Gastroprotective action of the ethanol extract of Leonurus sibiricus L. (Lamiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.

Endogenous Cys-Assisted GSH@AgNCs-rGO Nanoprobe for Real-Time Monitoring of Dynamic Change in GSH Levels Regulated by Natural Drug.

Glutathione (GSH) levels are closely related to the homeostasis of redox state which directly affects human disease occurrence by regulating cell apoptosis. Hence, real-time monitoring of dynamic changes in intracellular GSH levels is urgently needed for disease early diagnosis and evaluation of therapy efficiency. In this study, an endogenous cysteine (Cys)-assisted detection system based on GSH@AgNCs and reduced graphene oxide (rGO) with high sensitivity and specificity was developed for GSH detection. Compared with GSH, GSH@AgNCs with weaker affinity and bonding force was quite easier to extrude from the rGO surface when competing against GSH, leading to the obvious change in fluorescence signal. This phenomenon was termed as "a crowding out effect". Furthermore, the presence of Cys can improve GSH assay sensitivity by enhancing the quenching efficiency of rGO on the GSH@AgNCs. In vitro assay indicated that the efficiency of fluorescence recovery was positively related with GSH concentration in the range from 0 to 10 mM. In addition, the method was employed for real-time monitoring of the dynamic changes in GSH levels regulated by natural drugs. The imaging results showed that the natural compound 3 (C3) can downregulate GSH levels in HepG2 cells, which was accompanied by reactive oxygen species (ROS) release and apoptosis induction. Finally, the method was used to monitor the change of GSH levels in serum samples with chronic hepatitis B (CHB) infection. The results demonstrated that the occurrence and development of CHB may be positively correlated with GSH levels to some extent. Overall, the above results demonstrate the potential application of this new nanosystem in anticancer natural drug screening and clinical assay regarding GSH levels.

Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls.

Peptic ulcers are currently treated with various drugs, all having serious side effects. The aim of this study was to evaluate the gastroprotective activity of calein D (from Calea urticifolia), a sesquiterpene lactone with a germacrane skeleton. Gastric lesions were induced in mice by administering ethanol (0.2 mL) after oral treatment with calein D at 3, 10 and 30 mg/kg, resulting in 13.15 ± 3.44%, 77.65 ± 7.38% and 95.76 ± 2.18% gastroprotection, respectively, to be compared with that of the control group. The effect found for 30 mg/kg of calein D was not reversed by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME, 70 mg/kg, ip), indomethacin (10 mg/kg, sc) or N-ethylmaleimide (NEM, 10 mg/kg, sc). Hence, the mechanism of action of calein D does not involve NO, prostaglandins or sulfhydryl compounds. Calein D was more potent than carbenoxolone, the reference drug. The findings for the latter are in agreement with previous reports.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Association of cardiac injury with iron-increased oxidative and nitrative modifications of the SERCA2a isoform of sarcoplasmic reticulum Ca(2+)-ATPase in diabetic rats.

The role of iron in the etiology of diabetes complications is not well established. Thus, this study was performed to test whether the iron-induced increase of oxidative/nitrative damage is involved in SERCA2a-related diabetic heart complication. Four randomly divided groups of rats were used: normal control group; iron overload group; diabetes group, and diabetic plus iron overload group. Iron supplementation stimulated cardiomyocyte hypertrophy and led to an increase in cardiac protein carbonyls, nitrotyrosine (3-NT) formation, and iNOS protein expression, thus resulting in abnormal myocardium calcium homeostasis of diabetic rats. The levels of SECA2a oxidation/nitration were significantly increased in the iron overload diabetic rats, along with a decrease in SECA2a expression and activity. In order to elucidate the possible role of iron in SERCA2a dysfunction, the effects of iron (Fe(3+) or hemin) on peroxynitrite (ONOO(-)) induced SERCA2a oxidation and nitration were further investigated in vitro. It was found that tyrosine nitration played more important role in SERCA2a inactivation than thiol oxidation. These results present a potential mechanism in which iron exacerbates the diabetes-induced oxidative/nitrative modification of SERCA2a, which may cause functional deficits in the myocyte associated with diabetic cardiac dysfunction. Our findings may help to further understand the role of iron in the pathogenesis of diabetic complications.

Competitive kinetics as a tool to determine rate constants for reduction of ferrylmyoglobin by food components.

