Characteristics of taurine release induced by free radicals in mouse hippocampal slices

Summary. The release of the inhibitory neuromodulator taurine in the hippocampus is markedly enhanced under various neural cell-damaging conditions, including ischemia and exposure to free radicals. The properties and regulation of the release evoked by a medium containing free radicals was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The free radical damage was induced by applying 0.01% H₂O₂. The release of [³H]taurine was in both adult and developing hippocampus partly Ca²⁺-independent, mediated by Na⁺-dependent transporters and probably resulting from disruption of cell membranes and subsequent ion imbalance. The release in developing mice appeared to be more susceptible to regulation than that in adults, the stimulation by free radicals being in the latter already maximal. The release was reduced by adenosine A₁ receptor agonist R(–)N⁶-(2-phenylisopropyl)adenosine, which effect was, however, abolished by the antagonist 8-cyclopentyl-1,3-dipropylxanthine only in the immature hippocampus, indicating a receptor-mediated process. Moreover, the evoked taurine release in developing mice was potentiated by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate in a receptor-mediated manner, since the effects were abolished by their respective antagonists. The metabotropic glutamate receptors are of only minor significance in the release, the agonists of group I and II receptors slightly reducing the release. Furthermore, NO may also be involved in this release, the NO-generating compounds hydroxylamine and S-nitroso-N-acetylpenicillamine being able to enhance the free-radical-evoked release. It seems that the free-radical-stimulated release, potentiated by ionotropic glutamate receptor activation and NO production, could constitute part of the neuroprotective properties of taurine, being important particularly in the developing hippocampus and hence preventing excitotoxicity.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

硫氧还蛋白的结构及在生物抗氧化中的功能

硫氧还蛋白(thioredoxin,Trx)是广泛存在于原核与真核生物体内的氧化还原调节蛋白。Trx通过对目标蛋白质进行还原,从而调节机体的氧化还原平衡。Trx与硫氧还蛋白还原酶(thioredoxin reductase,TrxR)及NADPH共同组成硫氧还蛋白系统参与众多生理过程。细胞中的活性氧是导致生物氧化胁迫的一个主要方面。Trx可以通过对细胞内被氧化的二硫键的还原来修复机体的氧化损伤,并通过这种方式防止机体衰老。同时,Trx系统可以与其它氧化还原系统如谷胱甘肽(GSH)系统协调配合,并消除体内过多的活性氧。     

Hypochlorite-induced oxidation of amino acids, peptides and proteins

Summary. Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl. This review summarizes information on the rate of reaction of HOCl with proteins, the nature of the intermediates formed, the mechanisms involved in protein oxidation and the products of these reactions. The predicted targets for reaction with HOCl from kinetic modeling studies and the consequences of HOCl-induced protein oxidation are also discussed.     

Betaine or taurine administration prevents fibrosis and lipid peroxidation induced by rat liver by ethanol plus carbon tetrachloride intoxication

Summary. The aim of this study was to investigate the effect of betaine or taurine on liver fibrogenesis and lipid peroxidation in rats. Fibrosis was induced by treatment of rats with drinking water containing 5% ethanol and CCl₄ (2×weekly, 0.2ml/kg, i.p.) for 4 weeks. Ethanol plus CCl₄ treatment caused increased lipid peroxidation and disturbed antioxidant system in the liver. Histopathological findings suggested that the development of liver fibrosis was prevented in rats treated with betaine or taurine (1% v/v in drinking water) together with ethanol plus CCl₄ for 4 weeks. When hepatic taurine content was depleted with -alanine (3% v/v in drinking water), portal-central fibrosis induced by ethanol+CCl₄ treatment was observed to proceed cirrhotic structure. Betaine or taurine was also found to decrease serum transaminase activities and hepatic lipid peroxidation without any change in hepatic antioxidant system in rats with hepatic fibrosis. In conclusion, the administration of betaine or taurine prevented the development of liver fibrosis probably associated with decreased oxidative stress.     

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells

Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H₂O₂ and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H₂O₂. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.     

Inhibition of the L-dopa transport system in human epidermal Langerhans cells by omeprazole and its analogues

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) transport into human Langerhans cells (LC) occurs by a saturable mediation. This plasma membrane agency is, due to its characteristics, distinguishable from systems transporting other neutral, cationic and anionic amino acids into other cells and serves to catalyze the flow of L-dopa, only, into LC. The uphill operation of this L-dopa transport system is believed to occur by down-gradient countermigration of H⁺. Due to the uniqueness of the L-dopa transport system, the widely used analogue inhibition approach was not applicable. Instead we studied omeprazole and its analogues in our search for suitable inhibitory candidates. Omeprazole and most of its analogues were indeed inhibitory in the concentration range 1–100mol/L. Conspicuously, the compounds with strongest polarity were least inhibitory. The inhibitory pattern displayed by omeprazole and the other analogues on L-dopa uptake in LC corresponded to some extent to what has been observed previously for purified H⁺,K⁺-ATPase from tubulovesicles of the stomach. No effects of the inhibitors were registered on energy charge and lactate production of epidermal biopsies, nor were any gross alterations of ultrastructure of LC noticed.     

