of treatment." CONCLUSION: Patient-centered communication skills such as those presented in this intervention study can be taught. The ability to gain medical information and the readiness to communicate about aspects of medical treatment seem to improve with more professional experience; however, they also profit from the intervention.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: effect of membrane physical properties and S-adenosyl-L-methionine

Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.     

Oxidative stress: role of mitochondria and protection by glutathione

We have monitored estrous cycle and measured serum estradiol, GH, IGF-1, T4 and T3 levels in adult hypothyroid female rats which were divided into four groups: H group, hypothyroid rats without treatment; H-T4 group, hypothyroid rats injected daily with T4; HT4-PTU group, hypothyroid rats injected daily with T4 plus PTU (propylthiouracil), and H-T4-IOP group, hypothyroid rats injected daily with T4 plus IOP (iopanoic acid); Euthyroid rats (E group) were used as control. Our results indicate that the lack of sexual cycle in H animals was associated with lower values of estradiol, GH and IGF-1 in comparison to E group; the restoration of sexual cycle in H-T4 group was associated with values of estradiol, GH and IGF-1 higher than those of H group, whereas in H-T4-PTU and H-T-IOP groups the restoration was associated with higher values of GH and IGF-1 and values of estradiol similar to those of H group. These data could suggest a potential role of GH/IGF-1 axis, at least in part, in the lack of sexual cycle in H group and in the ovulation induction in H-T4, H-T4-PTU and H-T4-IOP groups.     

One electron reduction of metmyoglobin and methemoglobin and the reaction of the reduced molecule with oxygen

We have used the pulse radiolysis technique to reduce with solvated electrons (see article) a single Fe(III) site in methemoglobin and metmyoglobin. The reduction process was followed spectrophotometrically and the reactions rate constants were measured: (see article) =6.5 +/- 1-10(10) M-1-S-1. (see article)=2.5 +/- 0.3-10(10) M-1-S-1. Approx. 60% of the (see article) have reacted with the hemin group, and the rest of the (see article) have probably reacted with the globin moiety. We followed the reaction of the reduced proteins to yield the oxyderivatives and measured the rate constants of the oxygenation process k reduced methemoglobin + O2 = 2.6 +/- 0.6-10(7) M-1-S-1 and k myoglobin + O2 = 1.8 +/- 0.2-10(7) M-1-S-1, all the rate constants were measured at pH = 6.8, I = 0.004, T = 22 +/- 2 degrees C. The high rate constant for reduced methemoglobin indicates that one-site-reduced methemoglobin is probably in the R state, as predicted for methemoglobin from X-ray analysis. The spectra of the reduced and oxygenated species were measured under similar conditions at gamma = 450-650 nm. We were able to follow slight changes in the micro-second time scale, these changes were attributed to conformational changes. We were not able to detect any reaction between the radical (see article) and the hemin group (which would result in a complex such as heme O-2). This may be due to kinetic reasons.     

Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione

Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and gammaGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 microM for cysteine, CysGly, and gammaGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme gamma-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gamma-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.     

Kinetics of and preparation of nickel by Ni3S2−NiO reaction under reduced pressure

The reaction between Ni₃S₂ (liquid) and NiO (solid) resulting in the formation of Ni and SO₂ was investigated in the temperature range 800° to 1200°C under a reduced pressure of <0.1 mm Hg. From the kinetic studies in the temperature range 950° to 1150°C, the reaction was found to proceed in three stages: i) Up to about 25 pct reduction, the rate of reaction was high and followed approximately a cubic rate law. During this stage, the reaction is thought to be under mixed control. Activation energy for the first 10 pct reduction was found to be approximately 45 kcal per mole. ii) From about 25 to 90 pct reduction, it obeyed the parabolic rate law, with an activation energy of 86±6 kcal per mole. This value is in agreement with the activation energy reported in the literature for the diffusion of sulfur in nickel. iii) Beyond 90 pct reduction, the reaction was very sluggish owing to the poor availability of the reactants. Optimum conditions for preparing nickel sponge by the above reaction and its processing into thin strips have been standardized. Some of the properties of the metal thus produced have also been incorporated.     

Variation of reaction time can be reduced by the time locked application of magnetic stimulation of the motor cortex

It was the aim of this study to determine the influence of non-invasive magnetic stimulation on the performance of a visually induced reaction task. The experiment was performed in 8 healthy volunteers. After a visual stimulus subjects had to move their thumbs as soon as possible. The muscular reaction was recorded with surface EMG on both sides. After the visual go-signal a magnetic stimulus (Fig. 8 coil) was applied above the right or left motor cortex at varying time intervals (30 and 110 ms). The magnetic stimulus was randomly given or not. Short time intervals between visual and magnetic stimulus induced a shortening, long intervals induced a prolongation of the reaction time. The contralateral reaction time was always longer than the ipsilateral one. Compared with the reaction times without magnetic stimulation, the intraindividual standard deviation of the reaction times was markedly reduced by the application of the magnetic stimulation. This reduction was greater in ipsilateral than in contralateral recordings. The kind of the instruction had additional modifying effects.