RESUMEN
B cells orchestrate pro-survival and pro-apoptotic inputs during unfolded protein response (UPR) to translate, fold, sort, secrete and recycle immunoglobulins. In common variable immunodeficiency (CVID) patients, activated B cells are predisposed to an overload of abnormally processed, misfolded immunoglobulins. Using highly accurate transcript measurements, we show that expression of UPR genes and immunoglobulin chains differs qualitatively and quantitatively during the first 4 h of chemically induced UPR in B cells from CVID patients and a healthy subject. We tested thapsigargin or tunicamycin as stressors and 4-phenylbutyrate, dimethyl sulfoxide and tauroursodeoxycholic acid as chemical chaperones. We found an early and robust decrease of the UPR upon endoplasmic reticulum (ER) stress in CVID patient cells compared to the healthy control consistent with the disease phenotype. The chemical chaperones increased the UPR in the CVID patient cells in response to the stressors, suggesting that misfolded immunoglobulins were stabilized. We suggest that the AMP-dependent transcription factor alpha branch of the UPR is disturbed in CVID patients, underlying the observed expression behavior.
Asunto(s)
Linfocitos B/efectos de los fármacos , Inmunodeficiencia Variable Común/genética , Dimetilsulfóxido/farmacología , Fenilbutiratos/farmacología , Ácido Tauroquenodesoxicólico/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Cultivadas , Inmunodeficiencia Variable Común/metabolismo , Inmunodeficiencia Variable Común/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/inmunología , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Tapsigargina/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/genéticaRESUMEN
Regulated transcriptional readthrough during stress maintains genome structure and ensures access to genes that are necessary for cellular recovery. A broad number of genes, including of the bacterial sensor Toll-like receptor 4 (TLR-4), are markedly transcribed on initiating the systemic inflammatory response. Here we study the transcriptional patterns of tlr4 and of its modulator grp78 during human sepsis, and establish their correlations with the outcome of patients. We measured the daily tlr4 and grp78 RNA expression levels in peripheral blood of septic patients, immediately after admission to intensive care, and modeled these RNA values with a sine damping function. We obtained negative correlations between the transcription of tlr4 and grp78 RNA in the survivor group. In contrast, such relation is lost in the deceased patients. Loss of transcriptional homeostasis predicted by our model within the initial 4 days of hospitalization was confirmed by death of those patients up to 28 days later.
Asunto(s)
Proteínas de Choque Térmico/inmunología , Modelos Biológicos , Sepsis/inmunología , Receptor Toll-Like 4/inmunología , Transcripción Genética/inmunología , Adulto , Anciano , Supervivencia sin Enfermedad , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/sangre , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/sangre , ARN Mensajero/inmunología , Sepsis/sangre , Sepsis/mortalidad , Tasa de Supervivencia , Receptor Toll-Like 4/sangreRESUMEN
The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hand remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Receptor Toll-Like 4/metabolismo , Animales , Complejo II de Transporte de Electrones , Endotoxemia/inducido químicamente , Endotoxemia/veterinaria , Endotoxinas/toxicidad , Regulación de la Expresión Génica/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Enfermedades de los Caballos/inducido químicamente , Enfermedades de los Caballos/metabolismo , Caballos , Hipoxantina Fosforribosiltransferasa , Mucosa Intestinal/metabolismo , Receptores de Lipopolisacáridos/genética , Antígeno 96 de los Linfocitos/genética , Subunidades de Proteína , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptor Toll-Like 4/genéticaRESUMEN
Chaperone production is an essential step for proper folding of certain proteins. Accumulation of misfolded/unfolded proteins within the endoplasmic reticulum (ER) lumen triggers a signalling pathway named unfolded protein response (UPR). Upon activation, the UPR pathway augments transcription of ER chaperones increasing protein folding, decreases protein translation to ameliorate the ER overload, increases protein degradation, and activates the apoptotic programme if all previous measures fail. In this review, we will cover the chaperones involved in folding of proteins related to the immune response, followed by an overview of the UPR pathway. Lastly, we will discuss data from this last decade that demonstrate how the improper activation of the UPR pathway has been uncovered as a mechanism responsible for failure to mount a proper immune response, both innate and adaptive.
