RESUMEN
Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (pâ¯<â¯0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.
Asunto(s)
Antioxidantes/farmacología , Ditiotreitol/farmacología , Insecticidas/toxicidad , Ivermectina/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , alfa-Tocoferol/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Humanos , Ivermectina/toxicidad , Células K562 , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Purpose: The purpose of this study was to investigate the effects of bilberry extract with its anthocyanins on retinal photoreceptor cell damage and on the endoplasmic reticulum (ER) stress induced by exposure to blue light-emitting diode (LED) light. Methods: Cultured murine photoreceptor cells (661W) were exposed to blue LED light with or without bilberry extract or its anthocyanins in the culture media. Aggregated short-wavelength opsin (S-opsin) in murine photoreceptor cells was observed with immunostaining. The expression of factors involved in the unfolded protein response was examined with immunoblot analysis and quantitative real-time reverse transcription (RT)-PCR. Furthermore, cell death was observed with double staining with Hoechst 33342 and propidium iodide after dithiothreitol (DTT) treatment. Results: Bilberry extract and anthocyanins suppressed the aggregation of S-opsin, activation of ATF4, and expression of the mRNA of the factors associated with the unfolded protein response (UPR). In addition, bilberry extract and the anthocyanins inhibited the death of photoreceptor cells induced by DTT, an ER stress inducer. Conclusions: These findings suggest that bilberry extract containing anthocyanins can alter the effects of blue LED light and DTT-induced retinal photoreceptor cell damage. These effects were achieved by modulating the activation of ATF4 and through the suppression of the abnormal aggregation of S-opsin.
Asunto(s)
Antocianinas/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Luz/efectos adversos , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Extractos Vegetales/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Vaccinium myrtillus/química , Animales , Apoptosis , Western Blotting , Línea Celular , Ditiotreitol/farmacología , Immunoblotting , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Agregación Patológica de Proteínas , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & control , Opsinas de Bastones/metabolismoRESUMEN
The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same ß hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent ß hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Receptores Colinérgicos/química , Reactivos de Sulfhidrilo/farmacología , Sustitución de Aminoácidos , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , Cisteína/química , Cisteína/genética , Ditiotreitol/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Receptores Colinérgicos/genéticaRESUMEN
Cholestasis is a disorder characterized by impaired bile flow and accumulation of cytotoxic bile acids in the liver. On the other hand, oxidative stress and its deleterious consequences seem to have a significant role in cholestasis-induced organ injury. Hence, antioxidants and thiol-reducing agents could have potential protective effect against this complication. The current investigation was designed to evaluate the effect of dithiothreitol (DTT) as a safe and clinically applicable thiol-reductant in cholestatic animals. DTT is a dithiol compound which effectively reduces disulfide bonds in glutathione molecule or different proteins and preserves cellular redox environment. Bile duct ligated (BDL) mice were supplemented with DTT-containing drinking water (0.25% and 1% w: v) for 14 days. Blood, liver, kidney, and spleen samples were collected at scheduled time intervals (3, 7, and 14 days after BDL operation). Significant elevation in plasma biomarkers of liver and kidney injury was detected in BDL animals. Liver and kidney injury was also histopathologically evident by necrosis, inflammation, and fibrosis. Furthermore, high levels of reactive oxygen species in addition to lipid peroxidation, depleted glutathione reservoirs, and impaired tissue antioxidant capacity was detected in the liver and kidney of cholestatic animals. It was found that DTT supplementation (0.25% and 1% w:v) alleviated markers of oxidative stress in the liver and kidney. Moreover, liver and kidney histopathological changes and collagen deposition were markedly attenuated by DTT treatment. The beneficial effects of DTT administration in cholestasis and its associated complications might be linked to its ability for preserving cellular redox environment and preventing oxidative stress.
