RESUMO
Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.
Assuntos
Metionina , Humanos , Orexinas , Ligantes , Receptores de Orexina/genética , Receptores de Orexina/química , Espectroscopia de Ressonância MagnéticaRESUMO
Annular erythema is one of the cutaneous manifestations of Sjögren's syndrome (SS). Topical corticosteroids and tacrolimus, and oral corticosteroids, have been used as treatments for this condition. However, the safety and efficacy of these treatments remains unsatisfactory, and further development of therapies are desired. In this study, we performed a retrospective analysis of 16 annular erythema associated with SS (AESS) patients treated with hydroxychloroquine (HCQ). Disease activity was assessed using a modified version of the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI), which we termed the modified CLASI (m-CLASI). HCQ treatment improved AESS lesions in all 16 patients. The mean m-CLASI score was reduced by 85.6% at the 12-week follow-up relative to baseline (p < 0.01). Notably, 60% (6/10 cases) of patients with AESS lesions limited to the facial area achieved complete remission within 4 weeks. In the analysis of six patients who had taken oral prednisolone before starting HCQ, all were able to reduce the dose within 52 weeks without relapse. Particularly, 75% (3/4 cases) of patients with prednisolone dose of more than 5 mg/day could reduce their dose to less than 5 mg/day in combination with HCQ. For the safety concerns, two patients experienced grade 1 diarrhea during the 52-week observation period. However, neither serious adverse events nor adverse events requiring discontinuation of treatment occurred. The results of the present study suggest that HCQ may not only be highly effective as a single agent, but may also be useful as a steroid-sparing agent in refractory case requiring long-term steroid administration, making it a good treatment option for AESS.
Assuntos
Síndrome de Sjogren , Dermatopatias Genéticas , Eritema/tratamento farmacológico , Eritema/etiologia , Humanos , Hidroxicloroquina/uso terapêutico , Estudos Retrospectivos , Síndrome de Sjogren/complicações , Síndrome de Sjogren/tratamento farmacológico , Dermatopatias Genéticas/complicações , Dermatopatias Genéticas/tratamento farmacológicoRESUMO
We have previously reported that ischemic animal models treated with a respiratory inhibitor, rotenon, show an increased voluntary alcohol intake. Although it is clear that ischemic brain, as a result of reduced-blood flow, shows pathological events and/or neuro-degenerations apparently, little is known of causal relationship between the mechanism of neural dysfunction and voluntary alcohol consumption. Authors have investigated effects of permanent two-vessel occlusion (p2VO) on rat voluntary alcohol drinking behavior. In first experiment the p2VO-treated rats showed an increase of voluntary alcohol drinking behavior, as compared with sham controls. Using brain microdialysis technique, increases of only nucleus accumbens (ACC) dopamine (DA) releases were suppressed in the p2VO-treated rats significantly, following the high K+ (40 mM) perfusion through the microdialysis probe membrane. Alcohol (200 mM) perfusion-induced DA and serotonin (5-HT) releases in the ACC of the p2VO-treated rats were suppressed significantly in the second experiment, as compared with the sham-treated rats. In third experiment p2VO-treated rats showed significant decreases of the contents of DA, not 5-HT, in the ACC, caudate-putamen (C/P), ventral tegmental area-substantia nigra (VT/SN) and lateral hypothalamus (LH). Dopaminergic neurons in the ACC showed more functional vulnerability against the p2VO treatments, as compared with the serotonergic neurons. An increase of alcohol intake in the p2VO-treated rats means the compensation for the neural degeneration of the dopaminergic system in the ACC consisted brain rewarding system. It was likely suggested that neural disturbance of higher functions involved with incomplete global brain ischemia leads the risk of an abnormal alcohol drinking in human.
