RESUMEN
Eucommia ulmoides Oliver, a single extant species of Eucommiaceae, is an endemic dioecious tree in China. The natural resources of E. ulmoides have rapidly declined in recent years because of the over-collection of its cortex. To design a suitable protection strategy, it is necessary to develop a set of molecular markers to investigate genetic diversity and population structure of E. ulmoides. Pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform was used to isolate simple sequence repeats (SSRs) for E. ulmoides. A total of 1568 SSRs that contained enough flanking sequences for primer pair design were identified from 45,236 raw sequence reads. One hundred SSRs were randomly selected to design primer pairs and polymerase chain reaction was performed. Among these 100 tested primer pairs, 16 were polymorphic across 18 individuals from three E. ulmoides populations. The number of alleles ranged from 3 to 8, with an average of 5.1. The expected heterozygosity ranged from 0.110 to 0.830, with an average of 0.648, and the observed heterozygosity ranged from 0.111 to 0.833, with an average of 0.524. The inbreeding coefficient ranged from -0.349 to 0.547. This set of microsatellite markers could be valuable for landscape genetic structure assessment and molecular marker-assisted breeding in E. ulmoides.
Asunto(s)
Eucommiaceae/genética , Repeticiones de Microsatélite , Alelos , Especies en Peligro de Extinción , Heterocigoto , EndogamiaRESUMEN
OBJECTIVE: To investigate the expression of human long non-coding ribonucleic acid (RNA) small nucleolar RNA host gene 1 (SNHG1) in laryngeal carcinoma tissues, and to study the effect of SNHG1 on biological functions of laryngeal carcinoma HEp-2 cells. PATIENTS AND METHODS: The expression levels of SNHG1 in 20 pairs of laryngeal carcinoma tissues and para-carcinoma tissues were detected via Real-time fluorescence quantitative polymerase chain reaction (PCR). Laryngeal carcinoma cells were transfected with small interfering (si)-SNHG1 transiently using the RNA interference technique. The effects of si-SNHG1 on proliferation, apoptosis, invasion, and migration of laryngeal carcinoma HEp-2 cells were detected via cell counting kit-8 (CCK-8), colony formation assay, flow cytometry, and wound healing and Transwell assay, respectively. RESULTS: Results of PCR showed that the expression of SNHG1 in carcinoma tissues was increased compared with that in para-carcinoma tissues. Results of CCK-8 and colony formation assay revealed that SNHG1 knockdown could significantly inhibit the proliferation of laryngeal carcinoma HEp-2 cells. Flow cytometry showed that transfection with si-SNHG1 could promote the apoptosis of HEp-2 cells. Moreover, results of wound healing and Transwell assay showed that SNHG1 knockdown could inhibit invasion and migration of HEp-2 cells through inhibiting the epithelial-mesenchymal transition (EMT) process and expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. CONCLUSIONS: The expression of SNHG1 in laryngeal carcinoma tissues is significantly higher than that in para-carcinoma tissue. Patients with high expression of SNHG1 have a poor prognosis. SNHG1 knockdown in HEp-2 cells can inhibit cell proliferation, invasion, and metastasis, and can promote apoptosis.
Asunto(s)
Carcinoma de Células Escamosas/patología , Proliferación Celular , Neoplasias Laríngeas/patología , ARN Largo no Codificante/metabolismo , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/mortalidad , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Pronóstico , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
Protein-tyrosine phosphatases (PTPases) play an integral role in the regulation of cellular insulin action. LAR, a transmembrane PTPase expressed in insulin-sensitive tissues, acts as a negative regulator of insulin signaling in intact cell models. The physiological role of LAR was studied in mice in which LAR expression was eradicated by insertional mutagenesis. In the fasting state, adult male homozygous LAR (-/-) mice had significantly lower plasma levels of insulin and glucose, as well as a reduced rate of hepatic glucose production compared with wild-type controls, suggesting a heightened level of insulin sensitivity. In euglycemic clamp studies, the LAR (-/-) mice exhibited a significant resistance to insulin-stimulated glucose disposal and suppression of hepatic glucose output. Examination of hepatic insulin action demonstrated that the major alteration involved a 47% reduction in insulin-stimulated phosphatidylinositol 3'-kinase (PI 3-kinase) activity in the knockout mice, indicating a post-receptor signaling defect. Taken together with previous work on the cellular effects of LAR, the present results are consistent with a physiological role for LAR in the negative regulation of insulin action, with secondary abnormalities that contribute to the resistance to insulin-stimulated signaling in the knockout mice. Overall, these data provide further evidence for an important role for LAR in the regulation of insulin action and glucose homeostasis in intact animals.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/genética , Glucosa/metabolismo , Hígado/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Estudios de Cohortes , Técnica de Clampeo de la Glucosa , Homeostasis , Proteínas Sustrato del Receptor de Insulina , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/efectos de los fármacos , Ratas , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismoRESUMEN
Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of reversible tyrosine phosphorylation of cellular proteins that mediate insulin action. In order to explore the potential role of the transmembrane PTPase (LAR) in insulin receptor signal transduction, we overexpressed the full-length LAR protein in McA-RH7777 rat hepatoma cells and found that modest increases in the abundance of LAR protein expression downregulated a number of insulin-stimulated cellular responses closely related to the activation of the receptor kinase. An increase in LAR protein of 2.4-fold over the level in control cells caused a 40% reduction in insulin receptor autophosphorylation in intact cells, without an alteration in insulin receptor mass or a change in the insulin-stimulated receptor kinase activity measured with partially purified receptors in vitro. In addition, insulin-stimulated tyrosine phosphorylation of the endogenous insulin receptor substrates IRS-1 and Shc were decreased to 57% and 73% of control, respectively, and IRS-1 associated phosphatidylinositol 3'-kinase activity was reduced to 47% of control of the cells overexpressing LAR. The present results, taken with our recent data demonstrating that reducing the abundance of LAR by expression of antisense mRNA enhances insulin receptor signal transduction (Kulas D. T., et al. J. Biol. Chem. 270:2435, 1995), supports the hypothesis that LAR acts as a physiological modulator of insulin action in insulin-sensitive hepatoma cells.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superficie Celular , Animales , Carcinoma Hepatocelular , Regulación hacia Abajo , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Conejos , Ratas , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Transducción de Señal , Transfección , Células Tumorales Cultivadas , Dominios Homologos srcRESUMEN
Protein-tyrosine phosphatases (PTPases) regulate insulin signaling by catalyzing the tyrosine dephosphorylation of the insulin receptor and its substrate proteins. Previous studies have implicated a PTPase localized to a cell membrane fraction in the regulation of the insulin receptor in situ. LAR (leukoyte antigen related) is a transmembrane PTPase in insulin-sensitive tissues with in vitro catalytic specificity for the insulin receptor kinase domain. When transfected into Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-hlR), the LAR protein was processed as expected into an 85-kDa subunit containing the transmembrane and cytoplasmic domains. LAR was increased an average of 6-fold in clonal lines of stably transfected cells, and cell fractionation confirmed its localization in the cell membrane. After stimulation with 100 nM insulin, tyrosine phosphorylation of the insulin receptor was decreased by 31% at 1 min (P < 0.01) and by 42% at 10 min (P < 0.01), and that of IRS-1 was decreased by 34% (P < 0.01) at 1 min and by 56% (P < 0.01) at 10 min in the LAR-overexpressing cells compared with empty vector transfectants. LAR overexpression also blocked insulin-stimulated receptor kinase activation as well as thymidine incorporation into DNA. Quantitatively similar results were obtained in populations of CHO-hlR cells transfected transiently by electroporation. In contrast, overexpression of recombinant LAR cytoplasmic domain, detected as a 72-kDa protein in the cell cytosol, did not significantly affect the insulin-stimulated tyrosine phosphorylation of the insulin receptor or IRS-1 (99% and 93% of control at 10 min, respectively). These studies provide the first evidence that increased expression of LAR has negative regulatory effects at a proximal site in the insulin-signaling pathway. Since this effect occurs only when LAR is eutopically expressed at the cell membrane, these data further suggest that LAR requires a transmembrane localization to directly interact with the insulin receptor in situ.
Asunto(s)
Insulina/metabolismo , Proteínas del Tejido Nervioso , Proteínas Tirosina Fosfatasas , Receptores de Superficie Celular/genética , Transducción de Señal/fisiología , Animales , Células CHO , Cricetinae , ADN/biosíntesis , Humanos , Insulina/farmacología , Fosforilación , Fosfotirosina/metabolismo , Ratas , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Recombinantes/metabolismo , TransfecciónAsunto(s)
Pruebas Genéticas , Enfermedades del Recién Nacido , Tamizaje Neonatal , Humanos , Recién NacidoRESUMEN
The highly polysialylated form of neural cell adhesion molecule (PSA-NCAM) is important for neurite outgrowth. With this molecule as a marker of plastic change in neurons, we investigated its temporal expression in rat brain after transient middle cerebral artery (MCA) occlusion. In sham-control brain, only subependymal neurons showed a positive immunoreactivity for PSA-NCAM. After 90 min of transient MCA occlusion, neurons in the piriform cortex began to be positively stained at 1 h, while neurons in the cortex and caudate of the MCA territory became positive after 8 h. The stainings persisted for 1 and 3 days after reperfusion. The present results indicate that neurons in the cerebral cortex and caudate have the capability of plastic change in the adult brain, and that those in the piriform cortex rapidly undergo plastic change probably in response to transneuronal injury.