Asunto(s)
Conductos Biliares/patología , Colestasis/complicaciones , Colestasis/tratamiento farmacológico , Suplementos Dietéticos , Ditiotreitol/uso terapéutico , Riñón/patología , Hígado/patología , Animales , Biomarcadores/metabolismo , Colestasis/sangre , Colestasis/patología , Ditiotreitol/química , Ditiotreitol/farmacología , Hidroxiprolina/metabolismo , Ligadura , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos BALB C , Tamaño de los Órganos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Arsenic is naturally occurring toxic metalloid and drinking As2 O3 containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As2 O3 has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As2 O3 action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As2 O3 alone or combined with dithiothreitol (DTT). The results showed that 0.5 µM As2 O3 plus 20 µM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As2 O3 plus DTT upregulated Bax and Bak, downregulated Bcl-2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As2 O3 also triggered endoplasmic reticulum stress and increased the levels of glucose-regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As2 O3 on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 17-27, 2017.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ditiotreitol/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Enfermedades Mitocondriales/inducido químicamente , Neoplasias de la Boca/tratamiento farmacológico , Óxidos/toxicidad , Reactivos de Sulfhidrilo/farmacología , Trióxido de Arsénico , Arsenicales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Sinergismo Farmacológico , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Neoplasias de la Boca/patologíaRESUMEN
BACKGROUND: Brea gum (BG) is an exudate from the Cercidium praecox tree that grows in semi-arid regions of Argentina. Some previous studies on BG have shown physicochemical characteristics and functional features similar to those of gum arabic. However, there is a need to elucidate the molecular structure of BG to understand the functionality. In this sense, BG was fractionated using hydrophobic interaction chromatography and the obtained fractions were analyzed by size exclusion chromatography. RESULTS: Analysis of the fractions showed that the bulk of the gum (approx. 84% of the polysaccharides) was a polysaccharide of 2.79 × 10(3) kDa. The second major fraction (approx. 16% of the polysaccharides) was a polysaccharide-protein complex with a molecular mass of 1.92 × 10(5) kDa. A third fraction consisted of protein species with a wide range of molecular weights. The molecular weight distribution of the protein fraction was analyzed by size exclusion chromatography. Comparison of the elution profiles of the exudates in native and reducing conditions revealed that some of the proteins were forming aggregates through disulfide bridges in native conditions. Further analysis of the protein fraction by SDS-PAGE showed proteins with molecular weight ranging from 6.5 to 66 kDa. CONCLUSIONS: The findings showed that BG consists of several fractions with heterogeneous chemical composition and polydisperse molecular weight distributions. © 2016 Society of Chemical Industry.
Asunto(s)
Fabaceae/química , Gomas de Plantas/química , Proteínas de Plantas/análisis , Polisacáridos/análisis , Argentina , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Clima Desértico , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Fabaceae/crecimiento & desarrollo , Aditivos Alimentarios/análisis , Aditivos Alimentarios/química , Goma Arábiga/química , Interacciones Hidrofóbicas e Hidrofílicas , Peso Molecular , Oxidación-Reducción , Fenoles/análisis , Fenoles/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/química , Polisacáridos/química , Agregado de Proteínas/efectos de los fármacos , Sustancias Reductoras/farmacología , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
Sulfasalazine is widely used for inflammatory-mediated disorders in human. Renal damage is a serious adverse effect accompanied sulfasalazine administration. No specific therapeutic option is available against this complication so far. Oxidative stress seems to play a role in sulfasalazine-induced renal injury. Current investigation was designed to evaluate the effect of N-acetyl cysteine (NAC) and dithiothreitol (DTT) as thiol reductants against sulfasalazine-induced renal injury in rats. Oral administration of sulfasalazine (600 mg/kg for 14 consecutive days) caused renal injury as judged by increase in serum level of creatinine and blood urea nitrogen. Furthermore, the level of reactive oxygen species and lipid peroxidation were raised in kidney tissue after sulfasalazine administration. Additionally, it was also found that renal glutathione reservoirs were significantly depleted in sulfasalazine-treated animals. Histopathological examination of kidney endorsed organ injury in drug-treated rats. Daily intraperitoneal administration of NAC (250 and 500 mg/kg/day) and/or DTT (15 and 30 mg/kg/day) effectively alleviated renal damage induced by sulfasalazine. Data suggested that thiol reductants could serve as potential protective agents with therapeutic capabilities against sulfasalazine adverse effect toward kidney.
