Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 154-169).

BEZERRA, Lady Clarissa Brito da Rocha. Purificação, caracterização bioquímica e potencial quimiopreventivo de um novo inibidor de quimotripsina de sementes de Enterolobium contortisiliquum (Vell.) Morong. 2014. 110 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-02T12:04:52Z No. of bitstreams: 1 2014_tese_lcbrbezerra.pdf: 2595573 bytes, checksum: 6f4356b55ad7ccae8f05a1adf548f991 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-05T20:31:04Z (GMT) No. of bitstreams: 1 2014_tese_lcbrbezerra.pdf: 2595573 bytes, checksum: 6f4356b55ad7ccae8f05a1adf548f991 (MD5) / Made available in DSpace on 2016-09-05T20:31:04Z (GMT). No. of bitstreams: 1 2014_tese_lcbrbezerra.pdf: 2595573 bytes, checksum: 6f4356b55ad7ccae8f05a1adf548f991 (MD5) Previous issue date: 2014 / Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 °C – EcCI; from 37 to 60 °C – EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM – EcCI; from 1 to 100 mM – EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. / Inibidores de proteases são proteínas que inibem a atividade catalítica de enzimas, sendo bastante comuns em sementes de plantas. As proteases desempenham papéis centrais no desenvolvimento de muitas doenças. O controle de sua atividade realizado por inibidores de proteases despertou o interesse sobre estas moléculas como agentes quimiopreventivos, especialmente sobre o câncer. Os efeitos anticarcinogênicos das sementes de leguminosas presentes comumente na dieta, bem como de espécies vegetais subexploradas, têm sido extensivamente investigados. O objetivo deste trabalho foi purificar, caracterizar bioquimicamente e avaliar o potencial quimiopreventivo in vitro de inibidores de proteases de sementes de Enterolobium contortisiliquum, utilizando como modelo células de adenocarcinoma colorretal humano. Foram purificados dois inibidores de proteases denominados EcCI e EcTI. Através da sequência N-terminal, EcCI foi identificado como um inibidor da família Kunitz. EcTI foi identificado, por meio de peptide mass fingerprinting e por análise da sequência N-terminal, como aquele descrito previamente na literatura (NCBI Protein BLAST; número de acesso: sp|P86451.1|ITRY_ENTCO). Suas massas moleculares determinadas por SDS-PAGE e espectrometria de massas são, respectivamente, 18,5 kDa e 19.710,4 Da (EcCI); 20,2 kDa e 19.813,22 Da (EcTI). Ambos os inibidores são formados de duas subunidades proteicas e apresentam isoformas cujos pI’s são ácidos, entre 5 e 6. Frente a quimotripsina, EcCI é um inibidor não competitivo e apresenta ki de 8 x 10-8 M, enquanto EcTI é inibidor competitivo com ki de 48 x 10-8 M. Frente à tripsina, EcTI apresenta inibição não competitiva e ki de 2,8 x 10-8 M. EcCI e EcTI apresentam atividade inibitória de quimotripsina alta (93%) e moderada (38%), respectivamente. Apenas EcTI foi capaz de inibir a tripsina (100%). EcCI e EcTI apresentam alta atividade inibitória de elastase neutrofílica (85 e 75%, respectivamente). Os dois inibidores inibiram sutilmente a elastase pancreática (ca. de 10%) e nenhum foi capaz de inibir papaína e bromelaína. Os dois inibidores apresentam alta estabilidade à variação de temperatura (de 37 a 70 °C para EcCI e de 37 a 60 °C para EcTI), pH (2 a 12) e concentração de DTT (de 1 a 10 mM para EcCI e de 1 a 100 mM para EcTI). EcCI e EcTI inibiram a proliferação de células de adenocarcinoma colorretal humano da linhagem HT29 com CI50 de 35,5 e 20,4 x 10-6 M, respectivamente, indicando que esses inibidores apresentam bom potencial quimiopreventivo e, portanto, são moléculas bastante interessantes do ponto de vista biotecnológico.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. / Inibidores de proteases sÃo proteÃnas que inibem a atividade catalÃtica de enzimas, sendo bastante comuns em sementes de plantas. As proteases desempenham papÃis centrais no desenvolvimento de muitas doenÃas. O controle de sua atividade realizado por inibidores de proteases despertou o interesse sobre estas molÃculas como agentes quimiopreventivos, especialmente sobre o cÃncer. Os efeitos anticarcinogÃnicos das sementes de leguminosas presentes comumente na dieta, bem como de espÃcies vegetais subexploradas, tÃm sido extensivamente investigados. O objetivo deste trabalho foi purificar, caracterizar bioquimicamente e avaliar o potencial quimiopreventivo in vitro de inibidores de proteases de sementes de Enterolobium contortisiliquum, utilizando como modelo cÃlulas de adenocarcinoma colorretal humano. Foram purificados dois inibidores de proteases denominados EcCI e EcTI. AtravÃs da sequÃncia N-terminal, EcCI foi identificado como um inibidor da famÃlia Kunitz. EcTI foi identificado, por meio de peptide mass fingerprinting e por anÃlise da sequÃncia N-terminal, como aquele descrito previamente na literatura (NCBI Protein BLAST; nÃmero de acesso: sp|P86451.1|ITRY_ENTCO). Suas massas moleculares determinadas por SDS-PAGE e espectrometria de massas sÃo, respectivamente, 18,5 kDa e 19.710,4 Da (EcCI); 20,2 kDa e 19.813,22 Da (EcTI). Ambos os inibidores sÃo formados de duas subunidades proteicas e apresentam isoformas cujos pIâs sÃo Ãcidos, entre 5 e 6. Frente a quimotripsina, EcCI à um inibidor nÃo competitivo e apresenta ki de 8 x 10-8 M, enquanto EcTI à inibidor competitivo com ki de 48 x 10-8 M. Frente à tripsina, EcTI apresenta inibiÃÃo nÃo competitiva e ki de 2,8 x 10-8 M. EcCI e EcTI apresentam atividade inibitÃria de quimotripsina alta (93%) e moderada (38%), respectivamente. Apenas EcTI foi capaz de inibir a tripsina (100%). EcCI e EcTI apresentam alta atividade inibitÃria de elastase neutrofÃlica (85 e 75%, respectivamente). Os dois inibidores inibiram sutilmente a elastase pancreÃtica (ca. de 10%) e nenhum foi capaz de inibir papaÃna e bromelaÃna. Os dois inibidores apresentam alta estabilidade à variaÃÃo de temperatura (de 37 a 70 ÂC para EcCI e de 37 a 60 ÂC para EcTI), pH (2 a 12) e concentraÃÃo de DTT (de 1 a 10 mM para EcCI e de 1 a 100 mM para EcTI). EcCI e EcTI inibiram a proliferaÃÃo de cÃlulas de adenocarcinoma colorretal humano da linhagem HT29 com CI50 de 35,5 e 20,4 x 10-6 M, respectivamente, indicando que esses inibidores apresentam bom potencial quimiopreventivo e, portanto, sÃo molÃculas bastante interessantes do ponto de vista biotecnolÃgico.