Asunto(s)
Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Ataque Isquémico Transitorio/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Plasticidad Neuronal , Ácidos Siálicos/biosíntesis , Animales , División Celular , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Regulación de la Expresión Génica , Técnicas para Inmunoenzimas , Infarto de la Arteria Cerebral Media/genética , Ataque Isquémico Transitorio/genética , Masculino , Moléculas de Adhesión de Célula Nerviosa/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Ácidos Siálicos/genéticaRESUMEN
The time dependent influence of adenovirus-mediated glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with Ad-GDNF significantly reduced the infarct volume when immediately administered after the reperfusion, but became insignificant when administered at 1 h after the reperfusion as were the cases treated with vehicle- and adenoviral vector containing the E. coli lacZ gene (Ad-LacZ)-treated groups. The protective effect of GDNF was related to the significant reduction of the number of TUNEL positive cells as well as immunohistochemical positive cells for active caspase-3 but not -9. These results showed that exogenous GDNF gene transfer successfully reduced the infarct size in a time-dependant manner by suppressing active caspase-3 but not active caspase-9. However, the therapeutic time window was shorter than the effect of GDNF protein itself previously reported.
Asunto(s)
Adenoviridae/genética , Isquemia Encefálica/terapia , Terapia Genética/métodos , Arteria Cerebral Media/fisiología , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Animales , Western Blotting , Isquemia Encefálica/patología , Caspasas/metabolismo , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/patología , Masculino , Ratas , Ratas Wistar , Factores de Tiempo , TransfecciónRESUMEN
Time dependent influence of glial cell line-derived neurotrophic factor (GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with GDNF significantly reduced the infarct volume stained with 2,3,5-triphenyltetrazolium chloride (TTC) when GDNF was topically applied at 0 and 1 h of reperfusion, but became insignificant at 3 h as compared to vehicle group. The protective effect of GDNF was closely related to the significant reduction of the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) positive cells as well as immunofluorescently positive cells for active forms of caspases, especially active caspase-3 but not -9. Thus, the present study showed that topical application of GDNF significantly reduced infarct size in a time-dependent manner, while the therapeutic time window was shorter than other chemical compounds such as an NMDA receptor antagonist (MK-801) and a free radical scavenger (alpha-phenyl-tert-butyl-nitrone, PBN). The effect of GDNF was stronger in suppressing active caspase-3 than active caspase-9.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Masculino , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas WistarRESUMEN
We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia.
Asunto(s)
División Celular/fisiología , Giro Dentado/metabolismo , Ataque Isquémico Transitorio/metabolismo , Degeneración Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Ácidos Siálicos/metabolismo , Células Madre/metabolismo , Animales , Bromodesoxiuridina/farmacocinética , Giro Dentado/patología , Giro Dentado/fisiopatología , Gerbillinae , Inmunohistoquímica , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Masculino , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Neuronas/patología , Células Madre/citologíaRESUMEN
In order to examine the effect of insulin-like growth factor-1 (IGF-1) on ischemic brain injury, IGF-1 was applied topically on the surface of reperfused rat brain after 60 min of transient middle cerebral artery occlusion (MCAO). In contrast to the cases treated with vehicle, the infarct area was greatly reduced at 24 h of reperfusion by treatment with IGF-1. Terminal deoxynucleotidyl transferase mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunoreactivity for glycogen synthase kinase 3beta (GSK3beta) were also markedly reduced in the brains with IGF-1 treatment. The present results suggest that the treatment with IGF-1 significantly ameliorates brain injury after transient focal brain ischemia associated with the reduction of TUNEL and GSK3beta stainings.
