Navegando por Palavras-chave "Kininogens"
Agora exibindo 1 - 3 de 3
Resultados por página
Opções de Ordenação
- ItemSomente MetadadadosEstudo da ação da pepsina sobre cininogênio plasmático equino(Universidade Federal de São Paulo (UNIFESP), 1978) Toffoletto, Odaly [UNIFESP]; Stella, Regina Celes de Rosa [UNIFESP]
- ItemAcesso aberto (Open Access)Influência dos glicosaminoglicanos na interação das proteínas do sistema calicreína-cinina plasmático na superfície celular(Universidade Federal de São Paulo (UNIFESP), 2006-02-22) Melo, Kátia Regina Brasil [UNIFESP]; Motta, Guacyara da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The kallirein-kinin system comprises low and high molecular weight kininogens (LK and HK), tissue and plasma kallikreins, and was first recognized as a plasma and tissue proteolytic system responsible for bradykinin (BK) release. The main function of kininogens has been described as precursors of BK, a peptide which is not active unless it is released from kininogens. The plasma kallikrein-kinin system is related to vascular biology and it is involved and interacts with different systems such as coagulation, fibrinolysis, complement and renin-angiotensin systems. HK is a multidomain protein with antithrombin, antiadhesive and profibrinolytic activities. Recent studies have revealed that HK free from BK (HKa) inhibits angiogenesis while BK promotes angiogenesis. In blood and vascular cells some different proteins have been described as HK binding receptors: Mac-1 integrin (aMb-2 or CD11b/CD18) on granulocytes, the receptor that binds to the globular “heads” of C1q (p33/gC1qR), cytokeratin 1 (CK1) and urokinase plasminogen activator receptor (uPAR) on HUVECs, and glycoprotein Ib-IX-V and thrombospondin on platelets. More recently both heparan sulfate and chondroitin sulfate have been described as HK putative receptors. The aim of this work was to study the influence of glycosaminoglycans (GAGs) on HK binding to cell surface and lysates and its processing using two different Chinese hamster ovary cell lines, CHO-wild type (CHO-K1) and CHO-745 (mutant) which is deficient in the synthesis of proteoglycans (PGs) due to lack of activity of xylosyl transferase. Biotin-HK (b-HK) bound to CHO-K1 cell surface in Zn2+ dependent manner. The b-HK level of binding was much higher on cell surfaces comparing to lysates from both cell lines and this binding was not reversible by 20 fold molar excess of unlabeled HK at both CHO-K1 and CHO-745. At 37°C the Bmax values for both CHO-K1 and CHO-745 are very similar but the Kdap for HK binding to CHO-K1 is 4.4 times higher comparing to CHO-745 suggesting that the presence of PGs/GAGs impair HK interaction with other putative receptors on cell surface. Considering the Bmax determinations at 4°C on CHOFor K1 it is 3.0 times lower comparing to Bmax at 37°C and on CHO-745 the Bmax value at 4°C is 1.4 times lower comparing to Bmax at 37°C. The difference of Bmax values at both temperatures for each cell line suggests that HK is internalized and confocal microscopy experiments detected HK internalized only on CHO-K1. Plasma prekallikrein (PK) bound to both cell lines in the presence of HK; however, in the absence of HK PK also bound only to CHO-K1 cell surface suggesting that PGs/GAGs may interact with PK. Once HK/PK complex is assembled on cell surface and lysates of both cell lines PK turns to plasma kallikrein (huPK) and both prolylcarboxypeptidase (PRCP) and cathepsins may participate in this process. The intact HK assembled to cell surface and lysates of both cell lines and in the absence of PK/huPK is cleaved in one site and probably PRCP or tissue kallikrein may process HK in this circumstances. The cell membrane interaction of plasma kallikrein-kinin system proteins through binding and internalization can result in liberation of HK fragments and huPK pericellular activity which may influence in pathological and physiological process.