Competitive kinetics were applied as a tool to determine apparent rate constants for the reduction of hypervalent haem pigment ferrylmyoglobin (MbFe(IV)O) by proteins and phenols in aqueous solution of pH 7.4 and I=1.0 at 25°C. Reduction of MbFe(IV)O by a myofibrillar protein isolate (MPI) from pork resulted in kMPI=2.2 ± 0.1 × 10(4)M(-1)s(-1). Blocking of the protein thiol groups on the MPI by N-ethylmaleimide (NEM) markedly reduced this rate constant to kMPI-NEM=1.3 ± 0.4 × 10(3)M(-1)s(-1) consistent with a key role for the Cys residues on MPI as targets for haem protein-mediated oxidation. This approach allows determination of apparent rate constants for the oxidation of proteins by haem proteins of relevance to food oxidation and should be applicable to other systems. A similar approach has provided approximate apparent rate constants for the reduction of MbFe(IV)O by catechin and green tea extracts, though possible confounding reactions need to be considered. These kinetic data suggest that small molar excesses of some plant extracts relative to the MPI thiol concentration should afford significant protection against MbFe(IV)O-mediated oxidation.

Sodium selenite/selenium nanoparticles (SeNPs) protect cardiomyoblasts and zebrafish embryos against ethanol induced oxidative stress.

Alcoholic cardiomyopathy is the damage caused to the heart muscles due to high level of alcohol consumption resulting in enlargement and inflammation of the heart. Selenium is an important trace element that is beneficial to human health. Selenium protects the cells by preventing the formation of free radicals in the body. In the present study, protein mediated synthesis of SeNPs was investigated. Two different sizes of SeNPs were synthesized using BSA and keratin. The synthesized SeNPs were characterized by scanning electron microscopy (SEM) with elemental composition analysis Energy Dispersive X-ray spectroscopy(EDX) and X-ray diffraction (XRD). This study demonstrates the in vitro and in vivo antioxidative effects of sodium selenite and SeNPs. Further selenium and SeNPs were evaluated for their ability to protect against 1% ethanol induced oxidative stress in H9C2 cell line. The selenium and SeNPs were found to reduce the 1% ethanol-induced oxidative damage through scavenging intracellular reactive oxygen species. The selenium and SeNPs could also prevent pericardial edema induced ethanol treatment and reduced apoptosis and cell death in zebrafish embryos. The results indicate that selenium and SeNPs could potentially be used as an additive in alcoholic beverage industry to control the cardiomyopathy.

Analysis of cationic amino acid transport activity in canine lens epithelial cells.

Cationic amino acid transport activity in a canine lens epithelial cells (LEC) line was investigated. The transporter activity of arginine was 0.424 ± 0.047 nmol/mg protein min, while the presence of N-ethylmaleimide, an inhibitor of the canine cationic amino acid transporter (CAT), reduced transport activity by 30%. A full-length cDNA sequence of canine CAT1 was 2558 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence of canine CAT1 showed similarities of 92.1% and 88.6% to those of the human and mouse, respectively. Western blot analysis detected a band at 70 kDa in a membrane protein sample of LEC. RT-PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined.

Gastroprotection of suaveolol, isolated from Hyptis suaveolens, against ethanol-induced gastric lesions in Wistar rats: role of prostaglandins, nitric oxide and sulfhydryls.

Hyptis suaveolens is a medicinal plant that is, according to traditional medicine, considered useful in the treatment of gastric ulcers. Although its gastroprotective activity was reported, the active compounds have not been identified. Therefore, the aim of the present study was to identify at least one active compound potentially responsible for the gastroprotective activity of H. suaveolens by using a bioassay guided study with an ethanol-induced gastric ulcer experimental model in rats. The results show that the hexane extract had protective activity (close to 70% when using doses between 10 and 100 mg/kg), and that the compound suaveolol, isolated from this extract, was one of the active gastroprotective agents. This is the first report about the gastroprotective activity of suaveolol. Rats treated with this compound at 3, 10, 30 and 100 mg/kg showed 12.6, 21.3, 39.6 and 70.2% gastroprotection respectively. The effect elicited by suaveolol (at 100 mg/kg) was attenuated by pretreatment with either N(G)-nitro-L-arginine methyl ester (70 mg/kg, i.p.), a nitric oxide (NO) synthase inhibitor, indomethacin (10 mg/kg, s.c.), a blocker of prostaglandin synthesis, or N-ethylmaleimide (10 mg/kg, s.c.), a blocker of sulfhydryl groups. This suggests that the gastroprotective mechanism of action of this compound involves NO, prostaglandins and sulfhydryl groups.

A thallium transport FLIPR-based assay for the identification of KCC2-positive modulators.

