RESUMO
Cardiovascular diseases account for more than 30% of all deaths worldwide and many could be ameliorated with early diagnosis. Current cardiac imaging modalities can assess blood flow, heart anatomy and mechanical function. However, for early diagnosis and improved treatment, further functional biomarkers are needed. One such functional biomarker could be the myocardium pH. Although tissue pH is already determinable via MR techniques, and has been since the early 1990s, it remains elusive to use practically. The objective of this study was to explore the possibility to evaluate cardiac pH noninvasively, using in-cell enzymatic rates of hyperpolarized [1-13 C]pyruvate metabolism (ie, moles of product produced per unit time) determined directly in real time using magnetic resonance spectroscopy in a perfused mouse heart model. As a gold standard for tissue pH we used 31 P spectroscopy and the chemical shift of the inorganic phosphate (Pi) signal. The nonhomogenous pH distribution of the perfused heart was analyzed using a multi-parametric analysis of this signal, thus taking into account the heterogeneous nature of this characteristic. As opposed to the signal ratio of hyperpolarized [13 C]bicarbonate to [13 CO2 ], which has shown correlation to pH in other studies, we investigated here the ratio of two intracellular enzymatic rates: lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), by way of determining the production rates of [1-13 C]lactate and [13 C]bicarbonate, respectively. The enzyme activities determined here are intracellular, while the pH determined using the Pi signal may contain an extracellular component, which could not be ruled out. Nevertheless, we report a strong correlation between the tissue pH and the LDH/PDH activities ratio. This work may pave the way for using the LDH/PDH activities ratio as an indicator of cardiac intracellular pH in vivo, in an MRI examination.
Assuntos
Coração/diagnóstico por imagem , L-Lactato Desidrogenase/análise , Espectroscopia de Ressonância Magnética/métodos , Miocárdio/enzimologia , Complexo Piruvato Desidrogenase/análise , Animais , Isótopos de Carbono , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Perfusão , Fósforo , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Vibrio alginolyticus is a waterborne pathogen that infects a wide variety of hosts including fish and human, and the outbreak of this pathogen can cause a huge economic loss in aquaculture. Thus, enhancing host's capability to survive from V. alginolyticus infection is key to fighting infection and this remains still unexplored. In the present study, we established a V. alginolyticus-zebrafish interaction model by which we explored how zebrafish survived from V. alginolyticus infection. We used GC-MS based metabolomic approaches to characterize differential metabolomes between survival and dying zebrafish upon infection. Pattern recognition analysis identified the TCA cycle as the most impacted pathway. The metabolites in the TCA cycle were decreased in the dying host, whereas the metabolites were increased in the survival host. Furthermore, the enzymatic activities of the TCA cycle including pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) also supported this conclusion. Among the increased metabolites in the TCA cycle, malic acid was the most crucial biomarker for fish survival. Indeed, exogenous malate promoted zebrafish survival in a dose-dependent manner. The corresponding activities of KGDH and SDH were also increased. These results indicate that the TCA cycle is a key pathway responsible for the survival or death in response to infection caused by V. alginolyticus, and highlight the way on development of metabolic modulation to control the infection.
Assuntos
Ciclo do Ácido Cítrico , Vibrioses/imunologia , Vibrioses/patologia , Vibrio alginolyticus/patogenicidade , Peixe-Zebra , Animais , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Complexo Cetoglutarato Desidrogenase/análise , Malatos/análise , Metabolômica , Complexo Piruvato Desidrogenase/análise , Succinato Desidrogenase/análise , Análise de SobrevidaRESUMO
Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for ß-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.