- ItemSomente MetadadadosInteração do cininogênio humano de alta massa molecular com a superfície celular: envolvimento dos processos de endocitose e proteólise(Universidade Federal de São Paulo (UNIFESP), 2010-06-30) Melo, Kátia Regina Brasil [UNIFESP]; Motta, Guacyara da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Angiogenesis and hemostasis are among the most consistent host responses associated with cancer, and these two pathways interrelate with blood coagulation and fibrinolysis influencing tumor angiogenesis directly, thereby contributing to tumor growth. Since the tumor survival depends on blood supplement it has inspired many researchers to search for anti-angiogenic factors and to draw anti-angiogenic strategies for cancer treatment. The plasma kallikrein-kinin system is related to vascular biology and interacts with many physiologic processes such as coagulation, fibrinolysis, complement and renin-angiotensin, it regulates local blood pressure by bradykinin release (BK) and has both anti-thrombotic and pro-fibrinolitic activities. High molecular weight kininogen (HK) plays two roles in angiogenesis process, because BK promotes angiogenesis and kinin free HK (HKa) inhibits angiogenesis. Heparan sulfate proteoglycans (HSPG) may act as structural constituents of extracellular matrix, and those HSPG on cell surface may participate on processes such as endocytosis and vesicular uptake, regulating molecule traffic inside and outside cellular compartments. Different vesicles contribute on endocytosis depending on cell type and environment conditions of growth. The signaling pathways regulate all cell processes activities. The effectors in the pathways include transcription factors that control gene expression, the regulation of secretion apparatus, the metabolic enzymes, the structural and motor elements on cytoskeleton, the cell surface receptors, the cell cycle regulating proteins and membrane ionic channels. Some signaling pathways converge on all these effectors systems. The integration of different signals determine cell behavior, for instance, if cell secretes, moves, growths, proliferates and differentiates. The aim of the present study was analyze the HK interaction with surface of epithelial cells from human breast and CHO. The specific objectives in our work were to study the possibility of HK endocytosis, its processing and activity, in non-metastatic comparing to metastatic cells. In both CHO-K1 and CHO-745 cell lines we confirmed that HSPG plays role in ATP dependent and caveolae mediated endocytosis driven to endosomes the HK assembled on cell surface; we verified kinin release activities of both serine and cysteine proteases on cell surface in pH 7,35 of both cells lines CHO-K1 and CHO-745; both BK and HSPG are important in HK endocytosis; in both cells lines, and absence of Zn2+ and presence of 2% or 10% serum in medium, HKa has proliferative action, and glycosaminoglycans influence in HK activity on CHO-K1 proliferation. Both HK and HKa interactions were evaluated in mammary epithelium cells with different degrees of metastasis. The MCF-10A (non metastatic) interact more with both HK and HKa comparing to other two tumor cells, probably because their higher chondroitin sulfate content in tumor cells surface; the ratio between BK release in medium/BK internalized is higher in MDA-MB-231 cells (high degree of metastasis). In the absence of Zn2+ and presence of serum 2% or 10% in medium, within 24 h, HKa increased the proliferation of both MCF-10A and MCF-7 (middle metastatic) cells, and within 48 h the proliferation decreased in MCF-7 cells; in MDA-MB-231 both HK and HKa decreased cell proliferation and the presence of the serum changes the effect of both in 24 h, in 48 h only HK decreased cell proliferation in the presence of serum 2%. In MCF-7 Akt expression increased compared to non treated cells (control), except in cells treated with HKa in 48 h, ERK1/2 was detected in all conditions tested and c-Myc expression increased compared to non treated cells (control); in MDA-MB-231, HK or its fragments, and HKa increased Akt expression in 24 h; although p-38 is present in these cells in all conditions, HK or its fragments, and HKa promoted changes in p-38 expression, in similar manner in the presence of serum 2% or 10% in medium. The MDA-MD-231 cells migrated after in incubation with either HK or HKa in medium containing 2% or 10% serum and absence of zinc. The present study confirms the HSPG participation in cellular uptake of HK/HKa assembled on cell surface and fused with endosomes; the enzyme action of both serine and cysteine proteases classes in kinin release in incubation buffer in pH 7,35; the importance of both BK and HSPG in HK endocytosis; the involvement of caveolae in this process, and suggests the importance of zinc ions in proliferation processes related to uPAR or suPAR participation. Future studies on cell signaling may help in evaluation of cell cycle under HK/HKa treatment.