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Studies using real-world data (RWD) can complement evidence from clinical trials and fill evidence gaps during different stages of a medicine's lifecycle. This review presents the experience resulting from the European Medicines Agency (EMA) pilot to generate RWE to support evaluations by EU regulators and down-stream decision makers from September 2021 to February 2023. A total of 61 research topics were identified for RWE generation during this period, covering a wide range of research questions, primarily generating evidence on medicines safety (22, 36%), followed by questions on the design and feasibility of clinical trials (11, 18%), drug utilization (10, 16%), clinical management (10, 16%), and disease epidemiology. A significant number of questions were related to the pediatric population and/or rare diseases. A total of 27 regulatory-led RWD studies have been conducted. Most studies were descriptive and aimed at estimating incidence and prevalence rates of clinical outcomes including adverse events or to evaluate medicines utilization. The review highlights key learnings to guide further efforts to enable the use and establish the value of real-world evidence (RWE) for regulatory decisions. For instance, there is a need to access additional fit-for-purpose and representative data, and to explore further means to provide timely evidence that meets regulatory timelines. The need for early interactions and close collaboration with study requesters, e.g., from the Agency's scientific Committees, to better understand the research question is equally important. Finally, the review provides our perspective on the way forward to maximize the potential of regulatory-led RWE generation.
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BACKGROUND: Clozapine is the most effective antipsychotic but requires careful titration to therapeutic blood levels. Methods to predict therapeutic doses are based on population data. AIMS: We aimed to construct a model based on genetic variants which accurately predicted plasma levels for clozapine. METHOD: We measured clozapine plasma levels in patients on a stable dose of clozapine who were known to be fully compliant. Measured plasma levels were adjusted for sampling time and dose. Hepatic enzyme variants were analysed and models were constructed to predict the required dose. These predictions were compared with a standard population-based algorithm. RESULTS: We measured plasma clozapine concentrations in 18 adherent patients on stable doses of clozapine and recorded the exact timing of sampling. For the algorithm-predicted dose, the mean difference was -49.9 mg/day ((SD 155.9), r = 0.36) from the actual dose required to give a plasma concentration of 0.35 ng/ml. The gene variant activity score predicted a dose for which the mean difference was -43.5 mg/day ((SD 140.1), r = 0.55). For the gene variant activity score with omeprazole correction predicted dose, the mean difference was -31.0 mg/day ((SD 140.8), r = 0.54), and with the gene variant CYP1A2 inducibility predicted dose the mean difference was 44.9 mg/day ((SD 160.8), r = 0.32). CONCLUSION: Our gene variant activity score with omeprazole correction gave the best estimate of the clozapine dose required to achieve a minimum therapeutic plasma concentration. The use of this model will allow safer titration of clozapine and may reduce the need for plasma-level monitoring during titration.
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Antipsicóticos , Clozapina , Esquizofrenia , Humanos , Clozapina/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Antipsicóticos/uso terapéutico , Omeprazol/uso terapéutico , AlgoritmosRESUMEN
PURPOSE: Caring for a patient with Parkinson's disease (PD) is stressful and has a significant impact on the carer's physical and mental health. This article is aimed at assessing which characteristics are most frequently associated with the stress symptoms of PD carers. METHOD: Data from the Parkinson's UK Members' Survey were drawn from 1881 valid self-completion surveys, filled in by carers of a patient with PD. RESULTS: The carers, 95.6% of whom were a first-degree relative, reported an average of 2.7 ± 3.1 stress-related symptoms. In multiple regression analysis, a greater number of symptoms were associated (p < 0.01) with: the more tasks that the carer carried out (ß = 0.35), an increased number of carer's co-morbidities (0.22) and worsening financial status (0.16). CONCLUSIONS: It is important to consider the health of both the patient with PD as well as that of the family carer to reduce the impact of caring activity on the carer's stress.
