RESUMEN
To study the impact of the enterovirus 71(EV71) on the nuclear transport mechanism,The pGFP-NLS vector with nuclear location signal(NLS) was constructed, RD cells transfected by the pGFP-NLS vector were inoculated with the EV71 or cotransfected by EV71-2A vector. The results showed that GFP protein with NLS was expressed in the cytoplasm due to the inhibition of nuclear transport. In order to further study the mechanism of the EV71 to prevent nuclear transport,Nup62 was detected by Western blotting after RD cells were infected with EV71 or transfected by EV71-2A vector. The results showed that decreased expression of Nup62 could be detected after infection with EV71 and transfection by EV71-2A vector. This study demonstrates that the cleavage of Nup62 by EV71 2A protease may be the mechanism of nuclear transport inhibition.
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Núcleo Celular/metabolismo , Enterovirus Humano A/enzimología , Infecciones por Enterovirus/virología , Glicoproteínas de Membrana/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Péptido Hidrolasas/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Enterovirus Humano A/genética , Enterovirus Humano A/metabolismo , Regulación Viral de la Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
OBJECTIVE: To detect the expression patterns and localization of the Tyro3, Axl, and Mer (TAM) receptor tyrosine kinases in the rat brain, and to understand the significance of this receptor family in the brain. METHODS: Sprague Dawley rats aged P3 (day 3 of postnatal development), P9, P15, P30 and P52 were anesthetized and their brains removed. Real time quantitative PCR was used to determine mRNA expression levels of TAM. Western blotting was applied to analyze protein expression levels of TAM peptides. The immunohistochemical stainings of Tyro3 protein in adult rat brains were detected by the avidin-biotin-peroxidase complex method. RESULTS: Tyro3 was the most highly expressed and widely distributed receptor among TAM family. Its mRNA level increased dramatically during the second postnatal week, reached at the highest level by P30, and remained at that level in the adult. The relative expression quantitative of Tyro3 mRNA in the adult rat brain increased about 3.2-fold than that at P15 stage. The expression of Tyro3 protein was detected faintly at early stage, gradually increased from P15, and reached maximal levels at adult stage. The relative density of immunoreactive product of Tyro3 peptide in the rat brain was up-regulated 1.3-fold from P15 to adult (P52). Immunohistochemical stainings demonstrated that Tyro3 protein was detected in the majority of cells of all the cortical layers. Strong signals were observed in the piriform cortex and the hippocampus. At the subcellular level, Tyro3 was detected both in the soma and dendrites of pyramidal neurons. CONCLUSION: The results imply that Tyro3 is the main TAM receptor tyrosine kinase expressed in adult rat brain that modulates signaling cascades influencing synaptic function in the brain.
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Encéfalo/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Corteza Cerebral/metabolismo , Femenino , Hipocampo/metabolismo , Masculino , ARN/genética , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
The organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) plays an important role in the transport of endogenous and xenobiotic compounds, such as bile acids and rifampin. In this study, the association between OATP1B1 polymorphisms and rifampin hepatotoxicity was investigated using integrated population genetic analysis and functional studies. A total of 273 unrelated patients treated with rifampin were recruited. The allele frequencies were examined in patients with drug (rifampin)-induced liver injury (DILI) (n = 118) and without (non-DILI) (n = 155). Functional analyses were conducted to determine whether the inhibition of bile acids by rifampin was associated with OATP1B1 variants. In the present study, 24 single nucleotide polymorphisms (SNPs) in OATP1B1 were detected in a Chinese population, with two of them causing an amino acid change (rs2306283 and rs4149056). The haplotypes constructed by these two SNPs were OATP1B1 *1a, *1b, *5 and *15, with their respective frequencies being 23.44, 66.30, 0.73 and 9.52% in a total of 273 individuals. The logistic regression analysis indicated that the *15 haplotype was associated with susceptibility to DILI (p = 0.03, OR = 2.04, 95% CI 1.05-3.96). The frequency of the *15 haplotype in DILI patients was significantly higher than that in non-DILI patients (p = 0.03). In the subgroup analysis, the *15 haplotype was associated with susceptibility to cholestatic/mixed injury (p = 0.03, OR = 2.31, 95% CI 1.06-5.02). Functional assessment of the OATP1B1 *15 haplotype revealed that the activity of bile acid uptake was markedly reduced compared to the three other haplotypes. In the inhibition study, the inhibition by rifampin in the *15 haplotype was greater compared to that in the other haplotypes. These results suggest that the OATP1B1 *15 haplotype is an important predisposing factor for rifampin-induced liver injury.
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Antibióticos Antituberculosos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Predisposición Genética a la Enfermedad , Haplotipos , Transportadores de Anión Orgánico/genética , Rifampin/efectos adversos , Adulto , Estudios de Casos y Controles , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Femenino , Expresión Génica , Frecuencia de los Genes , Genotipo , Células HEK293 , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado , Masculino , Persona de Mediana Edad , Transportadores de Anión Orgánico/metabolismo , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
The present study aimed to investigate the effects of humanin (HN) on primary cortical neuronal apoptosis induced by Abeta31-35, and explore the potential mechanisms. Cultured cortical neurons were pretreated with different concentrations of HN (5, 10, 20 micromol/L) for different time period (0, 8 and 16 h) respectively, and then exposed to Abeta31-35 (25 micromol/L) for additional 24 h and the neuronal apoptosis was examined by morphological analysis, flow cytometric assays and TUNEL staining. Caspase activities were measured using a spectrophotometer. Bax expression was measured by Western blot. The results were as follows. (1) Pretreatment with HN (20 micromol/L) for 16 h significantly prevented Abeta31-35-induced apoptosis in cortical neurons; (2) HN significantly decreased Abeta31-35-induced elevation of caspase-3 and -9 activities; (3) HN suppressed Abeta31-35-induced translocation of Bax from the cytosol to mitochondria, but had no effect on overall Bax expression. In conclusions, HN attenuated Abeta31-35-induced cortical neuronal apoptosis by blocking intrinsic caspase-dependent apoptotic pathways.
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Péptidos beta-Amiloides/toxicidad , Apoptosis/efectos de los fármacos , Corteza Cerebral/citología , Péptidos y Proteínas de Señalización Intracelular/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Células Cultivadas , Corteza Cerebral/patología , Neuronas/citología , Neuronas/patología , Fragmentos de Péptidos/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Objective: To investigate the relationship of TNF-A-863 and CGRP979 gene polymorphisms with the susceptibility to severe chronic periodontitis in Chinese. Methods: Buccal swabs were collected from 100 adult patients with severe chronic periodontitis and 118 healthy adult controls. DNA was extracted from each subjects of the two groups. PCR-LDR technique was used to identify the genotypes of TNF-A-863 and CGRP979. The difference in the genotypes between the two groups was analyzed by statistics software. Results: The genotype of TNF-A--863 was mainly TNF-A-863 A/C in patients with severe chronic periodontitis and TNF-A-863 C/C in healthy controls. There were significant differences in TNF-A-863 distribution between the two groups( P<0.05). We also found that there were significant differences in genotype distribution of CGRP979 between the two groups (P<0.05) ,with A/C predominating in patients with severe chronic periodontitis. Condusion: TNF-A-863 polymorphism is associated with severe chronic periodontitis; A/C of the CGRP979 loci might be a factor for severe chronic periodontitis.
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OBJECTIVE: To investigate whether JNK-caspase-dependent apoptotic pathway is involved in Abeta(31-35)-induced apoptosis of cultured cortical neurons. METHODS: Cultured cortical neurons were treated with Abeta(31-35) (25 micromol/L) for 4 h, 8 h, 16 h and 24 h, respectively. Caspase activities were measured using a spectrophotometer. Levels of c-Jun phosphorylation (p-c-Jun) and Fas ligand (FasL) expression were assessed by immunocytochemistry method and quantified using Image-pro plus11.0 image processing and analysis software. RESULTS: Treatment with Abeta(31-35) (25 micromol/L) for 24 h induced significant increases in the activities of caspase-3 and caspase-8 in the cortical neurons. Besides, Abeta(31-35) could time-dependently enhance the expression of p-c-Jun protein. Moreover, SP600125 application (100 nmol/L) could completely abolish Abeta(31-35) neurotoxicity. The increase in FasL expression was detected at 8 h, 16 h and 24 h after Abeta(31-35) treatment, and SP600125 (100 nmol/L) significantly inhibited FasL expression. CONCLUSION: JNK-c-Jun-FasL-caspase-dependent extrinsic apoptotic pathway plays a critical role in mediating Abeta(31-35)-induced apoptosis of cultured neurons.