Asunto(s)
Linfocitos B/inmunología , Retículo Endoplásmico/inmunología , Inmunidad Innata/inmunología , Chaperonas Moleculares/inmunología , Respuesta de Proteína Desplegada/inmunología , Animales , Humanos , Modelos Inmunológicos , Pliegue de ProteínaRESUMEN
Quantity and variety of environmental antigens, age, diet, vaccine protocols, exercising practice and mucosal cytokine microenvironment are factors that influence serum immunoglobulin (Ig) levels. IgA, IgG, IgG(T) and IgM were quantified in 60 horses, which were classified into two groups, 'intensive' or 'relaxed', according to sanitary standards of the facilities and physical exercise to which animals were subjected to. The 'intensive' group presented lower means for all isotypes, but only IgA presented a significant (P < 0.0064) difference when compared to the 'relaxed' group. This suggests that mucosal immunity found in the 'intensive' group is lower when compared to the 'relaxed' group. Our data suggest that athlete horses may be less poised to mount an effective mucosal immunity response to environmental challenges and should not be considered by the same perspectives as a free-ranging horse.
RESUMEN
To evaluate the role of IFN-gamma (interferon gamma) in IL-12- (interleukin-12)-induced inhibition of the inflammatory response in the eye during endotoxin-induced uveitis (EIU). C57BL/6 wild type mice and IFN-gamma-deficient (GKO) mice were injected with 250 microg of Salmonella typhymurium endotoxin as a model for EIU. Animals were then injected intraocularly with 100 ng of rIL-12 or the equivalent volume of Phosphate-buffer saline (PBS). Histopathologic grading of disease was performed 12, 36 and 72 h after endotoxin injection. Chemokine mRNA expression in the eye was evaluated by reverse transcriptase-polymerase chain reaction. Depletion of NK1.1+ cells in vivo was performed using a PK136 antibody. Depletion of IFN-gamma was performed using the R4-6A2 antibody. C57BL/6 mice treated with rIL-12 intraocularly were protected from the development of EIU. Neutralization of IFN-gamma with a monoclonal antibody abrogated such protection. The IL-12 protective effects were lost in NK1.1-depleted mice. Intraocular IL-12 decreased the expression of keratinocyte-derived chemokines (KC) gene but had no effect on macrophage inflammatory protein (MIP-2) gene. The protective effect of IL-12 during EIU occurs through production of IFN-gamma by NK1.1+ cells. IL-12-induced higher levels of IFN-gamma are also correlated with lower expression of the chemokine KC, resulting in diminished attraction of neutrophils to the inflammatory site.
Asunto(s)
Antígenos de Superficie/biosíntesis , Regulación hacia Abajo/inmunología , Interleucina-12/administración & dosificación , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C/biosíntesis , Lipopolisacáridos/administración & dosificación , Uveítis/inmunología , Uveítis/terapia , Animales , Cámara Anterior/inmunología , Antígenos Ly , Antígenos de Superficie/fisiología , Inmunofenotipificación , Interleucina-12/uso terapéutico , Lectinas Tipo C/deficiencia , Lectinas Tipo C/fisiología , Lipopolisacáridos/antagonistas & inhibidores , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microinyecciones , Subfamilia B de Receptores Similares a Lectina de Células NK , Uveítis/metabolismoRESUMEN
Objetivo: Avaliação da morte celular por ativação em linfócitos T de pacientes com imunodeficiência comum variável (CVID) e de outros parâmetros da resposta imune. Métodos: Células mononucleares obtidas a partir de sangue periférico (PBMC) de 32 pacientes com CVID e 32 indivíduos normais foram utilizadas para o estudo da expressão de CD40L, linfoproliferação e apoptose, Para a análise de marcadores de ativação (CD25 e CD69) e de interação entre células T e B (CD70) PBMC foram estimuladas por diferentes tempos (24, 48, 72 e 96 horas), Os sobrenadantes de cultura foram utilizados para quantificação de citocinas (IL-2, IL-4, IL-5 e IFN-y) por ELISA. Todos os testes laboratoriais foram aplicados no grupo controle de voluntários sadios. Os resultados foram analisados utilizando testes de diferença de proporções, ANOV A, Kruskal-Wallis e a prova de Mann-Whitney. Resultados: O grupo de pacientes com CVID demonstrou aumento percentual de linfócitos CD3/CD4 que sofreram apoptose em relação ao grupo controle (p< (Au) pacientes. destes anticorpos por mediada e celular resposta da prejuízo conseqüente com Th2, citocinas de diminuída síntese além CD70, CD69 CD25, CD40L, expressão circulantes, B T células nas decréscimo pelo responsável seja fenômeno este que sugere ativadas morte aumento O Conclusões: normais. indivíduos grupo o comparados quando CVID pacientes.