Assuntos
Hibernação , Complexo Piruvato Desidrogenase/metabolismo , Sciuridae/fisiologia , Animais , Ativação Enzimática , Glicólise , Coração/fisiologia , Metabolismo dos Lipídeos , Fígado/enzimologia , Fígado/fisiologia , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiologia , Miocárdio/enzimologia , Fosforilação , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Complexo Piruvato Desidrogenase/análiseAssuntos
Doenças Cardiovasculares/diagnóstico , Técnicas de Diagnóstico Cardiovascular , Espectroscopia de Ressonância Magnética/métodos , Animais , Bicarbonatos , Isótopos de Carbono , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Doenças Cardiovasculares/metabolismo , Metabolismo Energético , Previsões , Glicólise , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/metabolismo , Humanos , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Neoplasias Experimentais/metabolismo , Complexo Piruvato Desidrogenase/análise , Sensibilidade e Especificidade , UreiaRESUMO
PURPOSE: The study's purpose was to examine the effects of a short-term period with intensified training or training cessation of trained soccer players on VO(2) kinetics at 75% maximal aerobic speed, oxidative enzymes, and performance in repeated high-intensity exercise. METHODS: After the last match of the season, 18 elite soccer players were, for a 2-wk period, assigned to a high-intensity training group (n = 7) performing 10 training sessions mainly consisting of aerobic high-intensity training (8 × 2 min) and speed endurance training (10-12 × 30-s sprints) or a training cessation group (n = 11) that refrained from training. RESULTS: For the training cessation group, VO(2) kinetics became slower (P < 0.05) with a larger time constant (τ = 21.5 ± 2.9 vs 23.8 ± 3.2 s (mean ± SD, before vs after)) and a larger mean response time (time delay + τ = 45.0 ± 1.8 vs 46.8 ± 2.2 s). The amount of muscle pyruvate dehydrogenase (17%, P < 0.01) and maximal activity of citrate synthase (12%) and 3-hydroxyacyl-CoA (18%, P < 0.05) were lowered. In addition, the fraction of slow twitch fibers (56% ± 18% vs 47% ± 15%, P < 0.05), Yo-Yo intermittent recovery level 2 test (845 ± 160 vs 654 ± 99 m), and the repeated sprint performance (33.41 ± 0.96 vs 34.11 ± 0.92 s, P < 0.01) were reduced. For the high-intensity training group, running economy was improved (P < 0.05), and the amount of pyruvate dehydrogenase (17%) and repeated sprint performance (33.44 ± 1.17 vs 32.81 ± 1.01 s) were enhanced (P < 0.05). CONCLUSIONS: Inactivity slows VO(2) kinetics in association with a reduction of muscle oxidative capacity and repeated high-intensity running performance. In addition, intensified training of already well-trained athletes can improve mechanical efficiency and repeated sprint performance.
Assuntos
Desempenho Atlético/fisiologia , Consumo de Oxigênio/fisiologia , Futebol/fisiologia , Adulto , Atletas , Citrato (si)-Sintase/análise , Humanos , Masculino , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/fisiologia , Resistência Física/fisiologia , Complexo Piruvato Desidrogenase/análise , Corrida/fisiologia , Adulto JovemRESUMO
The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25-31% of that in hippocampus. White matter choline acetyltransferase activity was 10-50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20-25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8-4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.
Assuntos
Acetilcolina/biossíntese , Encéfalo/enzimologia , Fibras Colinérgicas/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Fibras Nervosas Mielinizadas/enzimologia , Acetilcoenzima A/análise , Acetilcoenzima A/metabolismo , Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Animais , Encéfalo/citologia , Mapeamento Encefálico , Colina/análise , Colina/metabolismo , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/ultraestrutura , Inibidores da Colinesterase/farmacologia , Feminino , Glucose/metabolismo , Imuno-Histoquímica , Interneurônios/metabolismo , Masculino , Proteínas de Membrana Transportadoras/análise , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/ultraestrutura , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/metabolismo , Soman/farmacologia , Sus scrofa , Degeneração Walleriana/induzido quimicamente , Degeneração Walleriana/enzimologia , Degeneração Walleriana/fisiopatologiaRESUMO
In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase.
Assuntos
Astrócitos/enzimologia , Química Encefálica/fisiologia , Encéfalo/enzimologia , Metabolismo Energético/fisiologia , Líquido Intracelular/enzimologia , Animais , Encéfalo/citologia , Isótopos de Carbono , Células Cultivadas , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/biossíntese , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Neuroquímica/métodos , Fosforilação Oxidativa , Piruvato Carboxilase/análise , Piruvato Carboxilase/metabolismo , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismoRESUMO
The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.