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Cuidadores/psicología , Enfermedad de Parkinson/rehabilitación , Estrés Psicológico/epidemiología , Actividades Cotidianas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Empleo , Salud de la Familia , Femenino , Estado de Salud , Encuestas Epidemiológicas , Humanos , Masculino , Persona de Mediana Edad , Esposos/psicología , Reino Unido , Adulto JovenRESUMEN
Parkinson's disease (PD) can be manifested in many different ways. Although motor dysfunction represents the best characterised of the symptoms, the non-motor symptoms (NMS) of the condition can be equally disabling for people. These have been highlighted as being an issue of particular importance by people with PD. A comprehensive postal survey of members of the charity Parkinson's UK took place in 2008. This resulted in returns from 10,101 people with PD. The self-completed Non-Motor Questionnaire (NMSQuest) and quality of life scale (PDQ-8) were contained within the survey. The results showed that the percentage of people with PD experiencing NMS increased with the duration of the disease. However, people who had the younger onset form of the condition reported a greater impact of NMS, particularly in the areas of memory, depression and sleep function. There is an inverse correlation between NMS and (PDQ-8 scale). A significant number of people with PD reported that they experienced problems with olfaction, taste, nocturia and constipation prior to diagnosis and these may help to serve as a future biomarker for the condition. Although our understanding of PD-associated NMS has increased considerably in the recent past, there is still a general lack of awareness of the importance of NMS for people with PD. Further research is required to identify the best treatments that should be employed to address them.
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Estreñimiento/complicaciones , Trastornos de la Memoria/complicaciones , Trastornos Mentales/complicaciones , Enfermedad de Parkinson/complicaciones , Calidad de Vida , Trastornos del Sueño-Vigilia/complicaciones , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estreñimiento/psicología , Estudios Transversales , Femenino , Encuestas Epidemiológicas , Humanos , Masculino , Trastornos de la Memoria/psicología , Trastornos Mentales/psicología , Persona de Mediana Edad , Enfermedad de Parkinson/psicología , Trastornos del Sueño-Vigilia/psicologíaRESUMEN
Previous studies have shown that administration of nicotine modifies the expression and secretion of amyloid precursor protein (APP) in various cell lines. The present study investigated the extent to which chronic subcutaneous nicotine administration influences APP levels and processing in cerebral cortex, striatum and hippocampus of young and old rat brains. The results showed that constant nicotine infusion (0.25 or 4.00mg/kg/day) increased the levels of particulate APP (APPp) but not secreted APP (APPs) in the hippocampus of young rats in vivo. This response to nicotine was not observed in the striatum or cerebral cortex of young rats or in any of the brain regions examined in old animals. Subsequent in vitro analysis demonstrated that nicotine enhanced the release of APPs from hippocampal slice preparations and that this increase was attenuated by mecamylamine, a non-selective nicotinic acetylcholine receptor (nAChR) antagonist. The in vitro effect of nicotine on APPs was age-related, being only detected from hippocampal slices derived from the young but not the older animals. These results suggest that nicotine modulates APP expression and secretion in the hippocampus and that the responses observed to the drug are age-dependent being only detected in younger rats.
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Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Factores de Edad , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Hipocampo/metabolismo , Masculino , Mecamilamina/farmacología , Antagonistas Nicotínicos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Amyloid precursor protein (APP) mis-processing and aberrant tau hyperphosphorylation are causally related to the pathogenesis and neurodegenerative processes that characterize Alzheimer's disease (AD). Abnormal APP metabolism leads to the generation of neurotoxic amyloid beta (Abeta), whereas tau hyperphosphorylation culminates in cytoskeletal disturbances, neuronal dysfunction and death. Many AD patients hypersecrete glucocorticoids (GC) while neuronal structure, function and survival are adversely influenced by elevated GC levels. We report here that a rat neuronal cell line (PC12) engineered to express the human ortholog of the tau protein (PC12-htau) becomes more vulnerable to the toxic effects of either Abeta or GC treatment. Importantly, APP metabolism in GC-treated PC12-htau cells is selectively shifted towards increased production of the pro-amyloidogenic peptide C99. Further, GC treatment results in hyperphosphorylation of human tau at AD-relevant sites, through the cyclin-dependent kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein kinases. Pulse-chase experiments revealed that GC treatment increased the stability of tau protein rather than its de novo synthesis. GC treatment also induced accumulation of transiently expressed EGFP-tau in the neuronal perikarya. Together with previous evidence showing that Abeta can activate cyclin-dependent kinase 5 and GSK3, these results uncover a potential mechanism through which GC may contribute to AD neuropathology.