Asunto(s)
Humanos , Anexinas , Inmunodeficiencia Variable Común , Citocinas , Técnicas In Vitro , Interleucinas , Linfocitos T , Muerte Celular , Inmunidad Celular , MétodosRESUMEN
Intracellular protozoan parasites are potent stimulators of cell-mediated immunity. The induction of macrophage proinflammatory cytokines by Trypanosoma cruzi is considered to be important in controlling the infection and the outcome of Chagas' disease. Here we show that the potent tumour necrosis factor-alpha-, interleukin-12- and nitric oxide-inducing activities of T.cruzi trypomastigote mucins were recovered quantitatively in a highly purified and characterized glycosylphosphatidylinositol (GPI) anchor fraction of this material. The bioactive trypomastigote GPI fraction was compared with a relatively inactive GPI fraction prepared from T. cruzi epimastigote mucins. The trypomastigote GPI structures were found to contain additional galactose residues and unsaturated, instead of saturated, fatty acids in the sn-2 position of the alkylacylglycerolipid component. The latter feature is essential for the extreme potency of the trypomastigote GPI fraction, which is at least as active as bacterial endotoxin and Mycoplasma lipopeptide and, therefore, one of the most potent microbial proinflammatory agents known.
Asunto(s)
Glicosilfosfatidilinositoles/aislamiento & purificación , Mediadores de Inflamación/aislamiento & purificación , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Enfermedad de Chagas/inmunología , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/farmacología , Técnicas In Vitro , Mediadores de Inflamación/química , Mediadores de Inflamación/farmacología , Interleucina-12/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mucinas/química , Mucinas/aislamiento & purificación , Mucinas/farmacología , Óxido Nítrico/biosíntesis , Trypanosoma cruzi/química , Trypanosoma cruzi/patogenicidad , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.
Asunto(s)
AMP Cíclico/fisiología , Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Trypanosoma cruzi/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Glicosilfosfatidilinositoles/farmacología , Interleucina-10/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mucinas/farmacologíaRESUMEN
Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serve as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.
Asunto(s)
Inmunidad Celular/fisiología , Infecciones por Protozoos/inmunología , Animales , Citocinas/fisiología , Leishmania , Toxoplasma , Trypanosoma cruziRESUMEN
Toxoplasma gandii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.