Assuntos
Complexo Piruvato Desidrogenase/análise , Sequência de Aminoácidos , Animais , Arilamina N-Acetiltransferase/análise , Arilamina N-Acetiltransferase/genética , Sequência de Bases , Galinhas , DNA/genética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/enzimologia , Proteínas Quinases/análise , Proteínas Serina-Treonina Quinases , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
The pyruvate dehydrogenase complex (PDC) is integral to metabolism and energetics. Congenital PDC deficiency leads to lactic acidosis, neurological degeneration and early death. An investigational compound for such defects is dichloroacetate (DCA), which activates the PDC (inhibiting reversible phosphorylation of the E1alpha subunit) and decreases its turnover. Here, primary human fibroblast cultures from five healthy subjects and six patients with mutations in the PDC-E1 component were grown in media+/-DCA, exposed to media containing (13)C-labeled glucose, and studied (as cell extracts) by nuclear magnetic resonance (NMR) spectroscopy. Computer modeling of NMR-derived (13)C-glutamate isotopomeric patterns estimated relative carbon flow through TCA cycle-associated pathways and characterized effects of PDC deficiency on metabolism and energetics. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. With the exception of one patient cell line expressing an unusual splicing mutation, PDC-deficient cells had significantly higher GCR, LPR and label-derived acetyl-CoA, indicative of increased glycolysis vs. controls. In all cells, DCA caused a major shift (40% decrease) from anaplerotic-related pathways (e.g., pyruvate carboxylase) toward flux through PDC. Ignoring the patient with the splicing mutation, DCA decreased average glycolysis (29%) in patient cells, but had no significant effect on control cells, and did not change LPR or the nucleoside triphosphate to diphosphate ratio (NTP/NDP) in either cell type. Maintenance of NTP despite reduced glycolysis indicates that DCA improves metabolic efficiency by increasing glucose oxidation. This study demonstrates that NMR spectroscopy provides insight into biochemical consequences of PDC deficiency and the mechanism of putative therapeutic agents.
Assuntos
Glucose/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Mitocôndrias/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/metabolismo , Complexo Piruvato Desidrogenase/análise , Células Cultivadas , Ácido Dicloroacético/farmacologia , Metabolismo Energético , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Lactente , Masculino , Mitocôndrias/enzimologia , Complexo Piruvato Desidrogenase/efeitos dos fármacos , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologiaRESUMO
AIMS: The aim of this study was to examine the chronic effects of different non-esterified fatty acids (NEFA) on insulin secretion by pancreatic islets of normal Wistar rats in vitro. METHODS: Pancreatic islets were isolated from normal Wistar rats, and were incubated with 0.2, 0.4, or 0.8 mmol/L palmitate (C16:0), stearate (C18:0), oleate (C18:1), or linoleate (C18:2) for 24 h, then the insulin secretion and pyruvate dehydrogenase (PDH) activity were examined. RESULTS: Neither islet insulin content nor islet DNA content differed among islets incubated with each kind of NEFA. Compared with control, linoleate significantly inhibited glucose-stimulated insulin secretion (GSIS) and PDH activity at each concentration (p < 0.05), while others inhibited GSIS and PDH activity significantly only at 0.4 and 0.8 mmol/L (p < 0.05). There was no significant difference in GSIS and PDH activity among islets pretreated by palmitate, stearate, and oleate at the same concentration (p > 0.05). However, linoleate decreased GSIS more than others at the same concentration (p < 0.05), while linoleate (0.4 or 0.8 mmol/L) inhibited PDH activity more than others at the same concentration (p < 0.05). CONCLUSIONS: Elevation of palmitate, stearate, oleate or linoleate decreases the beta-cell secretory response to glucose, through inhibiting PDH activity. Linoleate exerts more negative effect on GSIS than other NEFA.