Mechanistic investigations of the liver toxicity of the free fatty acid receptor 1 agonist fasiglifam (TAK875) and its primary metabolites

For fasiglifam (TAK875) and its metabolites the substance‐specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole‐cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short‐term treatment (24 hours or 48 hours) with TC₅₀ values of 56 to 68 µM (adenosine triphosphate endpoint). The long‐term treatment for 14 days in the hLiMT resulted in a slight TC₅₀ shift over time of 2.7/3.6 fold lower vs 24‐hour treatment indicating possibly a higher risk for cytotoxicity during long‐term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Protective effect of taurine on the detachment of cultured cardiac fibroblasts from the substratum induced by calcium depletion

Summary. Removal of Ca²⁺ from the incubation medium of cultured rat cardiac fibroblasts causes cellular morphological changes, such as the formation of blebs, the ballooning of the cell membrane and the detachment from the culture dish. A 24hr preincubation with 20mM taurine blocked the Ca²⁺ depletion-induced detachment of the cardiac fibroblasts. However, taurine treatment did not prevent other morphological changes induced by Ca²⁺ depletion. The data suggest that taurine plays an important role in cell adhesion in the heart.     

The effect of EGF application in gel form on histamine content of experimentally induced wound in mice

Summary. The factors participating to the wound healing are complex and still obscure. Among these factors, epidermal growth factor (EGF) and histamine by increasing reepithelization and reparation tissue strength via enhancing collagen deposition to the wound site have a beneficial effect. This study was performed to investigate the effect of EGF dosage forms on the histamine content of the experimentally induced wound and some wound healing criters in the mice.Histological investigation of reepithelization, wound tensile strength for healing and collagen maturation, and histamine levels were assessed in the present study. Thirty two mice were divided into control, and EGF treated groups. Controls included three subgroups; untreated (n=5), 0.9% NaCl applied (n=5), and gel applied (n=5). Experimental groups were treated with two forms of EGF; EGF, solution form in 0.9% NaCl (n=5) and the gel form in 0.2% w/w in carbopol 940 (n=7). The discrepancy between these forms were evaluated. This evaluation was done by the application of two forms of EGF for 15 days on experimentally induced wound healing.Gel form of EGF by sustained release from bioadhesive polymer is found to be more effective than the soluble form, on the healing of the wound, by acceleration of reepithelization and increment of wound tensile strength. The tensile strength of the wound indicates the rate of repair and collagen maturation. It has been observed that when physiological saline and carbopol 940 exposed to incision without EGF causes a significant increase in tissue histamine content.According to the results of the present investigation; the histamine content is found to be decreased by EGF gel dosage form treatment, therefore preventing abnormal collagen formation has a beneficial effect on wound healing.     

Monoamine metabolites,iron induced seizures,and the anticonvulsant effect of tannins

Intracortical injections of iron ions have been shown to induce recurrent seizures and epileptic discharges in the EEG. (–)-Epigallocatechin (EGC) and (–)-epigallocatecatechin-3-O-gallate (EGCG), isolated from green tea leaves, have been reported to prevent or diminish the occurrence of epileptic discharges induced by iron ions, and to inhibit catechol-O-methyltransferase. Iron ions significantly increased DOPAC and HVA levels in the intrastriatal perfusate 140 and 180 minutes, respectively, after injection. EGC and EGCG inhibited the increases induced by iron ions. Furthermore, EGCG decreased the HVA level in the perfusate 200 minutes after injection whether or not iron ions were injected. Iron ions had no effect on the 5-HIAA level, and EGC and EGCG raised it. These results suggest that formation of an epileptic focus induced by iron ions might be accompanied by activation of dopaminergic neurons, and that EGC and EGCG inhibit that hyperactivity.     