Asunto(s)
Acetilcisteína/uso terapéutico , Lesión Renal Aguda/prevención & control , Antirreumáticos/efectos adversos , Ditiotreitol/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Sulfasalazina/efectos adversos , Acetilcisteína/farmacología , Lesión Renal Aguda/inducido químicamente , Animales , Ditiotreitol/farmacología , Evaluación Preclínica de Medicamentos , Depuradores de Radicales Libres/farmacología , Masculino , Distribución Aleatoria , Ratas Sprague-DawleyRESUMEN
Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the µ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-ß-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.
Asunto(s)
Baclofeno/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo T/metabolismo , Agonistas de Receptores GABA-B/farmacología , Receptores de GABA-B/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Ditiotreitol/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Ganglios Espinales , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Células HEK293 , Humanos , Nocicepción/fisiología , Dolor/metabolismo , Dolor/fisiopatología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Tionucleótidos/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii (lei gong teng; Thunder of God Vine), which belongs to the Celastraceae family, has long been used in traditional Chinese medicine to treat inflammation and rheumatoid arthritis. Celastrol is a bioactive compound isolated from T. wilfordii. AIM OF THE STUDY: We investigated whether celastrol suppressed binding of lipopolysaccharides (LPS) to myeloid differentiation factor 2 (MD2), thereby downregulating Toll-like receptor4 (TLR4) activation in mouse primary macrophages. MATERIALS AND METHODS: Cytokine expression was determined by polymerase chain reaction analysis and enzyme-linked immunosorbent assay in bone marrow-derived primary macrophages (BMDMs). The kinase activity of tank-binding kinase 1 (TBK1) was examined by a luciferase reporter assay and an in vitro kinase assay. LPS binding to MD2 was examined by an in vitro binding assay and confocal microscopy analysis. RESULTS: Celastrol reduced LPS-induced expression of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-1ß, at both the mRNA and protein levels in BMDMs. Celastrol suppressed LPS binding to MD2, as shown by the in vitro binding assay, whereas it did not inhibit TBK1. In addition, co-localization of LPS with MD2 in BMDMs was blocked by celastrol. The inhibitory effects of celastrol on LPS binding to MD2 were reversed by thiol donors (N-acetyl-L-cysteine and dithiothreitol), suggesting that the thiol reactivity of celastrol contributes to its inhibitory effects on TLR4 activation in macrophages. CONCLUSION: Our results demonstrate that celastrol suppresses TLR4 activation through the inhibition of LPS binding to the TLR4/MD2 complex. These results provide a novel mechanism of action by which celastrol contributes to the anti-inflammatory activity of T. wilfordii.
Asunto(s)
Antiinflamatorios/farmacología , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Triterpenos/farmacología , Acetilcisteína/farmacología , Animales , Antiinflamatorios/aislamiento & purificación , Citocinas/metabolismo , Ditiotreitol/farmacología , Ensayo de Inmunoadsorción Enzimática , Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/metabolismo , Medicina Tradicional China , Ratones , Ratones Endogámicos C57BL , Triterpenos Pentacíclicos , Reacción en Cadena de la Polimerasa , Compuestos de Sulfhidrilo/química , Tripterygium/química , Triterpenos/aislamiento & purificaciónRESUMEN
This paper describes the role of α-subunit VISIT-DG sequence residue αThr-349 in the catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the highly conserved αThr-349 in the proximity (2.68 Å) of the conserved phosphate binding residue ßR182 in the phosphate binding subdomain. αT349A, -D, -Q, and -R mutations caused 90-100-fold losses of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes. Double mutation αT349R/ßR182A was able to partially compensate for the absence of known phosphate binding residue ßR182. Azide, fluoroaluminate, and fluoroscandium caused insignificant inhibition of αT349A, -D, and -Q mutants, slight inhibition of the αT349R mutant, partial inhibition of the αT349R/ßR182A double mutant, and complete inhibition of the wild type. Whereas NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) inhibited wild-type ATPase and its αT349A, -D, -R, and -Q mutants essentially completely, ßR182A ATPase and double mutant αT349A/ßR182A were inhibited partially. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ßE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in the wild type, αT349R, and double mutant αT349R/ßR182A but not in αT349A, αT349D, or αT349Q. The results demonstrate that αThr-349 is a supplementary residue involved in phosphate binding and transition state stabilization in ATP synthase catalytic sites through its interaction with ßR182.