Assuntos
Ácidos Graxos não Esterificados/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Animais , Células Cultivadas , DNA/análise , Glucose/farmacologia , Insulina/análise , Secreção de Insulina , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/fisiologia , Masculino , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Complexo Piruvato Desidrogenase/fisiologia , Ratos , Ratos WistarRESUMO
Our laboratory recently showed that six sessions of sprint interval training (SIT) over 2 wk increased muscle oxidative potential and cycle endurance capacity (Burgomaster KA, Hughes SC, Heigenhauser GJF, Bradwell SN, and Gibala MJ. J Appl Physiol 98: 1895-1900, 2005). The present study tested the hypothesis that short-term SIT would reduce skeletal muscle glycogenolysis and lactate accumulation during exercise and increase the capacity for pyruvate oxidation via pyruvate dehydrogenase (PDH). Eight men [peak oxygen uptake (VO2 peak)=3.8+/-0.2 l/min] performed six sessions of SIT (4-7x30-s "all-out" cycling with 4 min of recovery) over 2 wk. Before and after SIT, biopsies (vastus lateralis) were obtained at rest and after each stage of a two-stage cycling test that consisted of 10 min at approximately 60% followed by 10 min at approximately 90% of VO2 peak. Subjects also performed a 250-kJ time trial (TT) before and after SIT to assess changes in cycling performance. SIT increased muscle glycogen content by approximately 50% (main effect, P=0.04) and the maximal activity of citrate synthase (posttraining: 7.8+/-0.4 vs. pretraining: 7.0+/-0.4 mol.kg protein -1.h-1; P=0.04), but the maximal activity of 3-hydroxyacyl-CoA dehydrogenase was unchanged (posttraining: 5.1+/-0.7 vs. pretraining: 4.9+/-0.6 mol.kg protein -1.h-1; P=0.76). The active form of PDH was higher after training (main effect, P=0.04), and net muscle glycogenolysis (posttraining: 100+/-16 vs. pretraining: 139+/-11 mmol/kg dry wt; P=0.03) and lactate accumulation (posttraining: 55+/-2 vs. pretraining: 63+/-1 mmol/kg dry wt; P=0.03) during exercise were reduced. TT performance improved by 9.6% after training (posttraining: 15.5+/-0.5 vs. pretraining: 17.2+/-1.0 min; P=0.006), and a control group (n=8, VO2 peak=3.9+/-0.2 l/min) showed no change in performance when tested 2 wk apart without SIT (posttraining: 18.8+/-1.2 vs. pretraining: 18.9+/-1.2 min; P=0.74). We conclude that short-term SIT improved cycling TT performance and resulted in a closer matching of glycogenolytic flux and pyruvate oxidation during submaximal exercise.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Exercício Físico/fisiologia , Glicogenólise/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Resistência Física/fisiologia , Corrida/fisiologia , Adulto , Estudos de Casos e Controles , Citrato (si)-Sintase/análise , Citrato (si)-Sintase/fisiologia , Teste de Esforço , Glicogênio/análise , Glicogênio/metabolismo , Humanos , Lactatos/análise , Lactatos/metabolismo , Masculino , Mitocôndrias Musculares/enzimologia , Músculo Esquelético/química , Consumo de Oxigênio/fisiologia , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/fisiologia , Fatores de TempoRESUMO
During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44+/-1% peak oxygen consumption (mean+/-SE) until exhaustion (exhaustion at 3 h 23 min+/-11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P<0.05). PDH activity peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced (approximately 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P<0.05) was also associated with decreasing PDH activity (P<0.05) and increased PDH kinase 4 mRNA (P<0.05) during exercise and recovery. At 1 h of exercise, pyruvate production was greatest and was closely linked to glutamate, which was the predominant amino acid taken up during exercise and recovery. Alanine and glutamine were also associated with pyruvate metabolism, and they comprised approximately 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism in early exercise.
