RESUMO
Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0±1.1 µM(-1)s(-1) and 30.0±0.5 µM(-1)s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.
Assuntos
Catepsina L/química , Proteínas de Insetos/química , Saccharum/parasitologia , Gorgulhos/enzimologia , Gorgulhos/fisiologia , Animais , Catepsina L/metabolismo , Sistema Digestório/química , Sistema Digestório/enzimologia , Fenômenos Fisiológicos do Sistema Digestório , Proteínas de Insetos/metabolismo , Cinética , Gorgulhos/químicaRESUMO
BACKGROUND: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. RESULTS: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. CONCLUSION: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.
Assuntos
Catepsina B/antagonistas & inibidores , Cistatinas/genética , Cistatinas/farmacologia , Embaralhamento de DNA/métodos , Biblioteca Gênica , Modelos Moleculares , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual/genética , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de SequênciaRESUMO
Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P<0.001) is accompanied by an altered protein distribution within the cell. In papillary carcinomas, P27(KIP1) is preferentially cytoplasmic as opposed to goiter or normal thyroid tissue, where P27(KIP1) is preferentially located in the nucleus. The exploitation of the data presented here could contribute to the understanding of the molecular events related to thyroid diseases and gives support to the notion that common molecular events might be related to the frequent observation of areas of papillary carcinomas in the gland of patients with goiter.
Assuntos
Carcinoma Papilar/genética , Perfilação da Expressão Gênica , Bócio/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Proteínas de Transporte/análise , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de TecidosRESUMO
O câncer de mama é a neoplasia mais freqüente que acomete as mulheres do mundo todo. Embora exista um considerável progresso no diagnóstico precoce e no tratamento desse tipo de tumor, a mortalidade por essa doença ainda permanece relativamente alta. Tumores de mama com aparentemente o mesmo tipo histológico e estadiamento variam amplamente em sua resposta às terapias disponíveis. A identificação de genes diferencialmente expressos no câncer de mama em relação ao tecido ao normal é de extrema importância para o entendimento da biologia do processo de tumorigênese e na identificação de novos potenciais marcadores de diagnóstico, prognóstico e no desenvolvimento de alvos terapêuticos. Neste trabalho, a análise do perfil da expressão gênica por meio de utilização dos dados disponibilizados pelo banco de SAGE resultou na identificação de 175 genes, os quais apresentaram um aumento da expressão de pelo menos quatro vezes em relação ao tecido normal. Levando-se em consideração a função celular, o processo biológico em que o gene está envolvido, a disponibilidade de anticorpos comerciais, e a localização celular, 19 genes (ALPPL2, BIRC5, CAD, CELSR2, CTSF, DGCR6, EFNA4, EI24, FN3KR3, GATA3, GATA4, HMG20B, PTPRF, QSCN6, SCARB2, STARD10, ZC3HDC1, KIAA0141 e KIF22) foram selecionados para terem sua expressão diferencial avaliada por meio de RT-PCR em tempo real em vinte e duas amostras de tecidos tumorais de mama, em estágio inicial de desenvolvimento da doença (menores do que dois centímetros) e em uma mistura de amostras não tumorais. A quantificação da expressão desses genes revelou que dez deles (ALPPL2, CELSR2, FN3KR3, GATA3, GATA4, HMG20B, QSCN6, SCARB2, STARD10 e ZC3HDC1) apresentaram-se com aumento da expressão de pelo menos duas vezes em mais de 25% das amostras testadas, em relação à mistura de tecidos normais. Os genes ALPPL2, CELSR2, GATA 3 e HMG20B, apresentaram aumento de expressão em mais de 50% das amostras analisadas. A correlação entre o aumento de expressão dos genes CELSR2, GATA3, GATA4 e KIAA0141 e a positividade para a imunodetecção de receptor de estrógeno se mostrou estatisticamente significativa (com valores de p iguais a: 0,0270; 0,0223 e 0,0146 e 0,0325, respectivamente). A associação entre a expressão do gene CELSR2 e a positividade para a imunodetecção do receptor de progesterona se mostrou estatisticamente significativa (p=0,0278). Os resultados apresentados no presente estudo demonstraram que a estratégia de escolha de genes a partir da análise de banco de dados públicos disponíves on line foi adequada, por terem sido validados como apresentando aumento de expressão por meio de RT-PCR em tempo real mais da metade dos genes selecionados nas amostras analisadas. Embora os resultados obtidos forneçam indícios de uma provável correlação entre o aumento da expressão dos genes e o processo de tumorigênese de mama, um estudo mais amplo, com um maior número de amostras será necessário para a confirmação dos achados.
Breast cancer is one o f the most common malignancies and the main cause of death among women. Although there have been a considerable progress on early diagnosis and treatment, the rates of mortality due to breast cancer among women remain still high. Breast cancer is characterized by an important histoclinical heterogeneity, which makes difficult the selection of the most appropriate treatment for each case. The identification of genes differentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and in the identification of new potential diagnostic and prognostic markers and in the development of therapeutic targets. Expression profiling using the public serial analysis of gene expression (SAGE) database resulted in the identification of 175 genes, which were at least 4 fold overexpressed in breast cancer SAGE libraries when compared to the normal breast SAGE libraries. Considering the cell function, biological processes in which the genes are involved, cell localization and the commercial availability of antibodies, 19 candidate genes (ALPPL2, BIRC5, CAD, CELSR2, CTSF, DGCR6, EFNA4, E/24, FN3KR3, GATA3, GATA4, HMG20B, PTPRF, QSCN6, SCARB2, STARDJO, ZC3HDCI, KJAA0141 e KIF22) were selected to have the differential expression tested by Real Time RT-PCR in 22 breast cancer samples smaller than two centimeters compared to a pool of normal breast tissues. Eleven of these genes (ALPPL2 , CELSR2, FN3KR3, GATA3 , GATA4, HMG20B, PTPRF, QSCN6, SCARB2, STARDIO and ZC3HDCJ) showed a significant overexpression o f at least 2 fold in more than 25% o f the breast tumor samples tested. The genes ALPPL2, CELSR2, GATA 3 e HMG20B were found overexpressed in more than 50o/o of the samples analyzed. The statistical analysis showed a significant correlation between the overexpression of the genes CELSR2 , GATA3, GATA4 and KIAAOI41 and the estrogen receptor immunodetection (the p values were 0.0270; 0.0223 , 0.0146, and 0.0325 , respectively). The association between the overexpression of CELSR2 and the progesterone receptor immunodetection was also statistically significant (p=0.0278). These preliminary results suggest that data mining of SAGE database seems to be an appropriate approach to identify differentially expressed genes in breast tumors. The results also suggest that these genes may be implicated in breast tumorigenesis, but further studies with larger series are needed to confirm our findings.