Assuntos
Artéria Carótida Primitiva , Estenose das Carótidas , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Doenças do Sistema Nervoso Central , Dopamina , Núcleo Accumbens , Ratos , SerotoninaRESUMO
Central ghrelin is required for the rewarding properties of drug abuse. We investigated whether alcohol affects ghrelinergic, dopaminergic, and serotoninergic neurons and growth hormone secretagogue receptor 1A (GHS-R1A) levels in the reward system of the brain. Alcohol-naïve C57BL/6J mice received 2g/kg ethanol (EtOH) intraperitoneally (i.p.). Plasma ghrelin levels decreased between 1 and 4h. We investigated the effects of EtOH administration on plasma ghrelin levels in two different animal models at 1, 3, and 10months of age. Plasma ghrelin levels decreased following the EtOH treatment in 1- and 3-month-old short-term (1-day) alcohol vapor-exposed (STA) mice. In contrast, EtOH administration increased plasma ghrelin levels in 1- and 3-month-old long-term (20-day) alcohol vapor-exposed (LTA) mice. In vivo ghrelin release in the lateral hypothalamus (LH) increased in STA and LTA mice after the i.p. administration of EtOH. EtOH increased in vivo dopamine (DA), but not serotonin (5-HT) release in the LH of STA mice, and increased in vivo DA and 5-HT release in the LH of LTA mice. GHS-R1A mRNA expression and GHS-R1A protein levels in the LH were increased in LTA mice. The number of GHS-R1A-immunoreactive cells was greater in the LH and amygdala of LTA mice. These results support the neurobiological correlation between the development of drinking behavior and activation of ghrelinergic and serotonergic neurons in the LH. The activation of ghrelinergic systems in the amygdala may also induce an increase in 5-HT release in the LH during long-term alcohol intake.
Assuntos
Consumo de Bebidas Alcoólicas , Tonsila do Cerebelo/fisiologia , Grelina/fisiologia , Região Hipotalâmica Lateral/fisiologia , Neurônios Serotoninérgicos/fisiologia , Animais , Dopamina/metabolismo , Grelina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Grelina/análise , Receptores de Grelina/genética , Receptores de Grelina/fisiologia , Serotonina/metabolismoRESUMO
Bread is rich in dietary fibre and many phytochemical compounds, which may influence chemoprevention of colon cancer. In the present study, we evaluated the effect of three kinds of bread on DMH-induced colorectal tumours in F344 rats. F344 rats were divided into four groups (Steinmetz Three-Grain bread, Steinmetz Country bread, White bread, and MF). All groups were injected with 1,2-dimethylhydrazine (DMH, 20 mg/kg body weight) once a week for 8 consecutive weeks from 5 weeks of age. To investigate the antioxidant effect of bread, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging rate of bread and the serum levels of 8-hydroxy-deoxyguanosine (8-OHdG) in rats were examined. The number of colorectal aberrant crypt foci (ACF) and the incidence of colorectal tumours were studied after 34 weeks of DMH treatment. The Steinmetz Three-Grain and Steinmetz Country bread groups had higher scavenging rates of the DPPH free radical and lower serum levels of 8-OHdG and incidence of ACF, adenomas, and adenocarcinomas of colon than the White bread and MF group. Steinmetz Three-Grain bread and Steinmetz Country bread have various ingredient combinations that may inhibit colorectal cancer progression.
Assuntos
1,2-Dimetilidrazina/toxicidade , Pão , Neoplasias Colorretais , Focos de Criptas Aberrantes/induzido quimicamente , Focos de Criptas Aberrantes/dietoterapia , Focos de Criptas Aberrantes/metabolismo , Focos de Criptas Aberrantes/patologia , Animais , Antioxidantes/farmacologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/dietoterapia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
BACKGROUND: Diet is a major modifiable contributing factor in the etiology of dental caries. The purpose of this paper is to examine the reliability and cross-cultural validity of the Japanese version of the Food Frequency Questionnaire to assess dietary intake in relation to dental caries risk in Japanese. METHODS: The 38-item Food Frequency Questionnaire, in which Japanese food items were added to increase content validity, was translated into Japanese, and administered to two samples. The first sample comprised 355 pregnant women with mean age of 29.2 ± 4.2 years for the internal consistency and criterion validity analyses. Factor analysis (principal components with Varimax rotation) was used to determine dimensionality. The dietary cariogenicity score was calculated from the Food Frequency Questionnaire and used for the analyses. Salivary mutans streptococci level was used as a semi-quantitative assessment of dental caries risk and measured by Dentocult SM. Dentocult SM scores were compared with the dietary cariogenicity score computed from the Food Frequency Questionnaire to examine criterion validity, and assessed by Spearman's correlation coefficient (rs) and Kruskal-Wallis test. Test-retest reliability of the Food Frequency Questionnaire was assessed with a second sample of 25 adults with mean age of 34.0 ± 3.0 years by using the intraclass correlation coefficient analysis. RESULTS: The Japanese language version of the Food Frequency Questionnaire showed high test-retest reliability (ICC = 0.70) and good criterion validity assessed by relationship with salivary mutans streptococci levels (rs = 0.22; p < 0.001). Factor analysis revealed four subscales that construct the questionnaire (solid sugars, solid and starchy sugars, liquid and semisolid sugars, sticky and slowly dissolving sugars). Internal consistency were low to acceptable (Cronbach's alpha = 0.67 for the total scale, 0.46-0.61 for each subscale). Mean dietary cariogenicity scores were 50.8 ± 19.5 in the first sample, 47.4 ± 14.1, and 40.6 ± 11.3 for the first and second administrations in the second sample. The distribution of Dentocult SM score was 6.8% (score = 0), 34.4% (score = 1), 39.4% (score = 2), and 19.4% (score = 3). Participants with higher scores were more likely to have higher dietary cariogenicity scores (p < 0.001; Kruskal-Wallis test). CONCLUSIONS: These results provide the preliminary evidence for the reliability and validity of the Japanese language Food Frequency Questionnaire.