Assuntos
Aminoácidos/fisiologia , Metabolismo dos Carboidratos/fisiologia , Exercício Físico/fisiologia , Ácidos Graxos/fisiologia , Resistência Física/fisiologia , Complexo Piruvato Desidrogenase/fisiologia , Adulto , Alanina/análise , Alanina/metabolismo , Aminoácidos/análise , Velocidade do Fluxo Sanguíneo , Glicemia/análise , Ácidos Graxos/análise , Artéria Femoral/fisiologia , Veia Femoral/fisiologia , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Glutamina/análise , Glutamina/metabolismo , Humanos , Insulina/sangue , Masculino , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Consumo de Oxigênio/fisiologia , Proteínas Quinases/análise , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Complexo Piruvato Desidrogenase/análise , Piruvatos/análise , Piruvatos/metabolismo , RNA Mensageiro/análiseRESUMO
Lactic acidosis has been associated with a variety of clinical conditions and can be due to mutation in nuclear or mitochondrial genes. We performed mutations screening of all mitochondrial tRNA genes in 44 patients who referred as hyperlactic acidosis. Patients showed heterogeneous phenotypes including Leigh disease in four, MELAS in six, unclassified mitochondrial myopathy in 10, cardiomyopathy in five, MERRF in one, pure lactic acidosis in six, and others in 12 including facio-scaplo-femoral muscular dystrophy (FSFD), familial cerebellar ataxia, recurrent Reye syndrome, cerebral palsy with mental retardation. We measured enzymatic activities of pyruvate dehydrogenase complex, and respiratory chain enzymes. All mitochondrial tRNA genes and known mutation of ATPase 6 were studied by single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods. We have found one patient with PDHC deficiency and six patients with Complex I+IV deficiency, though the most of the patients showed subnormal to deficient state of respiratory chain enzyme activities. We have identified one of the nucleotide changes in 29 patients. Single nucleotide changes in mitochondrial tRNA genes are found in 27 patients and one in ATPase 6 gene in two patients. One of four pathogenic point mutations (A3243G, C3303T, A8348G, and T8993G) was identified in 12 patients who showed the phenotype of Leigh syndrome, MELAS, cardimyopathy and cerebral palsy with epilepsy. Seventeen patients have one of the normal polymorphisms in the mitochondrial tRNA gene reported before. SSCP and PCR-RFLP could detect the heteroplasmic condition when the percentage of mutant up to 5, however, it cannot be observed by direct sequencing method. It is important to screen the mtDNA mutation not only by direct sequence but also by PCR-RFLP and the other sensitive methods to detect the heroplasmy when lactic acidosis has been documented in the patients who are not fulfilled the criteria of mitochondrial disorders.
Assuntos
Acidose Láctica/genética , Doenças Mitocondriais/genética , Mutação Puntual , RNA de Transferência/genética , RNA/genética , Acidose Láctica/enzimologia , Acidose Láctica/etiologia , Adolescente , Adulto , Criança , Pré-Escolar , Transporte de Elétrons , Feminino , Genes/genética , Humanos , Lactente , Masculino , Doenças Mitocondriais/complicações , Doenças Mitocondriais/enzimologia , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Complexo Piruvato Desidrogenase/análise , RNA Mitocondrial , Análise de Sequência de DNARESUMO
A family presented with three affected children with Leigh syndrome, a progressive neurodegenerative disorder. Analysis of the OXPHOS complexes in muscle of two affected patients showed an increase in activity of pyruvate dehydrogenase and a decrease of complex V activity. Mutation analysis revealed the T9176C mutation in the mtATPase 6 gene (OMIM 516060) and the mutation load was above 90% in the patients. Unaffected maternal relatives were tested for carrier-ship and one of them, with a mutation load of 55% in blood, was pregnant with her first child. The possibility of prenatal diagnosis was evaluated. The main problem was the lack of data on genotype-phenotype associations for the T9176C mutation and on variation of the mutation percentage in tissues and in time. Therefore, multiple tissues of affected and unaffected carriers were analysed. Eventually, prenatal diagnosis was offered with understanding by the couple that there could be considerable uncertainty in the interpretation of the results. Prenatal diagnosis was carried out twice on cultured and uncultured chorion villi and amniotic fluid cells. The result was a mutation percentage just below the assumed threshold of expression (90%). The couple decided to continue the pregnancy and an apparently healthy child was born with an as yet unclear prognosis. This is the first prenatal diagnosis for a carrier of the T9176C mutation. Prenatal diagnosis for this mutation is technically reliable, but the prognostic predictions are not straightforward.