Asunto(s)
Inmunidad Celular/fisiología , Infecciones por Protozoos/inmunología , Infecciones por Protozoos/fisiopatología , Citocinas/fisiología , Leishmania , Toxoplasma , Trypanosoma cruziRESUMEN
Components of Trypanosoma cruzi able to induce the production of IL-12 and other proinflammatory cytokines by macrophages were identified. Murine inflammatory macrophages were cultured with live parasites or with cellular components from different developmental forms of T. cruzi (i.e., trypomastigotes, amastigotes, metacyclic trypomastigotes, and epimastigotes), and the cytokine levels were measured after 24 and 48 h. Our results indicate that live trypomastigotes or live amastigotes (but not live epimastigotes or live metacyclic trypomastigotes) as well as trypomastigote extracts (but not extracts derived from epimastigotes) induce IL-12 and TNF-alpha synthesis by macrophages. Such biological activity is enhanced in membrane preparations from trypomastigotes. Further enrichment of the trypomastigote-derived monokine-inducing factor was obtained by solvent extraction and hydrophobic-interaction chromatography. The resultant purified molecules are a family of closely related glycoconjugates with predominant species at 70 to 80 and 120 to 200 kDa. These molecules are composed of carbohydrate chains O-linked to a polypeptide backbone that is anchored to the trypomastigote membrane via a glycosylphosphatidylinositol structure. The trypomastigote-derived glycoconjugates are active in inducing cytokine synthesis by macrophages at concentrations of 100 ng/ml. These effects are highly potentiated by IFN-gamma. Mapping of the glycoconjugate molecules to characterize the structural requirements for macrophage activation suggested that nonsaturated acyl fatty acid chains and periodate-sensitive units from the glycosylphosphatidylinositol anchor are important elements for the infective trypomastigote form to initiate cytokine synthesis by macrophages.
Asunto(s)
Glicoproteínas/inmunología , Glicosilfosfatidilinositoles/inmunología , Interleucina-12/biosíntesis , Macrófagos/inmunología , Mucinas/inmunología , Trypanosoma cruzi/inmunología , Animales , Línea Celular , Glicoproteínas/química , Glicosilfosfatidilinositoles/aislamiento & purificación , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C3H , Ratones SCID , Mucinas/química , Trypanosoma cruzi/químicaRESUMEN
In the present study, we investigated the role of glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) from Trypanosoma cruzi trypomastigotes in triggering the synthesis of nitric oxide as well as the microbicidal activity in murine macrophages. Our results show that GPI-mucins isolated from trypomastigote membranes are potent inducers of nitric oxide synthesis by IFN-gamma-primed macrophages, even at concentrations as low as 10 ng/ml. Our data also indicate the important role of glycosylphosphatidylinositol anchors from GPI-mucins as the second signal responsible for induction of nitric oxide synthesis by macrophages. To further investigate the role of these parasite molecules in inducing parasiticidal function, we cultured macrophages in the presence or absence of trypomastigote GPI-mucins and/or IFN-gamma and then infected these cells with either Leishmania spp. or T. cruzi. IFN-gamma was sufficient to induce microbial activity in macrophages infected with T. cruzi trypomastigotes. In contrast, killing of different species of Leishmania was further enhanced when macrophages exposed to IFN-gamma were also costimulated with trypomastigote-derived GPI-mucins. Our results also indicate that different glycolipids obtained from Leishmania major or Leishmania donovani (i.e., lipophosphoglycans or glycoinositolphospholipids) were unable to potentiate nitric oxide synthesis and/or microbicidal activity displayed by IFN-gamma-primed macrophages.