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Péptidos beta-Amiloides/metabolismo , Apoptosis/fisiología , Caspasas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/fisiología , Fragmentos de Péptidos/metabolismo , Animales , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Espectrofotometría , Factores de TiempoRESUMEN
The disruption of the intracellular Ca(2+) homeostasis has been reported to be one of the mechanisms of beta-amyloid (Abeta) neurotoxicity in Alzheimeros disease (AD). Abeta(31-35), a small active fragment of Abeta, is believed to possess the similar biological activities of full-length Abeta molecule. Humanin (HN) is a recently identified peptide that suppresses neuronal death initiated by AD-related insults. The present study was to investigate the effects of HN on Abeta(31-35)-induced elevation of [Ca(2+)](i) in cultured cortical neurons by real-time fluorescence imaging technique using the Ca(2+)-sensitive dye, Fura-2/AM. The elevation of [Ca(2+)](i) was observed in cultured neurons exposed to Abeta(31-35) (25 mumol/L) (F340/F380: 1 042.56+/- 83.54, compared with control group: 804.73+/- 48.230, P<0.05, n=10). Pretreatment of HN (10 mumol/L) for 10 min significantly decreased the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) (F340/F380: 918.788+/- 50.73, compared with Abeta(31-35) group, P<0.05, n=10). When neurons were treated with HN and Abeta(31-35) simultaneously, HN (10 mumol/L) could not change the elevation of [Ca(2+)](i) induced by Abeta(31-35) (F340/F380: 1 036.68+/- 88.96, compared with Abeta(31-35) group, P>0.05, n=10), while HN (20 mumol/L) diminished the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) significantly (F340/F380: 898.56+/- 76.46, compared with Abeta(31-35) group, P<0.05, n=10). The findings imply that: (1) the disruption of the calcium homeostasis induced by Abeta(31-35) is possibly the basis of the neurotoxicity of Abeta(31-35) in cultured cortical neurons; (2) HN suppresses the elevation of [Ca(2+)](i) induced by Abeta(31-35) in a dose- and time-dependent manner.
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Péptidos beta-Amiloides/farmacología , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Muerte Celular , Células Cultivadas , Homeostasis , HumanosRESUMEN
OBJECTIVE: To make a mechanical analysis on three-dimensional finite element models of the mandibular first molar with the maximum distal occlusal (DO) structure defect after the root canal therapy and filling and crown restoration under static and impact loads and to provide a guideline for planning restoration for the clinic. METHODS: The research adopted reverse engineering technology to build the model of three-dimensional finite element. The form of the intercuspal occlusion and cusp to cusp occlusion during the circulation of posterior teeth occlusion movement were simulated. Half-sine pulse/impact was chosen for the impact dynamic. The impact ratio was indicated to the stress change between impact loads and static loads. RESULTS: Under the two kinds of loads, the maximum Mohr stress values of the metal crowns were shown in all models. The restoration effects between the two kinds of models were compared, the maximum Mohr stress value of the crown metal and dentin was not obviously difference. The maximum Mohr stress values of dentin were all obviously smaller than the stretch limit strength of dentin. The impact ratio closed to 1. CONCLUSION: The impact loads accorded with the oral actual situation more than the static loads, but the suitable analysis of the static loads could be accepted. The restoration of metal crown is necessary. The effects between the amalgam filling and full crown restoration and composite resin filling and full crown restoration is not difference obviously.
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Análisis de Elementos Finitos , Diente Molar , Resinas Compuestas , Coronas , Dentina , HumanosRESUMEN
Alzheimer's disease (AD) is the leading cause of dementia for aging people, and far from control due to its obscure mechanism. Humanin, a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal region of AD brain, can specifically attenuate AD-related neurotoxicity. It protects neurons from insults of various AD genes, anti-APP antibodies and Abeta by forming a homodimer outside and interfering directly or indirectly with the activity of Abeta. Humanin seems, however, not to inhibit other toxic insults to neurons, such as Fas or etoposide, an agent against carcinomatous cells in clinical therapy. So Humanin rescues neurons from various AD-related toxicity specifically with efficiency.