Assuntos
Comparação Transcultural , Suscetibilidade à Cárie Dentária , Cárie Dentária/etiologia , Comportamento Alimentar , Inquéritos e Questionários , Adulto , Bebidas , Registros de Dieta , Dieta Cariogênica , Carboidratos da Dieta/administração & dosagem , Sacarose Alimentar/administração & dosagem , Análise Fatorial , Feminino , Humanos , Japão , Gravidez , Análise de Componente Principal , Kit de Reagentes para Diagnóstico , Medição de Risco , Saliva/microbiologia , Solubilidade , Streptococcus mutans/isolamento & purificação , Adulto JovemRESUMO
PURPOSE: To investigate whether the GLC1F locus is associated with normal tension glaucoma (NTG) in Japanese patients. METHODS: We recruited 242 unrelated Japanese subjects, including, 141 NTG patients and 101 healthy controls. The patients exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement. Genotyping and assessment of allelic diversity was performed on 11 highly polymorphic microsatellite markers in and around the GLC1F locus. RESULTS: Individuals carrying the 163 allele of D7S1277i had a statistically significant increased risk of NTG (p=0.0013, pc=0.016, OR=2.47, 95%CI=1.42-4.30). None of the other markers identified significant loci (pc>0.05) after Bonferroni's correction. CONCLUSIONS: These findings suggested that the genes in the GLC1F locus may be associated with the pathogenesis of NTG.
Assuntos
Povo Asiático/genética , Loci Gênicos , Glaucoma de Baixa Tensão/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Proteínas do Olho , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteínas Supressoras da Sinalização de CitocinaRESUMO
In order to evaluate the relationship between clinical markers of glycemia and glucose excursion, we performed 48-hour continuous glucose monitoring (CGM) in 43 diabetic patients. For the clinical markers, HbA(1c), glycoalbumin (GA), and 1,5-anhydroglucitol (1,5-AG) were measured, and for the parameters of glucose excursion from CGM, average glucose (AG), standard deviation of glucose (SD), the area under the curve for glucose levels >180 mg/dL (AUC(>180)), and the difference between the maximum and minimum glucose levels during 48 hours (DeltaG(48hr)) were analyzed. All patients were treated without any changes of the dosages of oral anti-diabetic agents or insulin for at least the previous 3 months with coefficient of variation (CV) of HbA(1c) less than 4 %. In results, while HbA(1c) did not show any single correlation with AG, SD, AUC(>180), or DeltaG(48hr), both GA and 1,5-AG were significantly related to all these parameters. Furthermore, GA significantly correlated to all CGM parameters, and SD significantly correlated to GA in multiple regression analyses. These results suggest that GA may be a different marker from HbA(1c) for diabetic complications, because GA, but not HbA(1c), may reflect not only short-term average glucose but also fluctuation of glucose.