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A great number of plant species produce latex, including Apocynacea, Sapotacea, Papaveracea and Euphorbiaceae. Thevetia peruviana (Pers.) Schum is a laticifer shrub belonging to Apocynaceae family popularly known as âchapÃu-de-napoleÃoâ. It is very limited the proteomic information about this specie. Thus, a proteomic analysis of protein fraction (TpLP) from T. peruviana latex was performed using two dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86%) were identified, including storage proteins, peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. This protein fraction showed strong proteolytic activity at pH 5.0 which was increased in the presence of low concentrations of the reducing agent DTT. The inhibition this activity in the presence of specific inhibitors E-64 and IAA and the high activity with BANA showed the predominance of cysteine peptidases in latex. A cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme was inhibited by E-64 and iodoacetamide (IAA) and follows the Michaelis-Menten kinetics, showing high affinity for azocasein, with Km value of 17.6 uM, exhibiting an optimal pH and temperature of 5.0-6.0 and 25-37 ÂC, respectively. Two-dimensional gel electrophoresis and mass spectrometry revealed that peruvianin-I (100 kDa) possesses a pI of 4.0 and five subunits (20 kDa). The N-terminal amino acid sequence of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) was similar to that of germin or germin-like proteins. High-resolution images from atomic force microscopy indicated the possible hexameric structure of peruvianin-I, which is organized as a trimer of dimers that form a central channel. TpLP and peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects on the spore germination of Fusarium solani. This study showed that T. peruviana latex are a rich source of pathogenesis-related proteins, including cysteine peptidases. Interestingly, these peptidases exhibit different structural and biochemical characteristics that may be related to their specific physiological functions. / Um grande nÃmero de espÃcies vegetais produzem lÃtex, incluindo Apocynacea, Sapotacea, Papaveracea e Euphorbiaceae. Thevetia peruviana (Pers.) Schum à um arbusto laticÃfero pertencente à famÃlia Apocynaceae, popularmente conhecido como "chapÃu-de-NapoleÃo". SÃo bastante limitadas as informaÃÃes proteÃmicas sobre esta espÃcie. Por tanto, uma anÃlise proteÃmica da fraÃÃo proteica (TpLP) do lÃtex de T. peruviana foi realizada a partir de eletroforeses bidimensionais e espectrometria de massas. Um total de 33 proteÃnas (86%) foi identificado, incluindo proteÃnas de reserva, inibidor de peptidase, peptidases cisteÃnicas, peroxidases e osmotinas. As proteÃnas desta fraÃÃo apresentaram uma forte atividade proteolÃtica no pH 5,0, a qual foi aumentada na presenÃa de baixas concentraÃÃes do agente redutor DTT. A inibiÃÃo desta atividade na presenÃa dos inibidores especÃficos E-64 e IAA e a alta atividade com o substrato BANA evidenciou a predominÃncia de peptidases cisteÃnicas no lÃtex. Uma peptidase cisteÃnica denominada peruvianina-I, foi purificada a partir do lÃtex atravÃs de um Ãnico passo cromatogrÃfico envolvendo filtraÃÃo em gel. A enzima foi inibida por E-64 e iodoacetamida (IAA) e seguiu a cinÃtica de Michaelis-Menten, apresentando alta afinidade à azocaseÃna, com um valor de Km de 17,6 ÂM, exibindo pH e temperatura Ãtimos de 5,0-6,0 e 25-37 ÂC, respectivamente. A peruvianina-I nÃo foi reconhecida por anticorpos anti-papaÃna. As Eletroforeses bidimensionais e a espectrometria de massas revelaram que a peruvianina-I (100 kDa) possui um pI de 4,0 e cinco subunidades (20 kDa). A sequÃncia de aminoÃcidos N-terminal da peruvianina-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) mostrou similaridade à germinas ou âgermin-like proteinsâ. Imagens de alta resoluÃÃo a partir da microscopia de forÃa atÃmica indicaram uma possÃvel estrutura hexamÃrica da peruvianina-I, que està organizada como um trÃmero de dÃmeros, formando um canal central. TpLP e Peruvianina-I nÃo exibiram atividade de oxalato oxidase e superÃxido dismutase ou efeitos antifÃngicos sobre a germinaÃÃo de esporos de Fusarium solani. Este estudo mostrou que o lÃtex de T. peruviana à uma fonte rica em proteÃnas relacionadas à patogÃnese, incluindo peptidases cisteÃnicas. Curiosamente, estas peptidases apresentam caracterÃsticas estruturais e bioquÃmicas diferentes, que podem estar relacionadas com as suas funÃÃes fisiolÃgicas especÃficas.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23433号 / 農博第2464号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5348(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 谷 史人, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Antisera to the basement membrane proteins laminin, fibronectin and type IV collagen were prepared, characterized and rendered monospecific by appropriate treatments. Methods were developed to allow the use of these antisera for inmunohistochemical staining of sections of tissue which had been preserved by several methods. In particular, the use of protease digestion {"unmasking") to allow staining of formalin-fixed, paraffin-embedded material was studied. The presence and intensity of staining was found to be highly dependent on the protease and the conditions of digestion, the type of basement membrane, and whether the tissue was normal or neoplastic. The distributions of the three proteins were studied in normal colorectal mucosa and in colorectal adenocarcinoma. A detailed retrospective study was made of the distribution of laminin in 158 cases of rectal carcinoma. Tumours fell into two groups: those which showed linear basement membrane-like laminin staining (laminin positive) and those which did not (laminin negative). Patients with laminin positive tumours had a reduced incidence of distant metastasis and an increased 5 yr survival rate; these correlations were statistically highly significant. Carparison by multivariate analysis with other widely used prognostic markers indicated that laminin status has considerable potential for use as a prognostic marker in the management of such patients. The antisera were also used in a study of the cellular origin and biosynthesis of basement membrane proteins in three systems. In a basement membrane-producing murine tumour, intracellular staining was seen, but it was found that different methods of tissue preparation and unmasking drastically affected the apparent distributions of the three antigens. In the developing rat intestine, no evidence was seen of basement membrane synthesis by the intestinal epithelial cells. However in isolated rat intestinal epithelial cells, some evidence was found for synthesis of laminin and fibronectin, but not type IV collagen.