Asunto(s)
Dominio Catalítico , Escherichia coli/enzimología , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Treonina/metabolismo , Adenosina Difosfato/farmacología , Aluminio/farmacología , Azidas/farmacología , Membrana Celular/enzimología , Diciclohexilcarbodiimida/farmacología , Ditiotreitol/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/citología , Flúor/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/genética , Modelos Moleculares , Mutagénesis , Mutación , Nitrobencenos/farmacología , Oxazoles/farmacologíaRESUMEN
Chromium occurs mostly in tri- and hexavalent states in the environment. Hexavalent chromium [Cr(VI)] compounds are extensively used in diverse industries, and trivalent chromium [Cr(III)] salts are used as micronutrients and dietary supplements. In the present work, we report that they both induce genetic mutations in yeast cells. They both also cause DNA damage in both yeast and Jurkat cells and the effect of Cr(III) is greater than that of Cr(VI). We further show that Cr(III) and Cr(VI) cause DNA damage through different mechanisms. Cr(VI) intercalates DNA and Cr(III) interferes base pair stacking. Based on our results, we conclude that Cr(III) can directly cause genotoxicity in vivo.
Asunto(s)
Cromo/química , Cromo/toxicidad , Daño del ADN , Mutágenos/química , Mutágenos/toxicidad , Tampones (Química) , División del ADN/efectos de los fármacos , ADN de Hongos/genética , ADN de Hongos/metabolismo , Ditiotreitol/farmacología , Humanos , Células Jurkat , Mutación/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , TemperaturaRESUMEN
Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents.
Asunto(s)
Biocatálisis/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Celulasa/antagonistas & inhibidores , Celulosa/metabolismo , Hierro/farmacología , Penicillium/enzimología , Celulasa/metabolismo , Ditiotreitol/farmacología , Ácido Edético/farmacología , Peróxido de Hidrógeno/farmacología , Hidrólisis/efectos de los fármacos , Iones , Penicillium/efectos de los fármacosRESUMEN
The present study was undertaken to establish mode of action, comparative therapeutic efficacy and safety evaluation of dithiothreitol (DTT) supplemented with Zn and Se against dimethylmercury in rats. Adult male albino rats of Sprague-Dawley strain (150±10 g, n=6 per group) were exposed a bolus dose of dimethylmercury (10 mg/kg, p.o.) for once only followed by DTT (15.4 mg/kg, i.p.) along with the combination of antioxidants Zn and Se (2 mmol/kg and 0.5 mg/kg, p.o.) after 72 h of toxicant administration for three days. The results showed a significant (P≤0.05) increase in the activities of AST, ALT, alkaline phosphatase, lactate dehydrogenase, in serum after toxicant administration. This was accompanied by histopathological observations. A significant rise was observed in lipid peroxidation level and mercury ion concentration however reduced glutathione content decreased in liver, kidney and brain. A significant (P≤0.05) decrease in the activity of acetyl cholinesterase was also seen in different regions of brain. Combined treatment of DTT along with Zn and Se significantly (P≤0.05) recouped the alterations in the enzymatic activities of serum and reversed the tissue biochemical and histopathological changes of liver, kidney and brain. Our results demonstrate that combined treatment of thiol chelator (DTT) along with antioxidants (Zn and Se) plays an important role against dimethylmercury induced tissue damage and hepatic, nephro and neurotoxicity.
