RESUMO
Composition and amount of 45Ca2+-binding proteins in the inner membrane fraction of rat liver and Zajdela hepatoma mitochondria were determined. In the inner membrane of liver mitochondria, three major 45Ca2+-binding polypeptides: a protein of approximately 130 kDa (carbamoyl-phosphate synthetase), a glycoprotein of 43-44 kDa (previously considered as the calcium uniporter), and 29-30 kDa protein were found. These components were absent (130 kDa component) or relatively reduced (43-44 kDa and 29-30 kDa components) in the inner membrane of hepatoma mitochondria. Previously unknown low molecular mass polypeptides, having very high Ca2+-binding ability, were found in the inner membrane of hepatoma mitochondria. One of them might be the natural Ca2+-binding inhibitor of H+-ATPase.
Assuntos
Proteínas de Ligação ao Cálcio/análise , Membranas Intracelulares/química , Fígado/química , Proteínas de Membrana/análise , Mitocôndrias/química , Peptídeos/análise , Animais , Carcinoma Hepatocelular , Células Cultivadas , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
Highly sensitive techniques are needed for the quantitative determination of mitochondrial oxidative phosphorylation function in single rat hippocampal slices or isolated hippocampal subfields. We determined the oxygen consumption of single hippocampal slices or subfields applying high-resolution respirometry adapted for slice measurements and measured the redox state of mitochondrial NAD(P)H in single hippocampal slices by laser-excited fluorimetry. These methods allow the sensitive detection of two parameters of mitochondrial oxidative phosphorylation which depend on supply of substrates and respiratory chain function.
Assuntos
Hipocampo/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Espectrometria de Fluorescência/métodos , Animais , Lasers , Masculino , NADP/análise , NADP/metabolismo , Técnicas de Cultura de Órgãos , Fosforilação Oxidativa , Ratos , Ratos Sprague-DawleyRESUMO
Blood levels of carcinoembryonic antigen (CEA), alpha-fetoprotein, ferritin, ACTH. triiodothyronine and thyroxin were measured by radioimmunoassay in 217 cases of lung, hepatopancreatoduodenal and colonic cancer, 61 patients with nontumor pathology of those sites and in 37 healthy controls. CEA proved the most reliable marker of lung and colonic cancer and tumor-related mechanical jaundice, its lowest concentration in 65-100% of cancer patients exceeding the highest levels observed in controls. In the colorectal group, CEA level returned to normal after radical surgery and rose again at recurrence or distant metastases. Ferritin, cortisol and ACTH appeared less efficient.
Assuntos
Biomarcadores Tumorais/sangue , Adenocarcinoma/diagnóstico , Adulto , Idoso , Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Diagnóstico Diferencial , Neoplasias do Sistema Digestório/diagnóstico , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Prognóstico , Tuberculose Pulmonar/diagnósticoRESUMO
Fifty-four patients with viral hepatitis B (VHB) were examined at the height of the disease as were 30 patients with mechanical jaundice (MJ) of tumorous etiology and 19 normal persons. Mechanical jaundice was mainly characterized by a considerable growth of the concentration of carcinoembryonic antigen, whereas VHB by an increase of the cholylglycine level. Concomitant detection of those markers can be used in differential diagnosis of parenchymatous jaundice and mechanical jaundice of tumorous etiology. Alterations of the alpha-fetoprotein level were of no information content.
Assuntos
Antígeno Carcinoembrionário/análise , Colestase/diagnóstico , Ácido Glicocólico/sangue , Hepatite B/diagnóstico , alfa-Fetoproteínas/análise , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Colestase/sangue , Diagnóstico Diferencial , Hepatite B/sangue , Humanos , Pessoa de Meia-IdadeRESUMO
There is compelling evidence for the direct involvement of mitochondria in certain neurodegenerative disorders, such as Morbus Parkinson, FRDA (Friedreich's ataxia), ALS (amyotrophic lateral sclerosis), and temporal lobe epilepsy with Ammon's horn sclerosis. This evidence includes the direct genetic evidence of pathogenic mutations in mitochondrial proteins in inherited Parkinsonism {such as PARK6, with mutations in the mitochondrial PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced kinase 1]} and in FRDA (with mutations in the mitochondrial protein frataxin). Moreover, there is functional evidence of impairment of the respiratory chain in sporadic forms of Parkinsonism, ALS, and temporal lobe epilepsy with Ammon's horn sclerosis. In the sporadic forms of the above-mentioned neurodegenerative disorders, increased oxidative stress appears to be the crucial initiating event that affects respiratory chain function and starts a vicious cycle finally leading to neuronal cell death. We suggest that the critical factor that determines the survival of neurons in neurodegenerative disorders is the degree of mitochondrial DNA damage and the maintenance of an appropriate mitochondrial DNA copy number. Evidence for a depletion of intact copies of the mitochondrial genome has been provided in all above-mentioned neurodegenerative disorders including ALS and temporal lobe epilepsy with Ammon's horn sclerosis. In the present study, we critically review the available data.
Assuntos
Mitocôndrias/fisiologia , Doenças Neurodegenerativas/fisiopatologia , HumanosRESUMO
Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria isolated from rat liver and Zaidelja hepatoma were compared. Cumene hydroperoxide at micromolar concentrations (0.3-10 microM) prevented the closing of the permeability transition pore in the inner mitochondrial membrane and, therefore, potentiated the Ca(2+)-induced Ca2+ efflux. This response was 10-100 times greater in hepatoma mitochondria than in rat liver mitochondria. Micromolar concentrations of cumene hydroperoxide induced the death of the hepatoma cells in vitro.
