RESUMEN
Neuroinflammation and increased oxidative stress are believed to contribute to the development of psychiatric diseases. Animal studies have implicated NADPH oxidases (NOX) as relevant sources of reactive oxygen species in the brain. We have analyzed the expression of NOX isoforms in post-mortem brain samples from patients with psychiatric disorders (schizophrenia, bipolar disorder) and non-psychiatric subjects. Two collections from the Stanley Medical Research Institute were studied: the Array Collection (RNA, 35 individuals per group), and a neuropathology consortium collection (paraffin-embedded sections, 15 individuals per group). Quantitative PCR analysis revealed expression of NOX2 and NOX4 in prefrontal cortex. No impact of psychiatric disease on NOX4 levels was detected. Remarkably, the expression of NOX2 was specifically decreased in prefrontal and cingulate cortices of bipolar patients, as compared with controls and schizophrenic patients. NOX2 expression was not statistically associated with demographic parameters and post-mortem interval, but correlated with brain pH. Immunostaining demonstrated that NOX2 was predominantly expressed in microglia, which was corroborated by a decrease in the microglial markers CD68 and CD11b in the cingulate cortex of bipolar disorder patients. The analysis of potentially confounding parameters showed association of valproic acid prescription and heavy substance abuse with lower levels of NOX2. Taken together, we did not observe changes of NOX2 in schizophrenic patients, but a marked decrease of microglial markers and NOX2 in the brain of bipolar patients. This might be an underlying feature of bipolar disorder and/or a consequence of valproic acid treatment and substance abuse.
Asunto(s)
Trastorno Bipolar/metabolismo , Encéfalo/metabolismo , NADPH Oxidasa 2/metabolismo , Esquizofrenia/metabolismo , Trastornos Relacionados con Sustancias/complicaciones , Ácido Valproico/efectos adversos , Adulto , Trastorno Bipolar/complicaciones , Trastorno Bipolar/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Femenino , Giro del Cíngulo/metabolismo , Humanos , Masculino , Microglía/metabolismo , Persona de Mediana Edad , NADPH Oxidasa 4/metabolismo , Corteza Prefrontal/metabolismo , ARN Mensajero/metabolismo , Esquizofrenia/complicaciones , Esquizofrenia/tratamiento farmacológico , Ácido Valproico/uso terapéutico , Adulto JovenRESUMEN
Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G(2)/M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G(2) phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclina B1/metabolismo , Ciclina B/metabolismo , Animales , Proteína Quinasa CDC2 , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , División Celular , Quinasas Ciclina-Dependientes , Proteínas del Ojo/metabolismo , Fase G2 , Células HeLa , Proteínas de Homeodominio/metabolismo , Humanos , Interfase , Mitosis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Primates/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cell cycle analyses of activated T lymphocytes from elderly humans generally show that the proportion of noncycling cells increases with age. T cells that are not definitively blocked in G0 usually strain to traverse the G1 phase and may still be arrested at the G1/S boundary. The molecular mechanisms underlying these cell cycle arrests are unknown. Because G0/G1 and G1/S transitions are regulated in part by cyclin-dependent kinase Cdk6, we investigated the possibility that a loss of activity of this kinase is implicated in the age-related dysfunction of the cell cycle in its initial phases. G0/G1 and G1/S blocks were first confirmed by [(3)H]uridine and [(3)H]thymidine incorporation studies in anti-CD3 activated T lymphocytes derived from elderly donors. In the same cell preparations, in vitro phosphorylation of recombinant truncated Rb protein by immunoprecipitated Cdk6 was significantly decreased. The reduced Cdk6 activity was not attributable to a low level of the protein since a 24-h activation resulted in a comparable expression of the kinase in T cells from young and old individuals. However, at least two other mechanisms might be incriminated in the loss of Cdk6 activity: (1) a poor induction of the associated cyclin D2 upon anti-CD3 stimulation and (2) a delayed downregulation of the Cdk inhibitor p27 following cell activation. The low Cdk6 activity observed in T lymphocytes from the elderly was associated with a defective phosphorylation of the endogenous Rb protein and an increased sequestration of the E(2)F-1 transcription factor, possibly resulting in early cell cycle arrest.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Portadoras , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Proteínas Supresoras de Tumor , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/inmunología , Anticuerpos Monoclonales/metabolismo , Complejo CD3/inmunología , Ciclo Celular/inmunología , Células Cultivadas , Ciclina D2 , Quinasa 6 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Regulación hacia Abajo , Factores de Transcripción E2F , Humanos , Activación de Linfocitos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Proteína 1 de Unión a Retinoblastoma , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Timidina/farmacocinética , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Uridina/farmacocinéticaRESUMEN
Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging.