KCC2, potassium chloride cotransporter 2, is expressed exclusively in the CNS (on inhibitory neurons) and plays a major role in maintaining appropriately low intracellular chloride levels that ensure inhibitory actions of GABA(A) and glycine receptors. As such, it plays a pivotal role in inhibitory mechanisms that control neuronal excitation in the CNS. KCC2 downregulation has been implicated in various excitatory disorders, such as epilepsy and neuropathic pain. Positive modulators of KCC2 expression or activity may thus provide effective therapy for these disorders. However, the identification of such agents is hindered by the lack of a high-throughput screening method. Here the authors report the development of a fluorescence-based thallium (Tl(+)) transport assay using a Fluorometric Imaging Plate Reader (FLIPR), in which KCC2 activity is assessed by measuring the initial rate of KCC2-mediated Tl(+) transport/influx. The authors demonstrate Tl(+)/Cl(-) cotransport by KCC2, which exhibits a high apparent affinity for Tl(+) and dependency on the presence of the Cl(-) ion. Pharmacological studies revealed anticipated effects and potencies of known KCC-positive (NEM, staurosporine) and KCC-negative (DIOA, furosemide) modulators. The authors demonstrate that the assay is robust and reproducible and can be employed in high-throughput screening for positive modulators of KCC2 as potential therapeutic agents.

Thiol-dependent membrane transport of selenium through an integral protein of the red blood cell membrane.

The molecular details of the selenium metabolism and transport in living systems are still not completely understood, despite their physiological importance. Specifically, little is known about the membrane transport of selenium from most of the selenium containing compounds. In the present study, we investigated the mechanism for the membrane transport of selenium from red blood cells (RBCs) to the blood plasma. When the selenium distribution in the RBC ghost membrane after treatment with selenious acid was analyzed, nearly 70% of the selenium in the membrane was found to bind to the anion exchanger 1 (AE1) protein, which suggested that the integral protein AE1 is responsible for the membrane transport of selenium. The thiol dependency of the selenium export from the RBC to the blood plasma was examined using membrane permeable thiol reagents, i.e., N-ethylmaleimide (NEM) and tetrathionate (TTN). Treatment of the RBC with NEM, a thiol-alkylating reagent, resulted in modification of the thiol groups in the amino-terminal cytoplasmic domain (N-CPD) of the AE1, but not those in the membrane domain. Such an NEM treatment provided a marked inhibition of the selenium export from the RBC to the blood plasma. In addition, the treatment with TTN, a thiol-oxidizing reagent that forms intermolecular disulfide bonds, appeared to oxidize thiol groups in both the N-CPD and the membrane domain of AE1, which resulted in complete inhibition of the selenium export even during the initial period in which the export had a maximum velocity when using the thiol reagent-free treatment. Such complete inhibition of the selenium export from the TTN-treated RBC appeared to be due to the oligomerized AE1 proteins resulting from the intermolecularly formed disulfide bonds. These inhibitory effects using NEM and TTN suggested that thiol groups in the integral protein AE1 play essential roles in the membrane transport of the selenium from the RBCs to the blood plasma.

Rheological and microstructural properties of porcine myofibrillar protein-lipid emulsion composite gels.

The objective of the study was to investigate the role of emulsified fat (lard) and oil (peanut oil) in the rheology and microstructure of porcine myofibrillar protein (MP) gels. Heat-induced composite gels were prepared from 2% MP with 0% to 15% pre-emulsified lipids at 0.6 M NaCl, pH 6.2. Dynamic rheological testing upon temperature sweeping (20 to 80 degrees C at 2 degrees C/min) showed substantial increases in G' (an elastic modulus) of MP sols/gels with the addition of emulsions. Gel hardness was markedly enhanced (P < 0.05) by incorporating >or=10% emulsions, and the composite gel with 15% lard was 33% more rigid (P < 0.05) than that with 15% peanut oil. Incorporation of both emulsions at 10% or higher levels improved the water holding capacity of the gels by 28% to 44% (P < 0.05). Light microscopy revealed a compact gel structure filled with protein-coated fat/oil globules that interacted with the protein matrix via disulfide bonds. The results indicated that both physical and chemical forces contributed to the enhancements in the rheology, moisture retention, and lipid stabilization in the MP-emulsion composite gels.

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1.

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.

Antioxidative action of methanolic extract and buthanolic fraction of Vochysia tucanorum Mart. in the gastroprotection.

