Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate

Summary. Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

Genetic study of glutathione accumulation during cold hardening in wheat

The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [³⁵S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat. Received: 24 March 1999 / Accepted: 19 July 1999     

Substrate specificity of the amino acid transporter PAT1

Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.     

Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco

 Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2′,5′-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources. Received: 9 November 1999 / Accepted: 28 February 2000     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

The effect of taurine on renal ischemia/reperfusion injury

Summary. Ischemia-reperfusion (I/R) injury is one of the most common causes of renal dysfunction. Taurine is an endogenous antioxidant and a membrane-stabilizing, intracellular, free beta-amino acid. It has been demonstrated to have protective effects against I/R injuries to tissues other than kidney. The aim of this study was to determine whether taurine has a beneficial role in renal I/R injury. Forty Wistar-Albino rats were allocated into four groups as follows: sham, taurine, I/R, and I/R + taurine. Taurine 7.5 mg/kg was given intra-peritoneally to rats in the groups taurine and I/R + taurine. Renal I/R was achieved by occluding the renal arteries bilaterally for 40 min, followed by 6 h of reperfusion. Immediately thereafter, blood was drawn and tissue samples were harvested to measure 1) serum levels of BUN and creatinine; 2) serum and/or tissue levels of malondialdehyde (MDA), glutathione (GSH), glucose 6-phosphate dehydrogenase (G-6PD), 6-phosphogluconate dehydrogenase (6-PGD) and glutathione reductase (GSH-red); 3) renal morphology; and 4) immunohistochemical staining for P-selectin. Taurine administration reduced I/R-induced increases in serum BUN and creatinine, and serum and tissue MDA levels (p < 0.05). Additionally, taurine lessened the reductions in serum and tissue glutathione levels secondary to I/R (p < 0.05). Taurine also attenuated histopathologic evidence of renal injury, and reduced I/R-induced P-selectin immunoreactivity (p < 0.05). Overall, then, taurine administration appears to reduce the injurious effects of I/R on kidney.     

Suppression of myofibrillar proteolysis in chick skeletal muscles by α-ketoisocaproate

Summary. We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, α-ketoisocaproate (α-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or α-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N^τ-methylhistidine concentration) in chicks while D-leucine and α-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and α-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and α-KIC. These results indicate that D-leucine and α-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to α-KIC.     

Decreases in taurine levels induced by β-alanine treatment did not affect the susceptibility of tissues to lipid peroxidation

Summary. We aimed to investigate the effect of decreased taurine levels on endogenous and induced lipid peroxide levels in liver, brain, heart and erythrocytes as well as prooxidant and antioxidant balance in the liver of rats administered β-alanine (3%, w/v) in drinking water for 1 month to decrease taurine levels of tissues. This treatment caused significant decreases in taurine levels of liver (86%), brain (36%) and heart (15%). We found that endogenous and ascorbic acid-, NADPH- and cumene hydroperoxide-induced malondialdehyde (MDA) levels did not change in the liver, brain and heart homogenates following β-alanine treatment. Also, H₂O₂-induced MDA levels remained unchanged in erythrocytes. In addition, we did not observe any changes in levels of MDA, diene conjugates, glutathione, α-tocopherol, ascorbic acid and the activities of superoxide dismutase, glutathione peroxidase and glutathione transferase in the liver. According to this, buffering or sequestering capacity of tissues to exogenous stimuli was not influenced by reduced taurine levels in tissues of rats.     

Differential responses to anxiogenic drugs in a mouse model of panic disorder as revealed by Fos immunocytochemistry in specific areas of the fear circuitry

Summary. Sensitivity to pharmacological challenges has been reported in patients with panic disorder. We have previously validated transgenic mice overexpressing the neurotrophin-3 (NT-3) receptor, TrkC (TgNTRK3), as an engineered murine model of panic disorder. We could determine that TgNTRK3 mice presented increased cellularity in brain regions, such as the locus ceruleus, that are important neural substrates for the expression of anxiety in severe anxiety states. Here, we investigated the sensitivity to induce anxiety and panic-related symptoms by sodium lactate and the effects of various drugs (the α2-adrenoceptor antagonist, yohimbine and the adenosine antagonist, caffeine), in TgNTRK3 mice. We found enhanced panicogenic sensitivity to sodium lactate and an increased intensity and a differential pattern of Fos expression after the administration of yohimbine or caffeine in TgNTRK3. Our findings validate the relevance of the NT-3/TrkC system to pathological anxiety and raise the possibility that a specific set of fear-related pathways involved in the processing of anxiety-related information may be differentially activated in panic disorder.     