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Citoprotección/efectos de los fármacos , Ditiotreitol/farmacología , Compuestos de Metilmercurio/toxicidad , Estrés Oxidativo/efectos de los fármacos , Selenito de Sodio/farmacología , Acetato de Zinc/farmacología , Animales , Antioxidantes/administración & dosificación , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Quelantes/administración & dosificación , Quelantes/farmacología , Terapia Combinada , Ditiotreitol/administración & dosificación , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Compuestos de Metilmercurio/administración & dosificación , Ratas , Ratas Sprague-Dawley , Selenito de Sodio/administración & dosificación , Acetato de Zinc/administración & dosificaciónRESUMEN
We have recently found that the dilution of purified potato Starch-Synthases (SS) and Starch-Branching-Enzymes (SBEs), by a glycine buffer (pH 8.5), containing 1.0mM dithiothreitol (DTT) and 0.04% (w/v) polyvinyl alcohol (PVA) 50K, produced a striking and significant increase in activity (mIU/mL and total mIU) when diluted 1â2 to 1â10. As an example, one SS fraction diluted 1â10 went from 259 to 1470 mIU/mL, giving a total of 14,700 mIU. Dilutions of 1â15 usually resulted in a complete loss of activity. Removal of both DTT and PVA also gave the complete loss of activity. Individual removal of just DTT and the removal of just PVA also produced lowered activities on dilution. The addition of the DTT and the PVA back to the diluted fractions did produce an increase in the activity, but never to the extent that occurred when the samples were diluted simultaneously with both DTT and PVA together in the diluting buffer. Dilution of SBE with buffer containing both DTT and PVA, gave moderate increases, with the exception of one fraction that diluted 1â20 gave a significant increase from 18 to 382 mIU/mL and a total of 7640 mIU. It is concluded that there are inactive starch synthesizing enzymes in the purified fractions that are significantly activated by DTT and PVA, giving much greater amounts of enzyme activities.
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Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Tampones (Química) , Ditiotreitol/farmacología , Alcohol Polivinílico/farmacología , Solanum tuberosum/enzimología , Almidón Sintasa/metabolismo , Activación Enzimática/efectos de los fármacosRESUMEN
The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.
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Bloqueadores del Canal de Sodio Epitelial , Ajo/química , Exudados de Plantas/farmacología , Compuestos de Sulfhidrilo/química , Ácidos Sulfínicos/farmacología , Animales , Cisteína/farmacología , Disulfuros , Ditiotreitol/farmacología , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Oocitos/fisiología , Técnicas de Placa-Clamp , Exudados de Plantas/química , Ácidos Sulfínicos/química , Ácidos Sulfínicos/aislamiento & purificación , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/fisiologíaRESUMEN
AIMS/HYPOTHESIS: It has been suggested that amylin amplifies leptin's effects and affects energy homeostasis synergistically with leptin in animals and humans. However, no previous study has reported on amylin and leptin signalling in hypothalamic, muscle and liver cells. METHODS: Leptin and amylin signalling studies were performed in vitro in mouse GT1-7 hypothalamic, C2C12 muscle and AML12 liver cell lines. RESULTS: Treatment of mouse GT1-7 and C2C12 cells with leptin or amylin increased signal transducer and activator of transcription 3 (STAT3) phosphorylation in a dose- and time-dependent manner. In mouse AML12 cells, leptin and amylin produced a biphasic response of STAT3 activity. Importantly, all leptin and amylin signalling pathways were saturable at leptin and amylin concentrations of â¼100 and â¼50 to 100 ng/ml, respectively. Leptin and amylin in combination activated STAT3, AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK) 1/2 and Akt signalling pathways in an additive manner, effects that were abolished under endoplasmic reticulum (ER) stress. Leptin, but not amylin, increased IRS-1 phosphorylation in GT1-7 hypothalamic, but not in C2C12 muscle and AML12 liver cell lines. CONCLUSIONS/INTERPRETATION: Our data suggest that leptin and amylin have overlapping and additive, but not synergistic, effects in the activation of intracellular signalling pathways. ER stress may induce leptin and amylin resistance in hypothalamic, muscle and liver cell lines. These novel insights into the mode of action of leptin and amylin suggest that these hormones may play an additive role in regulating energy homeostasis in humans.
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Hepatocitos/metabolismo , Hipotálamo/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Leptina/metabolismo , Células Musculares/metabolismo , Transducción de Señal , Animales , Línea Celular , Ditiotreitol/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Leptina/genética , Ratones , Células Musculares/efectos de los fármacos , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/agonistas , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Tunicamicina/farmacologíaRESUMEN
We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at amino acid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.