AIM OF THE STUDY: Vochysia tucanorum is an important medicinal plant used in the Cerrado of Brazil against gastric disorders and this study reveals the pharmacological action of this traditional medicine use. MATERIALS AND METHODS: The methanolic extract (E-MeOH) and buthanolic fraction (Fr-Bu) obtained from V. tucanorum were challenged by different necrotizing agents in rodents. NO-synthase inhibitor (L-NAME) and SH blocker (NEM) were used to evaluate the participation of cytoprotective factors in E-MeOH and Fr-Bu gastroprotection. Antiulcerogenic action of V. tucanorum was evaluated in rats and mice at doses 250, 500 or 1000 mg/kg (E-MeOH) and 37.5, 75 or 150 mg/kg (Fr-Bu). RESULTS: Both E-MeOH and Fr-Bu present elevated gastroprotective action in all in vivo experimental models, without signs of acute toxicity. The mechanisms involved in the gastroprotective action of E-MeOH and Fr-Bu are related to the antioxidant activity and protection to gastric mucosa NO levels. Phytochemical investigations of Fr-Bu identified different pentacyclic triterpenoids such as betulinic acid, erythrodiol, epi-betulinic acid and mixtures of ursolic acid and oleanolic acid derivatives as the major constituents. The presence of such triterpenoids in Fr-Bu is probably related to the potent gastroprotective action of this medicinal plant species. CONCLUSION: Effectiveness in gastroprotection and the absence of acute toxicity indicate this species as a promising herbal drug that is in accordance with ethnopharmacological use against gastric disorders.

The anti-ulcerogenic effects of Curatella americana L.

ETHNOPHARMACOLOGICAL RELEVANCE: Curatella americana L. (Dilleneaceae) is a medicinal plant very frequently cited as acting against gastrointestinal disorders in ethnopharmacological inventories of the Cerrado region of Brazil. AIM OF THE STUDY: The ethanolic extract (CEB) and infusion (BI) of Curatella americana bark were investigated for their ability to prevent and heal ulceration of the gastric mucosa. MATERIALS AND METHODS: The preventive and healing actions of Curatella americana were evaluated in experimental in vivo models in rodents that simulated this disease in human gastric mucosa. RESULTS: CEB significantly decreased the severity of gastric damage formation induced by the combination of several gastroprotective models (HCl/ethanol, indomethacin/bethanecol, absolute ethanol, stress and pylorus ligature). But, unlike CEB, the BI did not exert gastroprotective effect. The gastroprotective action of CEB involved antisecretory action, augmentation of gastric mucus (48%) and participation of endogenous sulfhydryl compounds that increase efficacy of barrier mucosa against injurious agents. CEB also presents effective healing action in chronic gastric disease (1.90+/-0.55 vs. 6.86+/-0.46 mm2)in the control) and its action mechanisms consisted of increasing the PGE2 (40%) and somatostatin levels (269%) while decreasing the gastrin level in rat plasma (79%). CONCLUSIONS: The gastroprotective effect and healing action of Curatella americana involved modulation of PGE2, somatostatin and gastrin levels, probably due to the presence of oligomeric and polymeric proanthocyanidins in the bark.

Bioassay-guided isolation of an anti-ulcer diterpenoid from Croton reflexifolius: role of nitric oxide, prostaglandins and sulfhydryls.

Croton reflexifolius H. B. K (Euphorbiaceae) is a very common medicinal plant in the Huastecan region of Mexico that, according to local folk medicine, is considered useful in the treatment of gastritis and gastric ulcer. We have aimed to test the validity of this practice by using the experimental model of an ethanol-induced gastric ulcer in male Wistar rats. The results showed that C. reflexifolius had gastroprotector activity, that the hexane extract had the highest protective activity (64.38+/-7.72%), and that polyalthic acid isolated from this extract was the main active gastroprotector agent. Rats treated orally with polyalthic acid showed a gastroprotective effect similar to that elicited by carbenoxolone. As with carbenoxolone, the effect elicited by polyalthic acid was attenuated by pretreatment with either N(G)-nitro-L-arginine methyl ester (70 mgkg(-1), i.p.), a nitric oxide (NO) synthase inhibitor, or N-ethylmaleimide (10 mgkg(-1), s.c.), a blocker of sulfhydryl groups. This suggested that the gastroprotective mechanism of this diterpenoid involved the participation of both NO and endogenous sulfhydryl groups. Contrary to carbenoxolone, the gastroprotective effect of polyalthic acid was not affected by the inhibition of prostaglandin synthesis with indometacin (10 mgkg(-1), s. c.). In conclusion, Croton reflexifolius contains compounds with gastroprotector activity. Polyalthic acid, which was isolated from this plant, was the main compound with gastroprotector activity, having effectiveness similar to that found with the use of carbenoxolone. Whereas NO and sulfhydryl groups were involved in the mechanisms of gastroprotective action of polyalthic acid, prostaglandins were not.

Selenium compounds counteract the stimulation of ecto-nucleotidase activities in rat cultured cerebellar granule cells: putative correlation with neuroprotective effects.