Assuntos
Glicemia/análise , Complicações do Diabetes/sangue , Diabetes Mellitus/sangue , Idoso , Área Sob a Curva , Biomarcadores/sangue , Desoxiglucose/sangue , Feminino , Hemoglobinas Glicadas/análise , Produtos Finais de Glicação Avançada , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Ambulatorial , Análise de Regressão , Albumina Sérica/análise , Albumina Sérica GlicadaRESUMO
Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to play pivotal roles in bile acid homeostasis by regulating the biosynthesis, conjugation, secretion and absorption of bile acids. Accumulating data suggest that FXR signaling is involved in the pathogenesis of liver and metabolic disorders. Here we show that FXR expression is significantly suppressed in HepG2 cells exposed to hypoxia. Concomitantly, the expression of the bile salt export pump, known as an FXR target gene product and responsible for the excretion of bile acids from the liver, is also decreased under hypoxia. Overexpression of hypoxia-inducible factor (HIF)-1alpha does not mimic the suppressive effect of hypoxia on FXR expression. Furthermore, simultaneous knockdown of HIF-1alpha, HIF-2alpha and HIF-3alpha fails to restore the FXR expression level under hypoxia, indicating that HIF is not involved in hypoxia-evoked FXR downregulation. Instead, we demonstrate that p38 mitogen-activated protein kinase is an indispensable factor for FXR downregulation under hypoxia. Thus, we propose a novel liver disorder model in which two signaling molecules, p38 mitogen-activated protein kinase and FXR, may contribute to the linkage of two pathogenic conditions, i.e. ischemia, a condition accompanying hypoxia, and cholestasis, a condition with intrahepatic accumulation of cytotoxic bile acids.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação para Baixo , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ácidos e Sais Biliares/metabolismo , Hipóxia Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Neoplasias/metabolismo , Preparações Farmacêuticas/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Interações Medicamentosas , Humanos , Metotrexato/farmacocinética , Microscopia Eletrônica de Varredura , Estrutura Molecular , Proteínas de Neoplasias/biossíntese , Preparações Farmacêuticas/metabolismo , Relação Quantitativa Estrutura-Atividade , Spodoptera/genéticaRESUMO
Although genetic alterations in proto-oncogenes, tumor-suppressor genes, cell cycle regulators, and cell growth factors have been implicated in the process of human gastric carcinogenesis, the principle carcinogenic mechanisms are not fully understood. In this study, we used a proteomic approach to search for genes that may be involved in gastric carcinogenesis and that might serve as diagnostic markers. We identified nine proteins with increased expression and 13 proteins with decreased expression in gastric carcinomas. The two most notable groups included proteins involved in mitotic checkpoint (MAD1L1 and EB1) and mitochondrial functions (CLPP, COX5A, and ECH1). This suggested that there are links between dysfunctions in these processes and gastric carcinogenesis. We also observed the differential expression of HSP27 and CYR61 proteins in gastric carcinoma, whose expression is known to be altered in other types of tumors. Furthermore, the study identified proteins whose function in gastric carcinomas was previously unsuspected and that may serve as new molecular markers for gastric carcinomas. Importantly, immunohistochemical analyses confirmed that the levels of expression of MAD1L1, HSP27, and CYR61 were altered in gastric carcinoma tissues. Therefore, our study suggested not only that the proteins identified in this study can be useful diagnostic markers but also that a proteomics-based approach is useful for developing a more complete picture of the pathogenesis and function of gastric carcinomas.
Assuntos
Carcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteômica/métodos , Neoplasias Gástricas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Regulação para Baixo , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Mitocôndrias/metabolismo , Mitose , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaAssuntos
Cromossomos Humanos Par 21/genética , Modelos Animais de Doenças , Síndrome de Down/genética , Epigênese Genética , Proteômica , Animais , Miosinas Cardíacas/genética , Miosinas Cardíacas/isolamento & purificação , Diferenciação Celular/genética , Eletroforese em Gel Bidimensional , Embrião de Mamíferos/citologia , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Camundongos , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/isolamento & purificação , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/isolamento & purificação , Superóxido Dismutase/genética , Superóxido Dismutase/isolamento & purificação , Superóxido Dismutase-1 , TrissomiaRESUMO
Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide (tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb alpha chain until 10 min, and immunoblotting study showed that membrane-bound Hb alpha chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb alpha chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb beta chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG.