Assuntos
DNA Mitocondrial/genética , Doença de Leigh/diagnóstico , ATPases Mitocondriais Próton-Translocadoras/genética , Diagnóstico Pré-Natal , Criança , Análise Mutacional de DNA , Feminino , Humanos , Doença de Leigh/genética , Masculino , Músculo Esquelético/enzimologia , Linhagem , Fenótipo , Mutação Puntual , Gravidez , Complexo Piruvato Desidrogenase/análiseRESUMO
The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281-1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.
Assuntos
Anticorpos Monoclonais , Doenças Mitocondriais/diagnóstico , Proteínas/análise , Proteômica/métodos , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Imuno-Histoquímica/métodos , Translocases Mitocondriais de ADP e ATP/análise , Translocases Mitocondriais de ADP e ATP/imunologia , Doenças Mitocondriais/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/imunologia , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/imunologiaRESUMO
BACKGROUND: Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic disease characterized by the presence of antibodies directed predominantly against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). What provokes tolerance breakdown in PBC remains to be established, though there is evidence to indicate that microbes may induce anti-mitochondrial antibodies (AMA) through a mechanism of molecular mimicry. METHODS: Having found that urease beta (UREB)(22-36) antigen of Helicobacter pylori (HELPY) shares extensive (87%) similarity with PDC-E2(212-226), the major mitochondrial autoepitope, it was hypothesized that this would also lead to cross-reactivity. The UREB/PDC-E2 mimics were thus constructed and tested by ELISA in 112 PBC patients and 114 controls. RESULTS: Reactivity to PDC-E2(212-226) was found in 104 patients but to UREB(22-36) in only 2. In these two patients, the double reactivity was not cross-reactive. The lack of surface antibody accessibility to UREB(22-36), as demonstrated through three-dimensional model prediction analysis, may explain this unexpected finding. There was some speculation on whether HELPY UREB(22-36) might act as a cross-reactive CD4 T-cell epitope. All seven PBC patients, tested in a standard proliferation assay against PDC-E2(212-226), gave a positive response. All seven were unresponsive to HELPY UREB(22-36). The pattern of reactivity to HELPY antigens by immunoblot was similar between anti-PDC-E2-positive and negative PBC cases, as well as between PBC patients and controls. CONCLUSION: Contrary to common belief, extensive sequence homology (molecular mimicry) between self and microbe does not necessarily result in cross-reactivity. It is therefore likely that, when present, cross-reactivity between self and microbes is of biological importance.
Assuntos
Helicobacter pylori/imunologia , Epitopos Imunodominantes/imunologia , Cirrose Hepática Biliar/imunologia , Mitocôndrias/imunologia , Complexo Piruvato Desidrogenase/imunologia , Urease/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/imunologia , Proteínas de Bactérias/imunologia , Estudos de Coortes , Reações Cruzadas , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cirrose Hepática Biliar/sangue , Masculino , Pessoa de Meia-Idade , Mimetismo Molecular , Complexo Piruvato Desidrogenase/análise , Estudos de Amostragem , Sensibilidade e Especificidade , Domínios de Homologia de src/imunologiaRESUMO
A number of antigens implicated in the pathogenesis of autoimmune diseases including Sjogren's syndrome and systemic lupus erythematosus (SLE) are expressed aberrantly by apoptotic cells. It is also known that apoptogenic proteins are released from the mitochondrial intermembrane space at an early stage during the induction and development of apoptosis. Combination of this evidence led us to test the hypothesis that apoptotic mechanisms provide an explanation for the abnormal expression of the inner mitochondrial enzyme, pyruvate dehydrogenase complex (PDC), observed on the surface of some cells in patients with the autoimmune liver disease primary biliary cirrhosis (PBC). Using one murine and two human cell lines it was found that the induction of apoptosis led to early detection of PDC within the cytoplasm. However, cytochrome c oxidase subunit 4 (COX 4), which is also present on the inner surface of the inner mitochondrial membrane, remained within the mitochondria. Immunoreactive PDC was also detected on the outer surface of the intact plasma membrane of cells sampled after the induction of apoptosis. Serial release of PDC to the cytoplasm and then onto the external surface of the plasma membrane provides direct evidence that the antigen on the cell surface is of mitochondrial origin. Immunoreactivity specific for PDC is strongly implicated in the pathogenesis of PBC, but this autoantigen is normally concealed from the immune system by three membrane systems. Release of PDC onto the cell surface during apoptosis provides a possible route for recognition of this antigen by the immune system which could contribute to both afferent and efferent phases of the disease process.