Expression of ABC-1 transporter is elevated in human glioma cells under irradiation and temozolomide treatment

Summary. Objective: Chemo-therapeutic treatment of glioma patients has minor success. Little is known about mechanisms of a pronounced resistance of gliomas towards actual therapies, yet. ABC-1 belongs to the group of transporters known to be involved in the export of hydrophobic substances and vascular regulation. This study investigates an effect of both temozolomide (TMZ) treatment and/or irradiation on the expression of the ABC-1 transporter in human U87-MG glioma cells.Material and methods: In parallel experiments U87-MG cells underwent either irradiation (RT), chemo-treatment (CT) using TMZ, and combined chemo/radiation-treatment (CT/RT). After each treatment the cells were incubated either 2 or 24 hours at 37°C and counted before protein analysis using Western-Blot technique.Results and conclusions: An exponential growth of cellular density was observed for both untreated and irradiated cells being, however, about 2-times slower in irradiated compared to untreated cells. In contrast the density increase of chemo-treated cells as well as that of cells, which underwent the combined CT/RT treatment was of linear nature. ABC-1 expression was detected in untreated as well as treated cells. Increasing cell density and all kinds of treatment resulted in a considerably enhanced ABC-1 expression. CT treatment resulted in highly up-regulated ABC-1 expression especially in non-confluent cultures compared to untreated cells. Irradiation had a comparable or even higher inducible effect on the ABC-1 expression rates depending, however, on cell density. The highest expression rates were observed in cultures with high cellular density 2 hours after application of the combined treatment. Strong up-regulation of ABC-1 expression under both irradiation and chemo-treatment might be a clue to multidrug and irradiation cross-resistance mechanisms of malignant glioma cells converting the ABC-1 transporter into an attractive pharmacological target for a clinical breakthrough in the therapy of malignant gliomas.     

Efficient preparation of L-cysteic acid and its esters

Summary. L-Cysteic acid and its esters were prepared in good yields from the oxidation of L-cystine by chlorine in water and in alcohols. When the reaction was carried out in alcohols the corresponding esters were produced.     

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum

Summary. Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.     

Effect of pH and salts on the binding of free amino acids to the corn protein zein studied by thin-layer chromatography

Summary. The interaction of free amino acids with the corn protein zein was studied by thin-layer chromatography carried out on cellulose layers covered with zein and the effect of pH and salts on the strength of interaction was elucidated. Only the binding of Arg, His, Lys, Orn and Trp to zein was verified, other amino acids were not retained. Retention of Arg, His, Lys and Orn decreased linearly with increasing concentration of salts the mobile phase indicating the hydrophilic character of amino acid–zein interaction. Both alkaline and acidic pH influenced the strength of binding. Principal component analysis indicated the different character of the influence of pH and salts on the interaction. The results suggest that these amino acid residues may account for the binding of other peptides and proteins to zein.     

Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide

Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4-, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4- oxidation of free methionine have been obtained (k1 = 0.48 +/- 0.08 M(-1)s(-1) by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood ( approximately 25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of alpha1-proteinase inhibitor (alpha1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4- oxidation of alpha1-PI (0.36 +/- 0.06 M(-1)s(-1)) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the alpha1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized alpha1-PI. These results strongly suggest that HCO4- should be considered a reactive oxygen species in aerobic metabolism.     

Activation of transient receptor potential ankyrin 1 by quercetin and its analogs

The agonistic activity of quercetin and its analogs towards the transient receptor potential ankyrin 1 (TRPA1) has been experimentally investigated. The human TRPA1 was expressed in HEK293T cells using a tetracycline-inducible system. The activation of TRPA1 was evaluated by a fluo-4 fluorescence assay based on calcium sensing. The results of a structure–activity relationship study led to the selection of six flavonoids, all of which activated the TRPA1 channel in a dose-dependent manner. Notably, the activation of TRPA1 by these flavonoid aglycones was completely inhibited by the co-treatment of the HEK293T cells with the TRPA1-specific antagonist, HC-030031. Several flavonoid glycosides and metabolites were also evaluated, but did not activate the TRPA1 except for methylated quercetin. On the other hand, TRPV1 (vanilloid receptor) did not respond to any of the flavonoids evaluated in this study. Therefore, these data suggest that the flavonoids would be promising ligands for the TRPA1.     

Effects of hypoxic and osmotic stress on the free D-aspartate level in the muscle of blood shell Scapharca broughtonii

Summary. Blood shell, Scapharca broughtonii, contains large quantities of free D-aspartate comparable to free L-aspartate in its tissues. When the shell was reared in hypoxic seawater, D-aspartate as well as L-aspartate in the foot muscle decreased rapidly, and their total level became about one-fourth within 24hr. None of the other amino acids examined showed a similar behavior, but many of them rather increased during the same period. The increase in L-alanine was especially remarkable and was almost equal to the sum of the decrease in aspartate enantiomers. When the shell that had been acclimated to hypoxic seawater for 96hr was transferred to normoxic seawater, all the amino acid levels mostly returned to the control levels within 96hr. In contrast to these effects of hypoxic stress, hyperosmotic stress of 150% seawater had no effect on the D- and L-aspartate levels in the same tissue. These results suggest that D-aspartate is involved in anaerobic energy metabolism of this bivalve as well as L-aspartate, whose vital role in anoxia-tolerant bivalves is well known.