Asunto(s)
Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/fisiopatología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Arteria Cerebral Media/patología , Arteria Cerebral Media/fisiopatología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/fisiopatología , Administración Tópica , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Encéfalo/irrigación sanguínea , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Etiquetado Corte-Fin in Situ , Masculino , Arteria Cerebral Media/cirugía , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas WistarRESUMEN
A phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene that suppresses cell growth, inhibits cell migration, and induces apoptosis. Phosphorylated form of PTEN (p-PTEN) is a key survival factor relating PI3K-Akt pathway and their downstream effectors. A spatiotemporal profiles of PTEN and p-PTEN expression were immunohistochemically examined after 90 min of transient middle cerebral artery occlusion in rats. In the ischemic core, PTEN progressively decreased by 3 days, whereas a rapid but transient increase of p-PTEN was found with a peak at 1 h after the reperfusion. In contrast, in the ischemic penumbra, PTEN showed a minor change and a gradual but sustained p-PTEN expression was observed in the ischemic penumbra with a peak at 12 h. In addition, the balance of population among strongly, moderately, and weakly stained cells was different between the ischemic core and penumbra at their peak time points. These results suggest an important role of p-PTEN for cell survival after ischemia as an upstream regulator for PI3K-Akt.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteínas Supresoras de Tumor/metabolismo , Animales , Supervivencia Celular , Inmunohistoquímica , Infarto de la Arteria Cerebral Media , Masculino , Fosfohidrolasa PTEN , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
In an attempt to elucidate whether glial cell line-derived neurotrophic factor (GDNF) receptors are induced after ischemic brain injury, possible expression of immunoreactive GDNF receptor-alpha1 (GFRalpha-1) and c-ret (RET) was examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Immunohistochemical study showed that both GFRalpha-1 and RET staining cells which were not detected in sham control brain, were present in the ipsilateral cortex and caudate at 3 to 8 h after permanent MCAO, and then decreased but remained to some extent at 24 h. Positive cells for both GDNF receptors were predominantly in cortical neurons of ischemic penumbral area. Western blot analysis confirmed the induction of those receptors after permanent MCAO. This rapid induction of GFRalpha-1 and RET, which correlates with the similar induction of GDNF under these conditions, may play a role in the early response to ischemic brain injury.
Asunto(s)
Arteriopatías Oclusivas/metabolismo , Arterias Cerebrales , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Proteínas de Drosophila , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Western Blotting , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica/métodos , Masculino , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Wistar , Coloración y Etiquetado , Factores de TiempoRESUMEN
In order to examine the effect of neurotrophin-3 (NT-3) on ischemic brain injury, NT-3 was topically applied to brain surface just after 90 min of middle cerebral artery occlusion (MCAO) in rats. NT-3 significantly reduced the infarct size at 24 h of reperfusion. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunohistochemical study for caspase-3 and heat shock protein 72 (HSP72) showed that NT-3 treatment decreased the number of cells with DNA fragmentation and caspase-3 and HSP72 expressions. These data suggest that NT-3 protects neuronal cells from ischemic injury, and it is possibly associated with inhibition of DNA fragmentation.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Arteria Cerebral Media/fisiología , Neurotrofina 3/uso terapéutico , Administración Tópica , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/biosíntesis , Circulación Cerebrovascular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/biosíntesis , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Neurotrofina 3/administración & dosificación , RatasRESUMEN
c-Jun response is involved in the development of ischemic brain injury, which is activated by c-Jun N-terminal kinase-1 (JNK-1). The activity of JNK-1 is strictly regulated, and only the phosphorylated form of JNK (phospho-JNK) which is translocated to the nucleus has an ability to activate c-Jun response. There is a protein which inhibits JNK-1 activation, and known as JNK interacting protein-1 (JIP-1). In this study, we investigated change in JNK-1, phospho-JNK, and JIP-1 immunoreactivity in rat brain after transient middle cerebral artery (MCA) occlusion. Immunoreactive JNK-1 was scant in the sham-control brain, but it was induced at 1 h after reperfusion, which was slightly increased at 3 h of reperfusion. By contrast, phospho-JNK remained negative till 3 h. At 8 h, JNK-1 and phospho-JNK became distinctly positive, and nuclei as well as cytoplasm were stained. Thereafter, immunoreactivity for JNK-1 and phospho-JNK became furthermore dense, and most neurons revealed positively stained nuclei. Immunoreactivity for JIP-1 remained negative till 8 h of reperfusion, but at 24 and 72 h, cytoplasm of cortical neurons at the MCA boundary area was positively stained. This JIP-1 induction got behind the JNK-1 activation, and therefore, may be a vain effort for neurons to survive. Inhibition of JNK-1 activation might become an innovative means of therapy for stroke treatment in the future.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Arterias Cerebrales/fisiopatología , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/metabolismo , Ataque Isquémico Transitorio/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Apoptosis , Proteínas Portadoras/inmunología , Núcleo Celular/metabolismo , Corteza Cerebral/patología , Citoplasma/metabolismo , Inducción Enzimática , Inmunohistoquímica , Ataque Isquémico Transitorio/fisiopatología , Masculino , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/inmunología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Ratas , Ratas Wistar , Reperfusión , Factores de TiempoRESUMEN
