RESUMO
Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψm) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψm in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1-50 nM), caused depolarisation of Δψm. Small decreases in complex I activity caused an immediate and partial Δψm depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψm. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψp) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψp were registered. These data indicate that in synaptosomes, both Δψm and Δψp are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson's disease in particular, where such reductions in complex I activity are present.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Animais , Antimicina A/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metacrilatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Cianeto de Potássio/farmacologia , Ratos Wistar , Rotenona/farmacologia , Sinaptossomos/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/química , Automação , Meios de Cultura/química , Oligossacarídeos/análise , Robótica , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ensaios de Triagem em Larga Escala , Humanos , Modelos MolecularesRESUMO
Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.
Assuntos
Antipsicóticos/farmacologia , Benzodiazepinas/farmacologia , Glicoproteínas/sangue , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Esquizofrenia Paranoide/sangue , Doença Aguda , Adulto , Antipsicóticos/uso terapêutico , Benzodiazepinas/uso terapêutico , Cromatografia Líquida , Feminino , Glicômica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Pessoa de Meia-Idade , Olanzapina , Orosomucoide/metabolismo , Polissacarídeos/sangue , Esquizofrenia Paranoide/tratamento farmacológico , Adulto Jovem , alfa 1-Antitripsina/sangueRESUMO
BACKGROUND: The activities of mitochondrial complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2) and complex IV (cytochrome c oxidase EC 1.9.3.1) are reduced by 30-70% in Huntington's disease and Alzheimer's disease, respectively, and are associated with excitotoxic cell death in these disorders. In this study, we investigated the control that complexes III and complex IV exert on glutamate release from the isolated nerve terminal. RESULTS: Inhibition of complex III activity by 60-90% was necessary for a major increase in the rate of Ca2+-independent glutamate release to occur from isolated nerve terminals (synaptosomes) depolarized with 4-aminopyridine or KCl. Similarly, an 85-90% inhibition of complex IV activity was required before a major increase in the rate of Ca2+-independent glutamate release from depolarized synaptosomes was observed. Inhibition of complex III and IV activities by ~ 60% and above was required before rates of glutamate efflux from polarized synaptosomes were increased. CONCLUSIONS: These results suggest that nerve terminal mitochondria possess high reserves of complex III and IV activity and that high inhibition thresholds must be reached before excess glutamate is released from the nerve terminal. The implications of the results in the context of the relationship between electron transport chain enzyme deficiencies and excitotoxicity in neurodegenerative disorders are discussed.
RESUMO
Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.
Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/química , Glicoproteínas/sangue , Glicoproteínas/química , Neoplasias Pulmonares/química , Neoplasias Pulmonares/diagnóstico , Polissacarídeos/análise , Resinas de Troca Aniônica/química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Glicosilação , Haptoglobinas/química , Haptoglobinas/metabolismo , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Neoplasias Pulmonares/sangue , Dados de Sequência Molecular , Curva ROC , Sensibilidade e EspecificidadeRESUMO
Glycosylation modifications have been reported in a number of disease states and, as a result, there is significant focus on the discovery and development of glycan-based biomarkers. Glyco-biomarkers have the potential to enhance the efficacy and efficiency of the diagnostic procedures for these diseases.
Assuntos
Biomarcadores Tumorais/metabolismo , Biomarcadores/metabolismo , Glicômica/métodos , Polissacarídeos/metabolismo , Glicosilação , Humanos , Neoplasias/diagnóstico , Neoplasias/metabolismoRESUMO
The development of glycoanalytical HPLC-based high-throughput technology has greatly enhanced the study of glycobiology, facilitating the discovery of disease-related solutions and providing an informative view of glycosylation and its relationship with other biological disciplines in a systems biology approach.
Assuntos
Glicômica/métodos , Biologia de Sistemas/métodos , Biomarcadores/metabolismo , GlicosilaçãoRESUMO
The effects of decylubiquinone, a ubiquinone analogue, on mitochondrial function and inhibition thresholds of the electron transport chain enzyme complexes in synaptosomes were investigated. Decylubiquinone increased complex I/III and complex II/III activities by 64 and 80%, respectively, and attenuated reductions in oxygen consumption at high concentrations of the complex III inhibitor myxothiazol. During inhibition of complex I, decylubiquinone attenuated reductions in synaptosomal oxygen respiration rates, as seen in the complex I inhibition threshold. Decylubiquinone increased the inhibition thresholds of complex I/III, complex II/III, and complex III over oxygen consumption in the nerve terminal by 25-50%, when myxothiazol was used to inhibit complex III. These results imply that decylubiquinone increases mitochondrial function in the nerve terminal during complex I or III inhibition. The potential benefits of decylubiquinone in diseases where complex I, I/III, II/III, or III activities are deficient are discussed.
