RESUMO
Epidemiological studies suggest that soy consumption may provide a protection in the development and progression of atherosclerosis. It is under debate, however, whether the soy isoflavones or other compounds are the "active principle". As apoptosis is a driving force in the process of atherosclerosis, we tested whether a soy extract or a combination of the two predominant isoflavones genistein and daidzein, in concentrations as found in the extract, exert similar or different effects on apoptosis in EA.hy 926 endothelial cells after exposure to the endothelial stressor homocysteine. Plasma membrane disintegration and nuclear fragmentation served as relevant apoptosis markers. To assess whether the extract and the genistein/daidzein mixture differently affect cellular target proteins changed in amount by homocysteine treatment, proteome analysis was performed by two-dimensional gel-electrophoresis and peptide mass fingerprinting of regulated protein spots. Homocysteine induced apoptosis in the cells, and both extract and genistein/daidzein inhibited apoptosis to a comparable extent. Whereas the extract prevented for 10 proteins the changes in expression levels as caused by homocysteine, the genistein/daidzein mixture reversed the homocysteine effects on the proteome for 13 proteins. The cytoskeletal protein matrin 3 and a U5 snRNP-specific 40-kDa protein were the only protein entities where both extract and genistein/daidzein reversed the homocysteine-induced changes in a common way. In conclusion, our studies provide evidence that an isoflavone containing soy extract and isolated isoflavones, despite similar effects on inhibition of homocysteine-induced apoptosis in endothelial cells, affect a quite different spectrum of cellular target proteins.
Assuntos
Células Endoteliais/efeitos dos fármacos , Glycine max/química , Homocisteína/farmacologia , Isoflavonas/farmacologia , Extratos Vegetais/farmacologia , Proteoma/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Catepsina D/análise , Linhagem Celular , Eletroforese em Gel Bidimensional , Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Homocisteína/antagonistas & inibidores , Isoflavonas/isolamento & purificação , Lamina Tipo A/análise , Proteínas Nucleares/análise , RNA Mensageiro/análise , Proteínas de Ligação a RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Canal de Ânion 1 Dependente de Voltagem/análiseRESUMO
Studies were undertaken to find out the effects of low frequency pulsed electromagnetic field (PEMF) in adjuvant induced arthritis (AIA) in rats, a widely used model for screening potential therapies for rheumatoid arthritis (RA). AIA was induced by an intradermal injection of a suspension of heat killed Mycobacterium tuberculosis (500 mug/0.1 ml) into the right hind paw of male Wistar rats. This resulted in swelling, loss of body weight, increase in paw volume as well as the activity of lysosomal enzymes viz., acid phosphatase, cathepsin D, and beta-glucuronidase and significant radiological and histological changes. PEMF therapy for arthritis involved optimization of three significant factors, viz., frequency, intensity, and duration; and the waveform used is sinusoidal. The use of factorial design in lieu of conventional method resulted in the development of an ideal combination of these factors. PEMF was applied using a Fransleau-Braunbeck coil system. A magnetic field of 5 Hz x 4 muT x 90 min was found to be optimal in lowering the paw edema volume and decreasing the activity of lysosomal enzymes. Soft tissue swelling was shown to be reduced as evidenced by radiology. Histological studies confirmed reduction in inflammatory cells infiltration, hyperplasia, and hypertrophy of cells lining synovial membrane. PEMF was also shown to have a membrane stabilizing action by significantly inhibiting the rate of release of beta-glucuronidase from lysosomal rich and sub-cellular fractions. The results indicated that PEMF could be developed as a potential therapy in the treatment of arthritis in humans.
Assuntos
Artrite Experimental/radioterapia , Campos Eletromagnéticos , Fosfatase Ácida/análise , Fosfatase Ácida/efeitos da radiação , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide , Peso Corporal , Catepsina D/análise , Catepsina D/efeitos da radiação , Diclofenaco/uso terapêutico , Edema/imunologia , Edema/patologia , Edema/radioterapia , Pé/patologia , Pé/efeitos da radiação , Glucuronidase/análise , Glucuronidase/efeitos da radiação , Membro Posterior/patologia , Membro Posterior/efeitos da radiação , Hiperplasia , Hipertrofia , Lisossomos/enzimologia , Lisossomos/efeitos da radiação , Masculino , Mycobacterium tuberculosis/imunologia , Ratos , Ratos Wistar , Membrana Sinovial/patologia , Membrana Sinovial/efeitos da radiaçãoRESUMO
Chronic administration of Senna occidentalis seeds induces an experimental toxic myopathy characterized by skeletal muscle fibers atrophy, decrease in histochemical activity of cytochrome oxidase, and increase of the acid phosphatase activity in muscle fibres at the light microscopic level. The mechanisms that lead to the increase of this lysosomal enzyme activity are not known and could be related to other biochemical disturbs than the mitochondrial function impairment. The main aim of the present study is to localize the acid phosphatase activity using a cytochemical method at transmission electron microscopy level and to quantify cathepsin D in muscle of rats chronically intoxicated with Senna occidentalis seeds by immunoblotting. Acid phosphatase was observed in lysosomes and over profiles of some organelles apparently not involved by lysosomal membrane. In addition immunoblotting demonstrated a decrease in the content of the precursor and of the mature form of cathepsin D in samples of muscles and liver of intoxicated animals. We concluded that there is a selective increase in acid phosphatase activity in muscle--and maybe in other tissues--of animals intoxicated with Senna occidentalis, that can be related to the skeletal muscle atrophy and the intense decrease in weight gain of these animals. Further studies should be performed to establish the mechanisms of selectivity in increase of lysosomal enzymes in different situations and pathological states.