Insulin-like growth factor-1 (IGF-1) was applied topically on the brain surface of reperfused rat brain after 60 min of transient middle cerebral artery (MCA) occlusion. In contrast to the cases treated with vehicle, the infarct volume was greatly reduced at 24 h of reperfusion by the treatment with IGF-1. Immunohistochemical analysis in the MCA territory showed that the increase of cyclin-dependent kinase 5 (cdk5) was greatly reduced, and that the decrease of the critical regulatory subunit of cdk5, p35, was preserved with treatment of IGF-1. The present results suggest that IGF-1 has a significant effect on ameliorating brain injury after transient focal brain ischemia with affecting the expressions of cdk5 and its activator p35.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Encéfalo/efectos de los fármacos , Quinasas Ciclina-Dependientes/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Lipoproteínas/efectos de los fármacos , Fosfotransferasas , Daño por Reperfusión/tratamiento farmacológico , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Encéfalo/metabolismo , Química Encefálica/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Lipoproteínas/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismoRESUMEN
In order to clarify the role of protein kinases in ischemic brain injury, the spatiotemporal expression of immunoreactive serine-threonine kinase Akt, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) were examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Weak staining for these protein kinases was found in both cortical and caudate neurons in sham controls. The staining for Akt-1 and PI3-K was increased at 3-8 h in the ischemic penumbral region and declined at 24 h. A slight induction of these kinases was observed in the ischemic core region. Robust expression of ERK was noted at 3-8 h in most neurons in the area of ischemia. At 24 h, ERK continued to be expressed in the ischemic penumbra, but decreased in the ischemic core. These findings suggest that the signaling for Akt and PI3-K are different from the ERK dependent signal transduction during ischemic brain injury.
Asunto(s)
Isquemia Encefálica/enzimología , Núcleo Caudado/enzimología , Corteza Cerebral/enzimología , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Fosfatidilinositol 3-Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Animales , Técnica del Anticuerpo Fluorescente Indirecta , Infarto de la Arteria Cerebral Media/enzimología , Masculino , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas WistarRESUMEN
Infarct volume and immunoreactivities for trkB and trkC in rat brain were compared at 24 h after 90 min of transient middle cerebral artery occlusion (MCAO) between animal groups with or without neurotrophin-3 (NT-3, 10 microg/250 g animal). Treatment of rat brain with topical application of NT-3 significantly reduced infarct volume (P = 0.02) and trkB and trkC inductions. These data suggest that NT-3 reduced the ischemic injury along with the reduction of trkB and trkC inductions.
Asunto(s)
Arteriopatías Oclusivas/enzimología , Arterias Cerebrales , Isoenzimas/metabolismo , Neurotrofina 3/farmacología , Proteínas Tirosina Quinasas/metabolismo , Administración Tópica , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Infarto Cerebral/patología , Inducción Enzimática/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Receptor trkB/antagonistas & inhibidores , Receptor trkC/antagonistas & inhibidoresRESUMEN
Human recombinant interleukin 1 beta (IL-1) administered intraperitoneally to rats produced the following gastric effects: 1. It was cytoprotective, preventing gastric mucosal necrosis produced by oral administration of one ml of absolute ethanol to fasted animals. The ED50 was 1200 units/kg (110 ng per animal). IL-1 was 125 times more potent than prostaglandin E2 (on a weight basis), and 6,000 times more potent (on a molar basis). 2. The cytoprotective effect of IL-1 was blocked by indomethacin (inhibitor of prostaglandin synthesis) and by IRAP (a specific interleukin-1 receptor antagonist protein). IRAP did not inhibit cytoprotection induced by PGE2. 3. IL-1 prevented the formation of gastric erosions induced by aspirin. 4. IL-1 inhibited gastric secretion (volume, acid concentration and output), in the pylorus-ligated rat, with an ED50 of 300 units/kg (3.2 ng per animal). 5. Indomethacin and IRAP blocked the antisecretory effect of IL-1. 6. IL-1 retarded gastric emptying, an effect blocked by IRAP, but not by indomethacin. 7. IL-1 increased synthesis of prostaglandin E2 by the gastric mucosa by 111%. IL-1 is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory, and delay gastric emptying. It appears to act mostly by stimulating the synthesis of prostaglandins by the stomach. These studies suggest that the stomach possesses IL-1 receptors. These are probably located on parietal cells (that produce acid), on prostaglandin-producing cells, on smooth muscle cells (responsible for gastric emptying), and on as yet unidentified cells involved in gastric cytoprotection. Both IL-1 and IRAP, being natural substances, may play a physiological role in the maintenance of gastric mucosal integrity, and in the regulation of acid secretion and gastric motility.