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Dexametasona/farmacología , Glucocorticoides/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 3 Dependiente de Ciclina , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Células PC12/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Sales de Tetrazolio , Tiazoles , Transfección , Proteínas tau/genéticaRESUMEN
The abnormal processing of the amyloid precursor protein (APP) is a pivotal event in the development of the unique pathology that defines Alzheimer's disease (AD). Stress, and the associated increase in corticosteroids, appear to accelerate brain ageing and may increase vulnerability to Alzheimer's disease via altered APP processing. In this study, rats were repeatedly exposed to an unavoidable stressor, an open elevated platform. Previous studies in this laboratory have shown that a single exposure produces a marked increase in plasma corticosterone levels but animals develop tolerance to this effect between 10 and 20 daily sessions. Twenty-four hours after stress, there was an increase in the ratio of the deglycosylated form of APP in the particulate fraction of the brain, which subsequently habituated after 20 days. The levels of soluble APP (APPs) tended to be lower in the stress groups compared to controls except for a significant increase in the hippocampus after 20 days of platform exposure. Since APPs is reported to have neurotrophic properties, this increased release may represent a neuroprotective response to repeated stress. It is possible that the ability to mount this response decreases with age thus increasing the vulnerability to stress-induced AD-related pathology.
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Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Estrés Fisiológico/patología , Animales , Conducta Animal , Encéfalo/patología , Masculino , Ratas , Ratas Sprague-Dawley , Estrés Fisiológico/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Soluble Alzheimer's Abeta oligomers autoinsert into neuronal cell membranes, contributing to the pathology of Alzheimer's Disease (AD), and elevated serum cholesterol is a risk factor for AD, but the reason is unknown. We investigated potential connections between these two observations at the membrane level by testing the hypothesis that Abeta(1-42) relocates membrane cholesterol. RESULTS: Oligomers of Abeta(1-42), but not the monomeric peptide, inserted into cholesterol-containing phosphatidylcholine monolayers with an anomalously low molecular insertion area, suggesting concurrent lipid rearrangement. Membrane neutron diffraction, including isomorphous replacement of specific lipid hydrogens with highly-scattering deuterium, showed that Abeta(1-42) insertion was accompanied by outward displacement of membrane cholesterol, towards the polar surfaces of the bilayer. Changes in the generalised polarisation of laurdan confirmed that the structural changes were associated with a functional alteration in membrane lipid order. CONCLUSION: Cholesterol is known to regulate membrane lipid order, and this can affect a wide range of membrane mechanisms, including intercellular signalling. Previously unrecognised Abeta-dependent rearrangement of the membrane sterol could have an important role in AD.
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Péptidos beta-Amiloides/metabolismo , Colesterol/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fragmentos de Péptidos/metabolismo , Transporte Biológico , Humanos , Difracción de Neutrones , Estructura Cuaternaria de Proteína , Solubilidad , Agua/metabolismoRESUMEN
RATIONALE: Nicotine is reported to improve learning and memory in experimental animals. Improved learning and memory has also been related to increased neurogenesis in the dentate gyrus (DG) of the hippocampal formation. Surprisingly, recent studies suggest that self-administered nicotine depresses cell proliferation in the DG. OBJECTIVE: To test the hypothesis that the effects of nicotine on cell proliferation in the DG and learning and memory depend upon the nicotine dose administered. METHODS: Rats were chronically infused from subcutaneous osmotic mini pumps with nicotine (0.25 or 4 mg kg(-1) day(-1)) or the saline vehicle for 10 days. Half the rats in each treatment group were trained to locate a hidden platform in a water maze task on days 4-7; a probe trial was performed on day 8. The remaining rats remained in their home cages. The effects of nicotine and of training in the water maze task on cell genesis in the DG were determined by measuring 5-bromo-2'-deoxyuridine (BrDU) uptake using fluorescence immunohistochemistry. RESULTS: Training in the water maze task increased cell proliferation in the DG. Infusions of nicotine at 4 mg kg(-1) day(-1), but not 0.25 mg kg(-1) day(-1), decreased cell proliferation in both untrained animals and animals trained in the maze and impaired spatial learning. CONCLUSIONS: The data suggest that learning in the water maze task is impaired by higher doses of nicotine tested, and that this response may be related to reduced cell genesis in the DG.
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Bromodesoxiuridina/metabolismo , Giro Dentado/efectos de los fármacos , Reacción de Fuga/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Orientación/efectos de los fármacos , Tabaquismo/fisiopatología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , Giro Dentado/fisiopatología , Relación Dosis-Respuesta a Droga , Reacción de Fuga/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Recuerdo Mental/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Orientación/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The processing of the amyloid precursor protein (APP) by the secretase family of protease enzymes can be influenced by a variety of diverse factors, including elements of the immune response. In this study, we have investigated the effect of the pro-inflammatory lipopolysaccharide (LPS) on APP processing in rat glial cell cultures derived from both cortex and cerebellum. LPS activation of the cells, as monitored by the induction of the pro-inflammatory nitric oxide synthase (iNOS) enzyme, elicited no change in the overall cellular expression levels of APP, although there was a marked concentration-related increase in the secretion of the soluble APPs following both short- (4 h) and long-term (18 h) drug treatment times. The stimulation of APPs secretion was blocked by the protein kinase C (PKC) inhibitor GF109203x, suggesting that LPS may act via a PKC-mediated pathway to increase APPs secretion.
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Precursor de Proteína beta-Amiloide/metabolismo , Lipopolisacáridos/farmacología , Neuroglía/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Animales , Cerebelo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Immunoblotting , Indoles/farmacología , Maleimidas/farmacología , Neuroglía/metabolismo , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The cellular prion protein, PrPc, is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a protease-resistant conformer of the protein may be the infectious agent in transmissible spongiform encephalopathies. PrPc is localized on growing axons in vitro and along fibre bundles that contain elongating axons in developing and adult brain. To determine whether the growth state of axons influenced the expression and axonal transport of PrPc, we examined changes in the protein following post-traumatic regeneration in the hamster sciatic nerve. Our results show (1) that PrPc in nerve is significantly increased during nerve regeneration; (2) that this increase involves an increase in axonally transported PrPc; and (3) that the PrPc preferentially targeted for the newly formed portions of the regenerating axons consists of higher molecular weight glycoforms. These results raise the possibility that PrPc may play a role in the growth of axons in vivo, perhaps as an adhesion molecule interacting with the extracellular environment through specialized glycosylation.
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Transporte Axonal/fisiología , Regeneración Nerviosa/fisiología , Proteínas PrPC/metabolismo , Neuropatía Ciática/metabolismo , Animales , Western Blotting/métodos , Corteza Cerebral/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Glucuronidasa/farmacología , Glicosilación , Masculino , Compresión Nerviosa/métodos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuraminidasa/farmacología , Proteínas PrPC/efectos de los fármacos , Neuropatía Ciática/fisiopatología , Factores de TiempoRESUMEN
Single O-linked N-acetylglucosamine (O-GlcNAc) sugar residues can compete with phosphate groups to occupy specific sites on certain nuclear and cytosolic proteins. Here we show that inhibiting cellular kinase activities resulted in changes in protein O-glycosylation levels in heat-stable cytoskeletal protein fractions derived from primary neuronal cells. As increased phosphorylation of the microtubule-associated protein tau is one of the pathological hallmarks of Alzheimer's disease and glycosylation may play an influential role in this process. We observed a significant decrease in the protein O-GlcNAc glycosylation of a tau-enriched cytoskeletal fraction generated from AD post-mortem brain samples as compared with control, suggesting an inverse relationship between the two post-translational modifications. Finally, cells transfected with the cDNA coding for O-GlcNAc transferase (OGT) displayed altered tau phosphorylation patterns as compared with control cells, suggesting that changes in tau glycosylation may influence its phosphorylation state. The specificity of the changes in the phosphorylation of individual amino acid residues provides evidence for a targeted O-glycosylation of tau.
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Enfermedad de Alzheimer/metabolismo , Glicosilación , Proteínas del Tejido Nervioso/metabolismo , Anciano , Enfermedad de Alzheimer/patología , Western Blotting , Demencia/metabolismo , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Humanos , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Sensibilidad y Especificidad , Lóbulo Temporal/metabolismo , Lóbulo Temporal/patología , Proteínas tauRESUMEN
The inducible form of nitric oxide synthase (iNOS) is an essential element of the immune response, which is expressed primarily in microglial cells within the CNS. Exposure of rat cortical neuronal cells to the pro-inflammatory bacterial endotoxin lipopolysaccharide (LPS) resulted in a significant increase in the expression of the cellular iNOS protein expression and NO generation (which serves as an indirect measure of NOS catalytic activity). These effects were potentiated by costimulation with interferon-gamma (IFNgamma) and the increase in NO generation was abolished by the iNOS selective inhibitor 1400W, although this did not attenuate the toxin-induced increase in the enzyme expression. As the cortex is one of the principal areas to be targeted in Alzheimer's disease (AD), the present findings may help to further our understanding of the biochemical events associated with the neurodegenerative process.