Asunto(s)
Antiprotozoarios/aislamiento & purificación , Glicosilfosfatidilinositoles/fisiología , Interferón gamma/farmacología , Leishmania/inmunología , Macrófagos/metabolismo , Mucinas/fisiología , Óxido Nítrico/biosíntesis , Trypanosoma cruzi/inmunología , Adyuvantes Inmunológicos/fisiología , Animales , Antiprotozoarios/farmacología , Membrana Celular/química , Membrana Celular/inmunología , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/aislamiento & purificación , Células L , Leishmania/efectos de los fármacos , Leishmania/crecimiento & desarrollo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos C3H , Mucinas/inmunología , Mucinas/aislamiento & purificación , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrollo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 microM cyclic AMP (cAMP), 1 mM calcium and 1 microM calmodulin (Ca2+/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 microM phorbol 12,13-dibutyrate (Ca2+/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca2+/calmodulin, respectively, but was unaffected in the presence of Ca2+/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 microM okadaic acid and 0.01 microM microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Corteza Cerebral/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoesqueleto/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Corteza Cerebral/ultraestructura , Polietilenglicoles , Ratas , Ratas Wistar , SolubilidadRESUMEN
We studied the effects of L-phenylalanine and alpha-methylphenylalanine on 32P in vitro incorporation into cytoskeletal proteins from cerebral cortex of 17-day-old rats. Slices of cerebral cortex were incubated in the absence or presence of increasing concentrations of L-phenylalanine, alpha-methylphenylalanine or L-phenylalanine plus alpha-methylphenylalanine for 1 h. The cytoskeletal fraction obtained from slices was incubated in the presence of the same drugs and the 32P in vitro incorporation into cytoskeletal proteins was measured. Addition of alpha-methylphenylalanine did not change 32P in vitro incorporation into the cytoskeletal proteins, but phenylalanine decreased the in vitro phosphorylation of beta tubulin. Furthermore, addition of L-phenylalanine plus alpha-methylphenylalanine decreased the in vitro phosphorylation of both 160 kDa neurofilaments and alpha-tubulin.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Animales , Corteza Cerebral/metabolismo , Técnicas In Vitro , Radioisótopos de Fósforo , Ratas , Ratas WistarRESUMEN
In this investigation we studied developmentally regulated endogenous protein kinase activity in cytoskeletal proteins in the cerebral cortex of rats and the effect of early malnutrition imposed on dams on the pattern of 32P incorporation into the cytoskeleton of pups. Our results indicated that in vitro incorporation was maximum in 7-day-old pups for both normal and malnourished groups, decreasing with development, and reaching minimum values in adult animals. However, 32P incorporation into NF-M and tubulin was significantly lower in 7-day-old malnourished pups than in normal pups.
Asunto(s)
Envejecimiento/metabolismo , Corteza Cerebral/metabolismo , Proteínas del Citoesqueleto/metabolismo , Trastornos Nutricionales/metabolismo , Proteínas Quinasas/metabolismo , Animales , Autorradiografía , Peso Corporal , Corteza Cerebral/anatomía & histología , Corteza Cerebral/crecimiento & desarrollo , Proteínas del Citoesqueleto/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Tamaño de los Órganos , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo , Fosforilación , Ratas , Valores de ReferenciaRESUMEN
Wistar rats from the same litter were randomly divided into four groups and received subcutaneously from the 6th to 28th day post partum one of the following drugs: L-proline, methylmalonate, L-phenylalanine plus alpha-methylphenylalanine, or equivalent volumes of 0.9% (w/v) saline (controls). On day 30, the animals were killed, the brain was removed and the cerebral cortex and cerebellum was immediately dissected. Total intermediate filament fraction (IF) was obtained from cerebral cortex and cerebellum by using a high-salt phosphate-buffered solution supplemented by 1% Triton X-100. The pellet contained the bulk of the IF proteins. Following SDS-polyacrylamide gel electrophoresis, these proteins were identified as the 200, 150 and 68 kD subunits of neurofilaments (NF-H, NF-M and NF-L, respectively), the 66 kDa associated protein, the 57 kDa intermediate filament-like protein and the 52 kDa glial fibrillary acidic protein (GFAP). They were further scanned through densitometry from enriched fractions of controls and of animals treated with the various drugs in order to determine the effects of the treatments on their concentration. Our results showed that the concentration of IF protein in cerebellum was not affected by the treatments, whereas chronic administration of all drugs significantly decreased NF-H subunit concentration in rat cerebral cortex.
Asunto(s)
Corteza Cerebral/metabolismo , Ácido Metilmalónico/farmacología , Proteínas de Neurofilamentos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Prolina/farmacología , Animales , Peso Corporal/efectos de los fármacos , Encéfalo/anatomía & histología , Corteza Cerebral/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
The possibility of false negative results from TLC when a diuretic is administered concomitantly with flunixin was studied. Samples were subjected to solvent extraction from acidic aqueous solutions; duplicate samples were also subjected to alkaline hydrolysis at pH 12.5. The internal standard was flufenamic acid. The quantification of flunixin was performed by HPLC and the results confirmed by GC/MS. The data show that furosemide influences the urinary concentration of flunixin.