No description available.

No description available.

Colon cancer occurs in over 150,000 men and women in the United States each year and is fatal about one third of the time. There are many well characterized genetic transformations which commonly occur during colon carcinogenesis, including mutations in the Adenometous Polyposis Coli (APC), Kirsten RAS (K-RAS) and p53 genes among many others. There are also many alterations in gene and protein expression which are not yet completely elucidated. We have identified kallikrein 6 (KLK6) as a gene whose expression is dependent on cancer associated proteins such as K-RAS, SRC and caveolin-1 (CAV-1). KLK6 is a member of the kallikrein protein family which consists of 15 secreted serine proteases. Like other types of proteases, kallikreins have been demonstrated to play a role in cancer progression and they hold promise as potential cancer biomarkers. The up-regulation of KLK6 was first identified by performing a microarray analysis on a mutant K-RAS transfected Caco2 cells. The activated K-RAS transfected cells expressed significantly more KLK6 than the mock transfected controls. Pathways downstream of K-RAS were found to induce KLK6 gene expression, including the p42/44 MAPK and the PI3-K/AKT pathways. Caveolae, plasma membrane associated structures, and their principle protein component, CAV-1, positively influence both KLK6 gene expression and KLK6 protein secretion in HCT116 colon cancer derived cells. Finally, it is shown that KLK6 increases the invasive potential of cells through laminin and Matrigel. Because KLK6 plays a role in cancer progression it may serves as a novel therapeutic target. Additionally, KLK6 holds potential for use as a serum biomarker for multiple types of cancer, including colon cancer for colon and other types of cancer.

No description available.