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Corteza Cerebral/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Neuronas/enzimología , Óxido Nítrico Sintasa/biosíntesis , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Neuronas/efectos de los fármacos , Óxido Nítrico/biosíntesis , Óxido Nítrico/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , RatasRESUMEN
OBJECTIVES: A decrease in cholinergic activity is a key event in the biochemistry of Alzheimer's disease (AD). The aim of the study was to investigate the expression levels of markers of cholinergic function in saliva, which is a readily accessible body fluid that can be obtained from subjects with minimal distress. DESIGN AND METHODS: Salivary samples were obtained from people with NINCDS-ARDRA "probable" Alzheimer's disease and age- and sex-matched controls. Salivary acetylcholinesterase enzyme (AChE) activity was determined colorometrically. RESULTS: Robust AChE catalytic activity was detected in the saliva samples that was stable for up to 6 h at room temperature following the provision of the salivary sample. The activity of the enzyme was significantly lower in people with AD than in age-matched controls. In addition, there were significant differences in activity between those who responded to acetylcholinesterase inhibitor (AChE-I) therapy and those who did not. CONCLUSIONS: Salivary enzyme activity may therefore prove to be a useful marker of central cholinergic activity.
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Acetilcolinesterasa/análisis , Enfermedad de Alzheimer/enzimología , Saliva/enzimología , Acetilcolinesterasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/tratamiento farmacológico , Biomarcadores , Inhibidores de la Colinesterasa/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Glucocorticoids modify gene expression via the translocation of receptors from the cytosol to the nucleus following agonist-associated receptor activation. In this study, we have characterized mitochondrial glucocorticoid (GR) localization and associated translocation kinetics in the C6 mouse glioma cell line. Treatment of the cells, which were cultured in steroid-depleted culture medium, with the GR agonist dexamethasone (dex) resulted in a dramatic decrease in mitochondrial GR levels in parallel with those of the cytosolic receptor. The effect was not observed in isolated intact mitochondria suggesting that the effect is unlikely to be direct but is rather a component of the combined cellular response to GR activation. A marked stimulation of the expression of the mitochondrially-encoded cytochrome oxidase-1 (COX-1) gene was found following GR activation and its export from mitochondria. The effects were inhibited by RU486. Therefore, GR is likely to have a functional role at the level of the mitochondria within intact cells.
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Glioma/química , Mitocondrias/química , Receptores de Glucocorticoides/análisis , Animales , Western Blotting , Línea Celular Tumoral , Ciclooxigenasa 1 , Técnica del Anticuerpo Fluorescente , Glioma/patología , Isoenzimas/genética , Cinética , Luteolíticos/farmacología , Proteínas de la Membrana , Ratones , Mitocondrias/fisiología , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/ultraestructura , Fracciones Subcelulares/químicaRESUMEN
Sialoglycoproteins play a key role in both brain development and neuronal plasticity with their sialylation state being controlled by the sialyltransferase (STN) family of enzymes. In this study, we have determined the role of specific kinase enzymes in the expression and catalytic activity of the alpha2,6 STN (ST6N) isozyme. The catalytic activity was moderately decreased following the inhibition of GSK3beta with LiCl. However, there was a significant increase in catalytic activity following activation of protein kinase C (PKC) by phorbol ester. There was no change in the expression levels of the enzyme protein following any of the treatments. The changes in enzyme catalytic activity were also mirrored by the expression of both protein-bound sialic acid and the polysialic acid oligosaccharide group attached to the neural cell adhesion molecule, NCAM. These results provide further evidence for the role of second messenger-associated kinase enzymes in the modulation of the cell glycosylation potential.
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Sialiltransferasas/metabolismo , Sialiltransferasas/fisiología , Catálisis , Activación Enzimática , Glicosilación , Humanos , Cloruro de Litio/farmacología , Ésteres del Forbol/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Protein glycosylation, which is a key post-translational event, is catalysed by the glycosyltransferase family of enzymes. There is an increasing body of evidence to suggest that these enzymes may themselves be glycosylated, possibly as an autocatalytic event. Using a novel in vitro system, we have investigated the role of enzyme glycosylation in sialyltransferase catalytic activity. The enzyme activity is glycosylation dependent, with the penultimate galactose residue on complex N-linked oligosaccharides playing a pivotal role. These results serve to underline the complexity of the glycosylation process.