RESUMEN
Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.
Asunto(s)
Inflamación/patología , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ceruletida/toxicidad , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Pancreatitis/patología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Daño por Reperfusión/metabolismo , Daño por Reperfusión/mortalidad , Daño por Reperfusión/patología , Sepsis/etiología , Sepsis/metabolismo , Sepsis/patología , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficiencia , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/fisiología , Tretinoina/farmacología , Proteínas de Pez Cebra , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Cromosomas Humanos Par 15 , Neoplasias del Colon/metabolismo , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteínas Wnt , Proteína Wnt1RESUMEN
Erythropoietin (Epo)-responsive anemia is a debilitating complication of chronic renal failure and human immunodeficiency virus (HIV) infection that effects more than 150,000 Americans. Patients with Epo-responsive anemias are currently treated with repeated injections of recombinant human Epo. In the studies described in this report, we have examined the safety and efficacy of using a single intramuscular (i.m.) injection of replication-defective adenoviral vectors (RDAd) encoding Epo for the treatment of Epo-responsive anemias in both mice and non-human primates. Our results demonstrate that there is a threshold dose of virus (2.5-8 x 10(7) pfu/gram of body weight) which is required to obtain long-term Epo expression and polycythemia in both species. A single i.m. injection of mice with 10(9) pfu of an RDAd encoding murine Epo (AdmEpo) resulted in elevations in hematocrits from control values of 49 +/- 0.9% to treated values of 81 +/- 3%, which were stable for more than 1 year. Similarly, a single i.m. injection of a monkey with 4 x 10(11) pfu of an RDAd-encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of > or =70%, which were stable for 84 days. Intramuscular injection of monkeys with AdsEpo appeared to be safe in that we did not detect abnormalities in chest X-rays, serum chemistries, hematologic, or clotting profiles (apart from elevated hematocrits) or organ histologies during the 84-day time course of the experiment. Taken together, these results suggest the feasibility of using i.m. injection of RDAd for the treatment of Epo-responsive anemias in humans.
Asunto(s)
Adenoviridae/genética , Anemia/terapia , Virus Defectuosos , Eritropoyetina/genética , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Animales , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta a Droga , Eritropoyetina/biosíntesis , Eritropoyetina/uso terapéutico , Estudios de Factibilidad , Expresión Génica , Hematócrito , Inyecciones Intramusculares , Macaca fascicularis , Ratones , Roedores , Factores de Tiempo , Replicación ViralRESUMEN
The concentrations of 14 elements were determined in scalp hair samples from control, autistic and autistic-like children. Significant differences were noted between normal males and females for calcium, magnesium and mercury. The autistic population had significantly lower levels of calcium, magnesium, copper, manganese and chromium and higher levels of lithium as compared to sex- and age-matched controls. Children with autistic features (autistic-like), classified as having childhood-onset pervasive disorder, had lower levels of magnesium, cadmium, cobalt and manganese as compared to controls. Discriminant function analysis using the 14 trace elements correctly classified 90.5% of the normal and 100% of the autistic population. Using a stepwise procedure, the five elements with the greatest discriminatory power were calcium, copper, zinc, chromium and lithium. Analysis based on these five trace elements led to the correct classification of 85.7% of the normal and 91.7% of the autistic group. Results indicate that the concentrations of trace elements in hair from normal children differ from patterns observed in both autistic and autistic-like children. Furthermore, evidence suggests that hair analysis may have potential use as a diagnostic tool for autism.
Asunto(s)
Trastorno Autístico/metabolismo , Cabello/metabolismo , Oligoelementos/metabolismo , Niño , Trastornos Generalizados del Desarrollo Infantil/metabolismo , Preescolar , Femenino , Humanos , MasculinoRESUMEN
A high pumping-speed vacuum system, incorporating a mechanical cryo-pump, was designed and built to attach to the source of a spark-source mass-spectrometer to reduce the partial pressure of carbon- and oxygen-containing gases. Pressures in the 10(-10) mmHg range were obtained, which provide a suitable environment for the determination of carbon and oxygen in tungsten. The effect of the partial pressure of CO on carbon and oxygen determination has been studied and characterized. A determination limit of 1 ppm has been achieved for these elements in tungsten.