Assuntos
Fosfatase Ácida/análise , Catepsina D/análise , Lisossomos/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Sementes/intoxicação , Extrato de Senna/intoxicação , Animais , Biomarcadores/análise , Western Blotting , Microscopia Eletrônica de Transmissão e Varredura , RatosRESUMO
Proteinase activity is increased in psoriatic epidermis. To elucidate the involvement of enzymes in psoriatic epidermis, the expression of cathepsins, L, B and D was investigated by Western blotting and immunohistological studies. Normal epidermis contained abundant inactive precursors (39 kDa) of cathepsins L and B and an inactive intermediate form (45 kDa) of cathepsin D. Cathepsin L in psoriasis was processed to a variable extent from the precursor to a single-chain form (30 kDa) and a mixture of single- and heavy-chain (25 kDa) forms of the active mature enzyme, corresponding to the immunohistological staining patterns 'diffuse dense', 'small granular', and unevenly distributed 'condensed granular'. Cathepsin B showed a mixture of precursor form (39 kDa) and single-chain (30 kDa) forms and was expressed as a 'diffuse dense' staining pattern in the mid-spinous layer and as a 'condensed' pattern in the upper spinous and granular layers. Cathepsin D was processed to the heavy-chain (31 kDa) form of activated mature enzyme with small granular staining and a mixture of heavy-chain and degraded protein (28 kDa) with larger and more condensed granular staining. The distribution patterns of 'small granular' cathepsin L, and of cathepsins B and D expression in suprabasal keratinocytes were very similar to that of involucrin. After complete clinical resolution of psoriasis by 8-methoxypsoralen plus UVA treatment, the expression of the three cathepsins was normalized. These results suggest that cathepsins L, B and D in forms activated to a variable extent may be involved in the pathology of psoriasis.
Assuntos
Catepsina B/análise , Catepsina D/análise , Catepsinas/análise , Cisteína Endopeptidases/análise , Endopeptidases , Precursores Enzimáticos/análise , Psoríase/enzimologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Catepsina L , Criança , Epiderme , Proteínas Filagrinas , Humanos , Proteínas de Filamentos Intermediários/análise , Pessoa de Meia-Idade , Terapia PUVA , Precursores de Proteínas/análise , Psoríase/tratamento farmacológicoRESUMO
OBJECTIVE: To study the expression of Cathepsin D (Cath D) and CA 125 antigens and ER and PR receptors on freshly obtained surgical specimens of ovarian carcinomas and their relationship with menopausal status, tumor histology, primary tumor size and lymph node invasion. METHODS: The tumors obtained from 100 women were measured and cut in half. The cut surface of one half was pressed against glass slides which were air dried and stained using the Avidin-Biotin peroxidase method for Cath D and CA 125 antigens. The slides were viewed under the light microscope for the characteristic brown granules in the cytoplasm or membrane of the malignant cells. The other half of the tumor was subjected to routine histological examination and part used for the demonstration of ER and PR receptors. The results were analyzed using chi 2 analysis. RESULTS: Cath D positivity was as common as CA 125 positivity. Cath D positivity is more frequently associated with serous carcinomas than with others. No relationship was observed between ER/PR positivity and Cath D or CA 125 positivity. CONCLUSION: The high incidence of Cath D positivity makes it a possible complementary method for following up ovarian carcinoma patients especially those who are CA 125 negative.
Assuntos
Antígeno Ca-125/análise , Catepsina D/análise , Neoplasias Ovarianas/química , Cistadenocarcinoma Seroso/química , Cistadenocarcinoma Seroso/patologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Menopausa/fisiologia , Neoplasias Ovarianas/patologia , Prognóstico , Receptores de Estrogênio/análise , Receptores de Progesterona/análiseRESUMO
The oestrogen induced pS2 protein was measured in the cytosol of 446 breast cancer samples by an immunoradiometric assay. The relationships between pS2 and several clinical and biological parameters were evaluated. pS2 was not correlated to age, pT and nodal status, while it was higher in pre- than in peri- and post-menopausal women. A statistically significant positive association was found between pS2 and ER, PgR and cathepsin D. However, the frequency of pS2 negative values in ER+ (25.6%), PgR+ (21.7%) and cathepsin D-(19.0%) cases suggests that pS2 provides information independent of the above parameters in a fairly high percentage of patients. The prognostic role of pS2 was evaluated in 267 cases (follow up time 24-102 months). pS2+ showed longer RFS (P = 0.016) and OS (P = 0.004) than pS2-. pS2+ cases were significantly associated with a better prognosis in N+ but not in N- cases. Multivariate analysis showed that pS2 is an independent prognostic factor being the second most effective indicator for OS after nodal status and the third for RFS after nodal status and cathepsin D. From the present findings, we conclude that pS2 probably provides additional biological information to steroid receptor status and cathepsin D in patients with primary breast cancer.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Proteínas , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Adulto , Idoso , Análise de Variância , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Catepsina D/análise , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Estrogênios , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Humanos , Metástase Linfática , Menopausa , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Radioimunoensaio , Recidiva , Análise de Sobrevida , Tamoxifeno/uso terapêutico , Fatores de Tempo , Fator Trefoil-1 , Proteínas Supressoras de TumorRESUMO
The regional distribution and cellular localization of cathepsin D immunoreactivity was demonstrated at the light microscopic level in the CNS of rat and man by use of unlabelled immunoenzyme technique. A wide but uneven distribution was substantiated for the rat brain. Furthermore, we present evidence that antiserum produced against rat liver enzyme is capable of recognizing cathepsin D in human brain.