RESUMO
We investigated the cytoprotective effects of lithium, the mood-stabilizer, on thapsigargin-induced stress on the endoplasmic reticulum (ER) in rat PC12 cells. Protracted lithium pretreatment of PC12 cells elicited cytoprotection against thapsigargin-induced cytotoxicity. Lithium protection was concurrent with inhibition of thapsigargin-induced intracellular calcium increase and with elevated expression of the molecular chaperone GRP78. Moreover, lithium pretreatment upregulated the antiapoptotic protein Bcl-2, and blocked Bcl-2 downregulation elicited by thapsigargin. Prior to the induction of GRP78, lithium treatment alone increased the expression of c-Fos whose induction by ER stress is necessary for GRP78 induction. Curcumin, an inhibitor of transcription factor AP-1, blocked lithium cytoprotection against thapsigargin cytotoxicity. Thus, the induction of GRP78 and Bcl-2, and activation of AP-1 likely contribute to lithium-induced protection against cytotoxicity resulting from ER stress. Additionally, thapsigargin-induced cytotoxicity was suppressed by pretreatment with another mood-stabilizer, valproate, indicating that cytoprotection against ER stress is a common action of mood-stabilizing drugs.
Assuntos
Cálcio/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Proteínas de Choque Térmico/fisiologia , Lítio/farmacologia , Chaperonas Moleculares/fisiologia , Fármacos Neuroprotetores , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Tapsigargina/antagonistas & inibidores , Animais , Western Blotting , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Curcumina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tapsigargina/toxicidade , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Ácido Valproico/farmacologiaRESUMO
Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.
Assuntos
Fator VIII/administração & dosagem , Fator VIII/imunologia , Técnicas de Transferência de Genes/normas , Hemofilia A/tratamento farmacológico , Adenoviridae/genética , Animais , Coagulação Sanguínea/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Fator VIII/genética , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/normas , Vetores Genéticos/toxicidade , Hemofilia A/complicações , Hemofilia A/imunologia , Isoanticorpos/sangue , Hepatopatias/enzimologia , Hepatopatias/etiologia , Masculino , Modelos Animais , Fenótipo , Contagem de Plaquetas , Fatores de TempoRESUMO
The coding activity of bovine hypothalamic poly A+ mRNA for neurophysin I and II immunoreactive proteins was characterized with respect to size and 5' cap. The mRNA was fractionated by methylmercuric hydroxide agarose gel electrophoresis and subsequently translated in vitro in rabbit reticulocyte lysates. Alternatively, mRNA was fractionated by gel exclusion HPLC and translated in wheat germ extracts. Immunoprecipitated translation products were analyzed by gel exclusion HPLC. Neurophysin-immunospecific protein of approximately 17,000 daltons, the size expected for the neuropeptide hormone-neurophysin precursors, was encoded by mRNA species of two size classes. The smaller class of mRNA's was of the size expected from the size of the precursor proteins. The larger class was 5-10 times larger. The low K+ concentration optimum for translation of unfractionated mRNA encoding neurophysin I immunoreactive proteins and the inability of a cap analogue to inhibit this translation suggest that mRNA species encoding neurophysin I-immunoreactive translation products are incompletely capped. By contrast, the mRNA encoding neurophysin II-immunoreactive products appear to contain a normal cap structure.
Assuntos
Arginina Vasopressina/genética , Neurofisinas/genética , Ocitocina , Poli A/genética , Precursores de Proteínas/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , Animais , Complexo Antígeno-Anticorpo , Sequência de Bases , Bovinos , Hipotálamo/metabolismo , Soros Imunes , Peso Molecular , Poli A/isolamento & purificação , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , Coelhos , Reticulócitos/metabolismoRESUMO
Adult genetically obese (ob/ob) mice which are characterized by adrenal hypertrophy and increased secretion of corticosteroids have considerably increased levels of ACTH in the pituitary gland. At 5 weeks of age there is no difference in the pituitary ACTH content of lean and obese animals and dietary restriction, sufficient to maintain body weight at normal values, reduces the pituitary ACTH content of adult obese mice from 14 times the level found in lean litter-mate controls to almost normal values. Using an in-vitro perifusion system, the release of ACTH from isolated pituitary glands was studied. Pituitaries from lean and obese mice responded similarly to stimulation with a crude extract of hypothalamic tissue containing coticotrophin releasing factor (CRF). The CRF content of the hypothalamus in both groups appears to be similar. In contrast with the high pituitary content, plasma values for ACTH in unstressed obese mice are not increased. The results are discussed in relation to other evidence for a hypothalamic disorder in ob/ob mice.