Assuntos
Mitocôndrias/fisiologia , Sinaptossomos/metabolismo , Ubiquinona/análogos & derivados , Animais , Antimicina A/farmacologia , Transporte de Elétrons , Feminino , Metacrilatos/farmacologia , Modelos Biológicos , Doenças Neurodegenerativas/metabolismo , Consumo de Oxigênio , Ratos , Ratos Wistar , Rotenona/farmacologia , Tiazóis/farmacologia , Ubiquinona/farmacologia , Desacopladores/farmacologiaRESUMO
Metabolic control analysis was used to determine the spread of control exerted by the electron transport chain complexes over oxygen consumption rates in the nerve terminal. Oxygen consumption rates and electron transport chain complex activities were titrated with appropriate inhibitors to determine the flux control coefficients and the inhibition thresholds in rat brain synaptosomes. The flux control coefficients for complex I, complex II/III, complex III, and complex IV were found to be 0.30 +/- 0.07, 0.20 +/- 0.03, 0.20 +/- 0.05, and 0.08 +/- 0.05, respectively. Inhibition thresholds for complex I, complex II/III, complex III, and complex IV activities were determined to be approximately 10, approximately 30, approximately 35, and 50-65%, respectively, before major changes in oxygen consumption rates were observed. These results indicate that, of the electron transport chain components, complex I exerts a high level of control over synaptosomal bioenergetics, suggesting that complex I deficiencies that are present in neurodegenerative disorders, such as Parkinson disease, are sufficient to compromise oxygen consumption in the synaptosomal model of the nerve terminal.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Neurônios/metabolismo , Consumo de Oxigênio , Animais , Feminino , Cinética , Ligação Proteica , Ratos , Ratos Wistar , Sinaptossomos/metabolismo , TitulometriaRESUMO
Detrimental changes to mitochondrial function have been shown to occur with age. In this study we examined the levels of H(2)O(2) production, in situ mitochondrial membrane potential (Deltapsi(m)), oxygen consumption (JO(2)) and electron transport chain (ETC) enzyme activities in synaptosomes isolated from rats of two age groups, 6 and 18 months. The rate of H(2)O(2) production in synaptosomes was found to be higher in the 18-month old group compared to that of 6-month old. Deltapsi(m) was found to be significantly lower in synaptosomes from the older rats, which also correlated with a reduction in JO(2). Measurement of the individual electron transport chain enzyme activities revealed that reduced complex II/III and complex IV activities were the possible contributors to the reduced bioenergetic function in synaptosomes from the older rats. These data suggest that ageing may lead to increased nerve terminal H(2)O(2) production while simultaneous deleterious effects on bioenergetic function occur in in situ synaptosomal mitochondria. In addition, Ca(2+)-independent glutamate release was found to be increased at lower levels of complex I inhibition in the synaptosomes from older rats, suggesting that reduction of mitochondrial function may potentiate excitotoxic conditions in the ageing brain.
Assuntos
Envelhecimento/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Sinaptossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte de Elétrons , Ácido Glutâmico/metabolismo , Homeostase , Potenciais da Membrana , Ratos , Partículas Submitocôndricas/fisiologiaRESUMO
Mitochondria have been implicated in the pathogenesis of several neurodegenerative disorders and, in particular, complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) activity has been shown to be partially reduced in postmortem studies of the substantia nigra of Parkinson's disease patients. The present study examines the effect of partial inhibition of complex I activity on glutamate release from rat brain synaptosomes. Following a 40% inhibition of complex I activity with rotenone, it was found that Ca(2+)-independent release of glutamate increased from synaptosomes depolarized with 4-aminopyridine. Highest rates of glutamate release were found to occur between 60-90% complex I inhibition. A similar pattern of increase was shown to occur in synaptosomes depolarized with KCl. The increase in glutamate release was found to correlate to a significant decrease in ATP. Inhibition of complex I activity by 40% was also shown to cause a significant collapse in mitochondrial membrane potential (Deltapsi(m)). These results suggest that partial inhibition of complex I activity in in situ mitochondria is sufficient to significantly increase release of glutamate from the pre-synaptic nerve terminal. The relevance of these results in the context of excitotoxicity and the pathogenesis of neurodegenerative disorders is discussed.