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Metabolismo Energético/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Ubiquinona/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Ditiotreitol/farmacología , Evaluación Preclínica de Medicamentos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias Hepáticas/fisiología , Consumo de Oxígeno/efectos de los fármacos , Ubiquinona/farmacologíaRESUMEN
A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.
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Proteínas Bacterianas/química , Girasa de ADN/química , Precursores Enzimáticos/química , Inteínas , Fragmentos de Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Empalme de Proteína , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catálisis , Girasa de ADN/genética , Girasa de ADN/aislamiento & purificación , Girasa de ADN/metabolismo , Ditiotreitol/farmacología , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Estabilidad de Enzimas , Hidroxilamina/farmacología , Cinética , Mesna/farmacología , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Oligopéptidos/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Empalme de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Sustancias Reductoras/farmacología , Synechocystis/enzimología , Zinc/farmacologíaRESUMEN
Pneumolysin (PLY) is a key virulence factor contributes to the pathogenesis of Streptococcus pneumoniae. In this study we investigated the effect of allicin and aqueous garlic extracts on hemolytic activity of PLY both in prelysed and intact cells. Additionally the antimicrobial activity of allicin was tested against the bacteria. All tested materials potently inhibited the PLY hemolytic activity. Allicin neutralizes PLY in a concentration- and time-dependent manner. Twenty five minute incubation of PLY (2 HU/mL) with 0.61 µM/mL concentration of allicin, totally inhibited hemolytic activity of PLY (IC50 = 0.28 µM/mL). The inhibitory activity of old extract of garlic was similar to pure allicin (IC50 = 50.46 µL/mL; 0.31 µM/mL; P < 0.05). In contrast fresh extract of garlic inhibits the PLY hemolytic activity at lower concentrations (IC50 = 13.96 µL/mL; 0.08 µM/mL allicin). Exposure of intact cells to allicin (1.8 µM) completely inhibited hemolytic activity of PLY inside bacterial cells. The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 5 mM, proposing that allicin likely inhibits the PLY by binding to cysteinyl residue in the binding site. The MIC value of allicin was determined to be 512 µg/mL (3.15 µM/mL). These results indicate that PLY is a novel target for allicin and may provide a new line of investigation on pneumococcal diseases in the future.
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Antiinfecciosos/toxicidad , Ajo/química , Estreptolisinas/antagonistas & inhibidores , Ácidos Sulfínicos/toxicidad , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Disulfuros , Ditiotreitol/farmacología , Antagonismo de Drogas , Eritrocitos/química , Eritrocitos/efectos de los fármacos , Hemoglobinas/análisis , Hemólisis/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Oxidación-Reducción , Streptococcus pneumoniae/efectos de los fármacos , Estreptolisinas/metabolismoRESUMEN
BACKGROUND: Kidney bean (Phaseolus vulgris L.) seed is an underutilised plant protein source with good potential to be applied in the food industry. Phaseolin (also named G1 globulin) represents about 50 g kg(-1) of total storage protein in the seed. The aim of the present study was to characterise physicochemical, functional and conformational properties of phaseolin, and to compare these properties with those of kidney bean protein isolate (KPI). RESULTS: Compared with kidney bean protein isolate (KPI), the acid-extracted phaseolin-rich protein product (PRP) had much lower protein recovery of 320 g kg(-1) (dry weight basis) but higher phaseolin purity (over 950 g kg(-1)). PRP contained much lower sulfhydryl (SH) and disulfide bond contents than KPI. Differential scanning calorimetry analyses showed that the phaseolin in PRP was less denatured than in KPI. Thermal analyses in the presence or absence of dithiothreitol, in combination with SH and SS content analyses showed the contributions of SS to the thermal stability of KPI. The analyses of near-UV circular dichroism and intrinsic fluorescence spectra indicated more compacted tertiary conformation of the proteins in PRP than in KPI. PRP exhibited much better protein solubility, emulsifying activity index, and gel-forming ability than KPI. The relatively poor functional properties of KPI may be associated with protein denaturation/unfolding, with subsequent protein aggregation. CONCLUSION: The results presented here suggest the potential for acid-extracted PRP to be applied in food formulations, in view of its functional properties.