Solid-phase S-3CR generates N-substituted α-aminonitriles for the synthesis of α-phenyl-α-(1-piperazinyl) substituted amino acids

Summary. Structurally diverse amino acids were prepared as versatile synthons for combinatorial chemistry. Using an optimized solid-phase synthesis by Strecker-three-component-reaction (S-3CR), two different polymer linker constructs carrying piperazine were investigated. (a) Acrylate derived base-labile linker yielded α-aminonitriles with N-alkylated piperazines via Hofmann elimination after quarternisation with an alkyl halide. The crude product purities were in the range of 54–87%. (b) A urethane type linker yielded α-aminonitriles with the free piperazine nitrogen when cleaved with acid and the product purities were 72–93%. The α-aminonitriles were easily converted to novel N^ɛ – Fmoc-protected α-amino acids with α-(1-piperazinyl) and α-phenyl substituents.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.     

α-Amino acid behaves differently from β- or γ-amino acids as treated by trimetaphosphate

Summary. The condensation reactions of sodium trimetaphosphate with single amino acids, namely glycine, L-alanine, β-alanine and γ-aminobutyric acid or pairs of these amino acids were reinvestigated by electrospray ion-trap mass spectrometry and high performance liquid chromatography. It was found when mixtures were treated by sodium trimetaphosphate only in the presence of α-amino acid dipeptides were formed. Without addition of α-amino acids, the β-amino acid or γ-aminobutyric acid could not form peptide either by themselves or with their mixtures under the same conditions. From the data it is concluded that phosphate might select α-amino acids to produce the peptides being important precursors for the origin of life. Authors’ address: Dr. Pengxiang Xu, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemistry, Xiamen University, Xiamen 361005, China     

Kinetics of the photosensitized oxidation of chymotrypsin in different media

Summary. The kinetic aspects of the Perinaphthenone-sensitized photooxidation (singlet molecular oxygen [O₂ (¹Δ_g)]-mediated) of α-chymotrypsin (α-Chymo) have been studied at pH 8 and pH 11 as well in reverse micelles (RMs) of sodium 1, 4 bis (2-ethylhexyl) sulfosuccinate (AOT) in n-heptane. The rate constant values for both overall (k_t) and chemical (k_r) quenching of O₂ (¹Δ_g) by α-Chymo in homogeneous media were higher at pH = 11 than at pH = 8, indicating that the OH-ionized tyrosine (Tyr) residues, clearly dominate the quenching process. Besides, the rate constants in water were higher than those determined in RMs, demonstrating that the organized medium protects the protein against photooxidation, probably due to a diminution in both, the accessibility towards oxidizable amino acid residues and the polarity inside the aggregate, as compared to water. The protection effect of α-Chymo against the attack by the oxidative species O₂ (¹Δ_g) in RMs of AOT seems to be due to the increase of protein stability by the encapsulation within the micellar structure. The effect of both, surfactant concentration and variation of the ratio ([H₂O]/[AOT]) = W on the reactive rate constant was also investigated. The process does not depend significantly on micelles concentration while the k_r values increase as W increases. Furthermore, at W = 30, the highest W studied, k_r tends to the value obtained in aqueous medium. Authors’ address: M. A. Biasutti, Departamento de Química, Campus Universitario, Universidad Nacional de Río Cuarto, (X5804ALH) Río Cuarto, Argentina     

Synthesis,cytotoxicity and clastogenicity of novel α-aminophosphonic acids

Summary. α-Ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, α-ethyl-α-N-(hydroxyethylamino) methylphosphonic acid, α-ethyl-N-(phosphonomethyl) glycine, α-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator

 The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated from the C₄ plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C₃ plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more ¹⁴CO₂ in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the rate of CO₂ assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. Received: 26 March 1999 / Accepted: 21 August 1999     

Protective effects of GSH,vitamin E,and selenium on lipid peroxidation in cadmium-fed rats

Increased intake of Cd results in its retention and in peroxidative damage in soft tissues. Coadministration of antioxidants, viz., glutathione (GSH), α-tocopherol, and Se, restricted the uptake and distribution of Cd in liver and kidney of rats. Moreover, no rise in malondialdehyde was recorded. Although possible antioxidative mechanisms manifested by GSH, α-tocopherol, and Se have been discussed, it is hypothesized that GSH functions as a Cd chelator. Glutathione yielded favorable effects in comparison to Se and α-tocopherol.