Assuntos
Cirrose Hepática Biliar/imunologia , Complexo Piruvato Desidrogenase/análise , Linfócitos T/imunologia , Animais , Apoptose , Caspases/análise , Linhagem Celular , Membrana Celular/química , Citometria de Fluxo , Humanos , Immunoblotting , Células Jurkat , Cirrose Hepática Biliar/patologia , Camundongos , Microscopia ConfocalRESUMO
OBJECTIVES: To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2). METHODS: The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA. RESULTS: The expression vector pExSecI/PDC-E2 was successfully constructed. The products could be identified by the specific self-antibodies in the sera from the primary biliary cirrhosis patients. CONCLUSION: High efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.
Assuntos
Cirrose Hepática Biliar/sangue , Complexo Piruvato Desidrogenase/biossíntese , Clonagem Molecular , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Ensaio de Imunoadsorção Enzimática , Humanos , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/imunologia , Linfócitos/enzimologia , Reação em Cadeia da Polimerase , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/genéticaRESUMO
Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [(3)H]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V(max) for uptake of [(3)H]-D-aspartate, with no change in the K(m) value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [(3)H]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Leucina/análogos & derivados , Deficiência de Tiamina/metabolismo , Hidrolases Anidrido Ácido/análise , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Ácido D-Aspártico/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Glutationa/farmacologia , Immunoblotting/métodos , Complexo Cetoglutarato Desidrogenase/análise , Leucina/farmacologia , Piritiamina/efeitos adversos , Complexo Piruvato Desidrogenase/análise , Ratos , ATPase Trocadora de Sódio-Potássio/análise , Tiamina/análise , Tiamina/farmacologia , Deficiência de Tiamina/induzido quimicamente , Fatores de Tempo , Transcetolase/análise , Trítio/metabolismo , alfa-Tocoferol/farmacologiaRESUMO
The E2 subunit of pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen recognized by antimitochondrial Abs (AMA) in primary biliary cirrhosis (PBC). Recently, we replaced the lipoic acid moiety of PDC-E2 with a battery of synthetic structures designed to mimic a xenobiotically modified lipoyl hapten on a 12-aa peptide that was found within the immunodominant autoepitope of PDC-E2 and demonstrated that AMA in PBC reacted against several organic modified mimotopes as well as, or sometimes significantly better than, the native lipoyl domain. Based on this data, we immunized rabbits with one such xenobiotic organic compound, 6-bromohexanoate, coupled to BSA. One hundred percent of immunized rabbits developed AMA that have each and every characteristic of human AMAs with reactivity against PDC-E2, E2 subunit of branched chain 2-oxo-acid dehydrogenase, and E2 subunit of 2-oxoglutarate dehydrogenase complex. The rabbit AMA also inhibited enzymatic function of PDC-E2 and, importantly, binds to peptide sequences not present in the xenobiotic carrier immunogen. In contrast, BSA-immunized controls did not produce such activity. Our observation that animals immunized with a xenobiotic BSA complex produce autoantibodies that react not only with the xenobiotic, but also with mitochondrial autoantigens recognized by autoimmune PBC sera, suggests that environmental xenobiotic agents can be a risk factor for the induction of PBC.