Asunto(s)
Envejecimiento/inmunología , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/inmunología , Linfocitos T/citología , Linfocitos T/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Proteína Quinasa CDC2/biosíntesis , Ciclina B/biosíntesis , Ciclina B1 , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Fase G2/inmunología , Humanos , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Sustancias Macromoleculares , Mitosis/inmunología , Fosforilación , Linfocitos T/inmunologíaRESUMEN
Although transmembrane signaling defect has been recognized as one of the major functional alterations involved in immune senescence, its biochemical nature as well as its precise molecular localization are still unknown. The available data indicate that an early step in the signaling cascade may be affected during the aging process. Because protein tyrosine kinases (PTK) are ubiquitously implicated in the initiation of physiological signals, they appear as prime candidates for age-related changes. The present investigation examined the effect of age on the activity of PTK associated with CD3, CD4, CD8 or the IL-2 receptor (IL-2R) in human T lymphocytes. By comparison with cells derived from young individuals, anti-CD3-activated T lymphocytes from elderly donors were more susceptible to herbimycin A, a PTK inhibitor known to prevent signal transduction by the T cell antigen receptor. This increased sensitivity of cells from senescent organisms to PTK inhibitors is most likely related to a lesser PTK activity since a significant decrease in the tyrosine phosphorylation of particular endogenous substrates was observed as a consequence of either CD3, CD4, CD8 or IL-2R activation. However, no age-related difference in tyrosine phosphorylation could be demonstrated when T cells were activated by pervanadate, a pharmacological activator of PTK. These results suggest that the intrinsic activity of the enzymes is preserved and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. The defect in PTK activation could be the primary cause for the dysfunction of various components of the signaling cascade observed during the course of aging.
Asunto(s)
Envejecimiento/sangre , Antígenos CD/sangre , Proteínas Tirosina Quinasas/sangre , Receptores de Interleucina-2/sangre , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/inmunología , Benzoquinonas , Donantes de Sangre , Complejo CD3/sangre , Antígenos CD4/sangre , Inhibidores Enzimáticos/farmacología , Humanos , Lactamas Macrocíclicas , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Rifabutina/análogos & derivados , Vanadatos/farmacologíaRESUMEN
The contact of blood with some biomaterials results in complement activation, primarily by the alternative pathway (AP). Insoluble polystyrene derivatives bearing isolated sulphonate groups (PSSO3) deplete complement, whereas identical surfaces substituted with both sulphonate and hydroxymethyl groups (PSCH2OH-SO3) are non-activators. Polystyrene sulphonate derivatives possess high adsorptive properties, particularly for serine proteases of the coagulation cascade. Thus, we studied the interactions between polystyrene derivatives and factor D, an enzyme essential for AP activation. C3 was activated when normal human serum (NHS) was incubated with PSSO3, whereas PSCH2OH-SO3 did not induce any specific C3 activation. Both polymers adsorbed factor D from serum, as shown by the loss of haemolytic factor D from NHS incubated with the polymers and by the specific adsorption of radiolabelled factor D. When bound to the polymers, factor D was not functional. The disappearance of factor D was in contradiction to the observed complement activation induced by PSSO3. When other AP components were studied, it was evident that PSSO3 adsorbed factor H even more rapidly and efficiently than factor D. Thus, the net effect was an immediate deregulation of the AP resulting in C3 activation, followed by inhibition of the AP when factor D was finally depleted. Pre-exposure of PSSO3 to NHS prevented any complement activation because the polymer was saturated with factor H, but still adsorbed factor D. Such properties could be beneficial during haemodialysis with membranes for uremic patients who have increased levels of factor D in their serum.