Assuntos
Eritrócitos/metabolismo , Dissulfeto de Glutationa/metabolismo , Hemoglobinas/metabolismo , Actinas/química , Actinas/metabolismo , Cisteína/química , Cisteína/metabolismo , Citosol/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutationa/química , Glutationa/metabolismo , Dissulfeto de Glutationa/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hemoglobinas/química , Humanos , Immunoblotting , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mercaptoetanol/química , Metemoglobina/química , Metemoglobina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espectrina/química , Espectrina/metabolismo , terc-Butil Hidroperóxido/química , terc-Butil Hidroperóxido/farmacologiaRESUMO
Correlation of glycated hemoglobin (HbA(1c)) level with degrees of certain peroxidative changes in erythrocyte membrane lipids in diabetic patients have been reported. In the present study, peroxidation of erythrocyte lipids was assessed by changes in tocopherols (Toc), phospholipids (PL), and malondialdehyde (MDA). Membrane cholesterol, Toc, and PL were determined from the same lipid extract. Toc and cholesterol were measured simultaneously by high-performance liquid chromatography (HPLC), and each PL class was determined by a single HPLC elution with ultraviolet light (UV) detection. The detection of PL with UV depends primarily on double bonds in fatty acids and shows a decrease in fatty acids by peroxidation. Changes in Toc and each PL were calculated on the basis of cholesterol and SM, respectively, since cholesterol and sphingomyelin (SM) in the cell membrane are not prone to peroxidation. MDA was measured by an HPLC method with fluorescence detection. These methods for assessment for peroxidation of membrane lipids in intact erythrocytes were validated by experiments with 2, 2-azobis(2-amidinopropane)dihydrochloride (AAPH) and tert-butylhydroperoxide (tBHP); nevertheless, significant differences in the levels of Toc, each PL class, and MDA between a high-HbA(1c) group and a low-HbA(1c) group were not detected.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Membrana Eritrocítica/química , Hemoglobinas Glicadas/análise , Lipídeos/sangue , Adulto , Ácido Araquidônico/sangue , Colesterol/sangue , Cromatografia Líquida de Alta Pressão , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Graxos/sangue , Feminino , Humanos , Peroxidação de Lipídeos , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Fosfatidiletanolaminas/sangue , Fosfatidilserinas/sangue , Fosfolipídeos/sangue , Esfingomielinas/sangue , Tocoferóis/sangue , alfa-Tocoferol/sangue , gama-Tocoferol/sangue , terc-Butil Hidroperóxido/farmacologiaRESUMO
Intracellular signaling through Rho-associated coiled-coil forming kinase (ROCK) is a target of antimetastatic therapy and is proposed to be involved in carcinogenesis. Focal adhesion kinase (FAK) functions downstream of ROCK and participates in anti-apoptotic signaling. We hypothesized that a specific ROCK inhibitor, Y-27632, may exert a pro-apoptotic effect in combination with anticancer agents through the suppression of FAK. A549 lung carcinoma cells were treated with Y-27632 and cisplatin. The simultaneous combination did not exert any additional effect, whereas sequential treatment, in which cisplatin followed Y-27632, enhanced cytotoxicity in concentration- and time-dependent manner. Y-27632 did not suppress tyrosine phosphorylation of FAK in A549-FAK, the active form of FAK expressing A549 cells, as observed in parental cells. Nevertheless, the pretreatment of A549-FAK cells with Y-27632 still enhanced cisplatin-induced cytotoxicity. It was concluded that the ROCK inhibitor enhances cisplatin-induced cytotoxicity through FAK suppression-independent mechanism(s). These observations raise the possibility that the inhibition of the ROCK-mediated signal enhances the effect of anti-cancer agents in addition to its antimetastatic property.
Assuntos
Carcinoma/tratamento farmacológico , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Amidas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metástase Neoplásica , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Piridinas/farmacologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Tirosina/metabolismo , Quinases Associadas a rhoRESUMO
Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.
Assuntos
Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Metemoglobina/metabolismo , terc-Butil Hidroperóxido/farmacologia , Células Cultivadas , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Glutationa/metabolismo , Hemoglobinas/classificação , Humanos , Substâncias Macromoleculares , Oxirredução , Ligação ProteicaRESUMO
Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in alpha-tocopherol (alpha-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte alpha-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.