Glutamate is the main excitatory neurotransmitter in brain involved in pathophysiology of several brain injuries. In this context, glutamate showed to stimulate ecto-nucleotidase activities in cerebellar granule cells increasing extracellular adenosine levels, an important neuromodulator in the CNS able to prevent cell damage. The organoselenium compounds, such as ebselen and diphenyl diselenide [(PhSe)(2)], display neuroprotective activities mediated at least in part by their antioxidant and anti-inflammatory properties. Ebselen was described to prevent glutamate-induced lipid peroxidation and cell death in cerebellar granule cells and (PhSe)(2) modify glutamatergic synapse parameters in vitro and in vivo. In the present study, we investigated the effects of ebselen or (PhSe)(2) on glutamate-induced stimulation of ecto-nucleotidase activities in rat cultured cerebellar granule cells. Glutamate increased nucleotide hydrolysis at lower concentrations (10 and 100 microM) than described in the literature and this effect was counteracted by both organoselenium compounds tested. Based on these results, we investigated the association of organoselenium effects with their antioxidant properties searching for redox site modulation by using the alkylant agent N-ethylmaleimide (NEM). Our results suggest that selenium compounds, as well as the well-known antioxidant trolox, can avoid the increase on glutamate-induced stimulation of ecto-nucleotidase activities probably due to their antioxidant properties.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Molecular cloning, expression, biochemical characteristics, and biomarker potential of theta class glutathione S-transferase (GST-T) from the polychaete Neanthes succinea.

We cloned and sequenced the full-length cDNA of a theta class glutathione S-transferase (GST-T) from the polychaete Neanthes succinea. The open reading frame of N. succinea GST-T cDNA was 678bp and encoded 226 amino acid residues. We generated recombinant N. succinea GST-T by expression in transformed Escherichia coli and studied the kinetic properties as well as the effects of inhibitors, pH, and temperature on N. succinea GST-T. GST-T expression was studied using real-time RT-PCR in response to exposure to the model oxidative stress-inducing agent, CuCl(2). Copper induced a concentration-dependant increase in the expression of GST-T. Moreover, polychaetes collected from a heavily contaminated lake near an industrial complex showed significantly higher levels of GST-T expression. Interestingly, the site-collected polychaetes with the highest GST-T mRNA expression levels also showed the highest metallothioneins levels. These results suggest that GST-T in polychaetes may have an antioxidant role and that N. succinea GST-T expression may be a useful biomarker for exposure to environmental contaminants such as copper. Our findings provide a better understanding of the biochemical characteristics of N. succinea GST-T, and elucidate the potential role of GST-T in heavy metal-induced oxidative stress and as a biomarker for environmental contamination.

Evaluation of the anti-ulcerogenic effect of sesquiterpene lactones from Centaurea solstitialis L. ssp. solstitialis by using various in vivo and biochemical techniques.

The guaianolide type sesquiterpene lactones chlorojanerin, 13-acetyl solstitialin A and solstitialin A were identified as the anti-ulcerogenic components of the chloroform extract of the aerial parts of Centaurea solstitialis ssp. solstitialis (Asteraceae). In this study, these compounds were investigated by using various in vivo ulcer models in rats and mice. Chlorojanerin was shown to be significantly effective in preventing the induction of lesions by ethanol- (EtOH-) (both oral and subcutaneous administration), indomethacin-, indomethacin plus HCl/EtOH-, N(G)-nitro-l-arginine methyl ester plus EtOH-, N-ethylmaleimide plus EtOH-, water immersion and restraint stress, and serotonin, as well as inhibiting titratable gastric acidity and acid output, and increasing gastric pH, but was ineffective in the prevention of ulcers induced by pyloric ligation, diethyldithiocarbamate, and cysteamine, and had no effect on gastric secretion volume or peptic activity. A mixture of 13-acetyl solstitialin A (95%) and solstitialin A (5%) was found to be significantly effective against EtOH-induced lesions on oral administration but was ineffective when administered subcutaneously. This mixture was also found to be effective in preventing lesions induced by EtOH, indomethacin, indomethacin plus HCl/EtOH, N(G)-nitro-l-arginine methyl ester plus EtOH, N-ethylmaleimide plus EtOH, water immersion and restraint stress, serotonin and cysteamine, as well as inhibiting titratable gastric acidity and titratable acid output, and gastric pH, but was found ineffective against the pyloric ligation-induced and diethyldithiocarbamate-induced ulcerogenesis models, as well as gastric secretion volume and peptic activity. On the other hand, active compounds did not show any toxic effect on acute toxicity (3 days administration) evaluation tests in mice.