Assuntos
Derivados de Benzeno/farmacologia , Cálcio/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Animais , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Cinética , Medições Luminescentes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Oxidantes/farmacologia , Permeabilidade , Ratos , Ratos Wistar , Rotenona/farmacologiaRESUMO
The polypeptide composition of liver and Zajdela hepatoma mitochondria is compared. Polypeptides of mitochondria and submitochondrial fractions (the outer and the inner mitochondrial membranes, the intermembrane and the matrix spaces of mitochondria) were separated by electrophoresis in polyacrylamide gel. The percentage content of each polypeptide was evaluated after scanning gels using a differential spectrophotometer-densitometer. Significant changes of several proteins of the hepatoma mitochondria and submitochondrial fractions as compared with normal liver preparations have been found.
Assuntos
Neoplasias Hepáticas Experimentais/química , Mitocôndrias Hepáticas/química , Proteínas de Neoplasias/química , Peptídeos/química , Animais , Masculino , Peso Molecular , Proteínas de Neoplasias/isolamento & purificação , Peptídeos/isolamento & purificação , Ratos , Ratos Wistar , Partículas Submitocôndricas/químicaRESUMO
It was shown on pubertal albino rats that the intensity of excretion with the bile of radioactive Bengal rose was different at different seasons: the maximum and minimum levels were observed in winter and summer, respectively. When the liver was affected with tetracycline, this process was suppressed especially in summer. The use of antioxidants, such as tocopherol acetate in combination with sodium selenite promoted the recovery of liver excretion function in winter, spring and autumn. In summer, the recovery was only partial.
Assuntos
Bile/metabolismo , Doença Hepática Induzida por Substâncias e Drogas , Fígado/metabolismo , Selênio/uso terapêutico , Tetraciclina/antagonistas & inibidores , Vitamina E/uso terapêutico , Animais , Hepatopatias/metabolismo , Masculino , Ratos , Rosa Bengala , Estações do Ano , Ácido SeleniosoRESUMO
In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Músculo Esquelético/enzimologia , Oftalmoplegia Externa Progressiva Crônica/enzimologia , Cianeto de Potássio/farmacologia , Difosfato de Adenosina/metabolismo , Adulto , Southern Blotting , Permeabilidade da Membrana Celular , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Cinética , Pessoa de Meia-Idade , Mitocôndrias Musculares/enzimologia , Miopatias Mitocondriais/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Oftalmoplegia Externa Progressiva Crônica/genética , Oftalmoplegia Externa Progressiva Crônica/patologia , Consumo de Oxigênio/efeitos dos fármacos , Deleção de SequênciaRESUMO
Mitochondrial function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or Kearns-Sayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. The citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a 'metabolic threshold' which can be assessed by functional studies of muscle fibres providing the link to the phenotypic expression of the mtDNA mutation in skeletal muscle.
Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Miopatias Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Adulto , Transporte de Elétrons/genética , Humanos , Síndrome de Kearns-Sayre/genética , Síndrome de Kearns-Sayre/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Pessoa de Meia-Idade , Mutação , NADH NADPH Oxirredutases/metabolismo , Oftalmoplegia Externa Progressiva Crônica/genética , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Consumo de OxigênioRESUMO
Zajdela hepatoma mitochondria were able to accumulate two to five times more Ca2+ than rat liver mitochondria before the permeability transition was induced. Pulses of Ca2+ were given in series to determine the Ca2+ threshold by recording changes in [Ca2+] and membrane potential, the permeability transition causing the release of accumulated Ca2+ and collapse of the membrane potential. Hepatoma mitochondria had lower Ca2+ efflux rates, higher net Ca2+ uptake rates and lower phosphorylation rates than liver mitochondria. Since the differences in regard to induction of the permeability transition might be due to higher expression of the Bcl-2 protein in hepatoma cells than in hepatocytes, the transcription of Bcl-2 and the proteins reacting with a Bcl-2 polyclonal antiserum were estimated by Northern and Western blotting, respectively. Hepatoma cells had two Bcl-2 specific mRNA bands of 7 and 2.4 kb, and substantial amounts of the Bcl-2 protein, whereas in liver cells and mitochondria these were not detected. Both cell lines had a reactive band at 19-20 kDa, and hepatocytes a small band at 31-32 kDa. Bcl-2 antibodies stimulated the permeability transition potently in hepatoma mitochondria.
Assuntos
Cálcio/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Sequência de Bases , Primers do DNA , Neoplasias Hepáticas Experimentais/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
A study of the blood levels of gonadotropic and steroid hormones in 321 breast cancer patients has shown that the basal levels of gonadotropin concentration in them exceed the control values (p less than 0.05); those of the follicle-stimulating hormone--in 54.1% of patients at reproductive age and in menopause less than 5 years and in 33.8% of patients in menopause over 5 years; those of luteotropin--in 50 and 93.5% of patients, respectively. Low basal levels of estradiol and progesterone were found more than 70% of breast cancer patients. A significant decrease in the level of the above hormones (p less than 0.05) was noted after polychemotherapy. An increase in the levels of corticotropin (in 54.5-65.2% of patients) and cortisol (in 81.6-84.3% of patients) was noted with progression of breast cancer. Data on the above hormones can be used as a diagnostic and prognostic test.
Assuntos
Hormônio Adrenocorticotrópico/sangue , Neoplasias da Mama/sangue , Gonadotropinas Hipofisárias/sangue , Progesterona/sangue , Adulto , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Hormônio Luteinizante/sangue , Pessoa de Meia-Idade , Prognóstico , RadioimunoensaioRESUMO
Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE.