Navegando por Palavras-chave "proteoglycan"
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- ItemSomente MetadadadosAnálise do proteoglicano glipicam e a via de sinalização wnt: seu papel no câncer de próstata(Universidade Federal de São Paulo (UNIFESP), 2016-06-30) Moraes, Gabrielle Ferrante Alves de [UNIFESP]; Toma, Leny Toma [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Prostate cancer development proceeds through a multiple steps to reach advanced metastasis. Identifying the signaling pathway involved in this cascade, which initiates at the cell surface microenvironment, could lead to the interruption of this cancer progresso Glypicans are cell surface proteoglycans linked to the membrane through glycosyl-phosphatidylinositol anchor. Interactions with specific ligants have been reported to trigger diverse signaling, including Wnts. In this study we sought to investigate whether there is a correlation between the expression of glypicans in prostate cancer cell lines and the Wnt signaling pathway. For this purpose, tumor cell lines PC-3, DU-145 e LNCaP were used compared to the normal RWPE-1 cells. Glypican expression pattern was analyzed by RT-PCR and showed that glypican-1 isoform was expressed at high levels by the cells, with exception of LNCaP cell line which express mainly glypican-5. Glypican-1 was investigated by immunocytochemistry, whose subcellular localization was achieved at the cell surface in RWPE-1 and PC-3 cells. Interestingly, in DU-145 cells glypican-1 was found in cytoplasmic compartment. Analysis by flow cytometry confirmed glypican in the cell lines, as well as the presence of the Wnt-3a ligant and activated [3-catenin. In order to investigate the involvement of glypican proteoglycan in this signaling pathway, co-immunoprecipitation with Wnt-3a was done. Increased levels of these proteins were detected in cancer cells, suggesting the link of glypican with this pathway. Translocation of [3-catenin to the nucleus has been associated with progression to malignant prostate carcinogenesis. So, nuclear and cytoplasm [3-catenin was investigated and a markedly increase in nuclear [3-catenin was observed in cancer cell lines, compared to RWPE-1 cells. In conclusion, glypican expression in prostate cancer cell lines correlates with activated nuclear [3-catenin via Wnt-3a ligant and has the potential to interfere in events of the canonical Wnt signaling pathway which can control neoplastic growth and disease progression.
- ItemSomente MetadadadosCharacterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ions effects(Blackwell Publishing, 2005-10-01) Borges, Fernanda Teixeira [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Aguiar, Jair Adriano Kopke [UNIFESP]; Dalboni, Maria Aparecida [UNIFESP]; Garofalo, Andrezza S.; Schor, Nestor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Background. the interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines.Methods. Glycosaminoglycan synthesis was analyzed by metabolic labeling with S-35-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry.Results. the main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of S-35-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased S-35-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium.Conclusion. Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.
- ItemSomente MetadadadosA comparative analysis of structure and spatial distribution of decorin inhuman leiomyoma and normal myometrium(Elsevier B.V., 2003-01-02) Berto, AGA; Sampaio, L. O.; Franco, CRC; Cesar, R. M.; Michelacci, Y. M.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. the data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio D-glucuronate/L-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. the relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix. (C) 2002 Elsevier Science B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Effect of epithelial debridement on human cornea proteoglycans(Associação Brasileira de Divulgação Científica, 2001-03-01) Soriano, Eduardo Sone [UNIFESP]; Campos, Mauro Silveira de Queiroz [UNIFESP]; Aguiar, Jair Adriano Kopke [UNIFESP]; Michelacci, Yara Maria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Corneal transparency is attributed to the regular spacing and diameter of collagen fibrils, and proteoglycans may play a role in fibrillogenesis and matrix assembly. Corneal scar tissue is opaque and this opacity is explained by decreased ultrastructural order that may be related to proteoglycan composition. Thus, the objectives of the present study were to characterize the proteoglycans synthesized by human corneal explants and to investigate the effect of mechanical epithelial debridement. Human corneas unsuitable for transplants were immersed in F-12 culture medium and maintained under tissue culture conditions. The proteoglycans synthesized in 24 h were labeled metabolically by the addition of 35S-sulfate to the medium. These compounds were extracted by 4 M GuHCl and identified by a combination of agarose gel electrophoresis, enzymatic degradation with protease and mucopolysaccharidases, and immunoblotting. Decorin was identified as the main dermatan sulfate proteoglycan and keratan sulfate proteoglycans were also prominent components. When the glycosaminoglycan side chains were analyzed, only keratan sulfate and dermatan sulfate were detected (~50% each). Nevertheless, when these compounds were 35S-labeled metabolically, the label in dermatan sulfate was greater than in keratan sulfate, suggesting a lower synthesis rate for keratan sulfate. 35S-Heparan sulfate also appeared. The removal of the epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by the stroma. The increased deposit of dermatan sulfate proteoglycans in the stroma suggests a functional relationship between epithelium and stroma that could be related to the corneal opacity that may appear after epithelial cell debridement.
- ItemSomente MetadadadosEnzyme and integrin expression by high and low metastatic melanoma cell lines(Lippincott Williams & Wilkins, 2003-02-01) Staquicini, F. I.; Moreira, C. R.; Nascimento, F. D.; Tersariol, ILS; Nader, H. B.; Dietrich, C. P.; Lopes, J. D.; Universidade Federal de São Paulo (UNIFESP); Univ Mogi CruzesDissemination of a malignant tumour is the result of a cascade of events beginning with detachment of cells from primary tumour followed by extravasation and growth at secondary sites. the differences in metastatic ability could be attributed to properties intrinsic to the various tumour types. Thus the clonal selection of tumour cells from successive metastases apparently results in cells better equipped for survival and formation of colonies in secondary sites, indicating that survival is not a random phenomenon. Many studies of malignant cells have correlated the overexpression of adhesion receptors such as integrins and the production of cysteine proteases and glycosidases with the progression of malignancy. the interaction of cysteine proteases with basement membrane components has been implicated in tumour invasion, activation of hormones and growth factors. On the other hand, the expression of the heparanase gene and its protein has been associated with the metastatic potential of several human and mouse tumour cell lines. This study aimed to investigate the correlations between the metastatic properties of clones with high and low metastatic potential and their ability to adhere to the extracellular matrix and to degrade proteins and sulphated glycosaminoglycans present there. Clonal selection of the B16F10 cell line was performed, and the clones were examined for the expression of an integrin-type laminin receptor. A significantly higher level was detected in a high metastatic clone. Enzymatic assays showed higher activity for alpha-D-N-acetylglucosaminidase, beta-D-N-acetylgalactosaminidase and beta-D-glucuronidase in conditioned medium from low metastatic clones compared with that from high metastatic clones. However, higher endopeptidase activity was observed in conditioned medium from high metastatic clones. in summary, these results showed a positive correlation between high metastatic potential and endopeptidase secretion. Similarly, a positive correlation was observed between low metastatic cells and the secretion of glycosaminoglycan-degrading glycosidases.
- ItemAcesso aberto (Open Access)Heparan sulfate and cell division(Associação Brasileira de Divulgação Científica, 1999-05-01) Porcionatto, Marimélia Aparecida [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Dietrich, Carl Peter [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Heparan sulfate is a component of vertebrate and invertebrate tissues which appears during the cytodifferentiation stage of embryonic development. Its structure varies according to the tissue and species of origin and is modified during neoplastic transformation. Several lines of experimental evidence suggest that heparan sulfate plays a role in cellular recognition, cellular adhesion and growth control. Heparan sulfate can participate in the process of cell division in two distinct ways, either as a positive or negative modulator of cellular proliferation, or as a response to a mitogenic stimulus.
- ItemSomente MetadadadosAn improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases(Wiley-Blackwell, 2003-04-01) Aguiar, JAK; Lima, C. R.; Berto, AGA; Michelacci, Y. M.; Universidade Federal de São Paulo (UNIFESP)Chondroitinases are very important tools for the identification and structural analysis of proteoglycans. Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes. Furthermore, it was recently shown that F. heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis. Due to the increasing importance of F. heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C. the Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F. heparinum. This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A(232), which measures the unsaturated products formed. Trypticase was the best culture medium, both for bacterial growth and enzyme induction. the chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space. Maximum chondroitinase induction occurred in the presence of 0.2-1.0 g/l chondroitin sulphate. Chondroitin sulphate-degraclation products were also inducers, but heparin and heparan sulphate were not. Chondroitinases AC, B and C were separated from each other by hydrophobic-interaction chromatography on Phenyl-Sepharose HP. When contaminant proteins were first removed from crude extract by Q-Sepharose, the chondroitinases could be purified to homogeneity in this phenyl-Sepharose chromatographic step.
- ItemSomente MetadadadosPresence of a laminin-binding chondroitin sulfate proteoglycan at the cell surface of a human melanoma cell Mel-85(Kluwer Academic Publ, 1999-07-01) Elias, MCQB; Veiga, S. S.; Gremski, W.; Porcionatto, M. A.; Nader, H. B.; Brentani, R. R.; Univ Fed Parana; Ludwig Inst Canc Res; Universidade Federal de São Paulo (UNIFESP)Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with beta-mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. the molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [S-35]-sulfate and chemical beta-elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. the location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.
- ItemSomente MetadadadosProteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats(Elsevier B.V., 1996-05-21) Hadad, S. J.; Michelacci, Y. M.; Schor, N.; Universidade Federal de São Paulo (UNIFESP)In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. the dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. in contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. the dermatan sulfate was secreted to the medium as proteoglycan, Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. the proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.
- ItemSomente MetadadadosPutative role of heparan sulfate proteoglycan expression and shedding on the proliferation and survival of cells after photodynamic therapy(Elsevier B.V., 2007-01-01) Pazos, Marcelo de Castro; Ricci, Ritchelli; Simioni, Andreza R.; Lopes, Carla C.; Tedesco, Antonio C.; Nader, Helena B.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)Introduction: Photodynamic therapy is based on the selective retention of a photosensitizer by highly proliferating cells and its activation with light at the appropriate wavelength. This combination generates reactive oxygen species that ultimately kill the cells. Some cells, however, may survive photodynamic therapy and the interaction of these cells with the extracellular matrix has profound effect in tumor biology. the knowledge of photodynamic therapy action on the extracellular matrix has not been fully explored. It has been focused mainly on integrins, matrix metalloproteinases and on growth factors and immunological mediators. Other important molecules involved in the regulation of many cell processes are the glycosaminoglycans, polymers of disaccharide units, present on the cell surface and in the extracellular matrix. in most cases, the glycosaminoglycans occur as proteoglycans.Aims: the purpose of the present investigation is to evaluate heparan sulfate proteoglycan expression and shedding, and its relation to the survival of the remaining cells, after a liposomal-AlClPc based photodynamic treatment.Materials: A wild-type endothelial cell derived from rabbit aorta and its counterpart transfected with EJ-ras oncogene were used.Results: Both cell lines presented augmented heparan sulfate proteoglycan syndecan-4 mRNA expression, augmented synthesis of heparan sulfate chains and increased shedding. Also, the formation of stress fibers on the border of the cells and the arrest in G, phase of the cell cycle was observed.Conclusions: These results show that surviving cells after photodynamic therapy exhibit changes in their morphology and cell processes that differ from that of non-treated cells, and these changes are probably hindering the cells from resuming normal proliferation. (C)2007 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosSulfation of intrinsic glycoproteins of the rabbit vitreous(Academic Press Ltd, 1998-09-01) Goes, R. M.; Laicine, E. M.; Mendes, MLO; Nader, H. B.; Haddad, A.; Universidade de São Paulo (USP); UNESP; Universidade Federal de São Paulo (UNIFESP)The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation. Rabbits were injected intravitreally with S-35-sodium sulfate and killed at several time intervals after injection. in another series of experiments, rabbits were injected either with S-35-sodium sulfate, H-3-fucose or H-3-tyrosine, associated or not associated with tunicamycin administration. Vitreous from the control eyes was also digested with N-glycosidase.. Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with S-35-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis. These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal. the experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins. (C) 1998 Academic Press.
- ItemSomente MetadadadosUltrastructural, immunohistochemical and biochemical analysis of glycosarninoglycans and proteoglycans in the mouse, pubic symphysis during pregnancy(Elsevier B.V., 2005-06-01) Pinheiro, M. C.; Mora, O. A.; Caldini, E. G.; Battlehner, C. N.; Joazeiro, P. P.; Toledo, OMS; Universidade Federal de São Paulo (UNIFESP); Universidade Estadual de Campinas (UNICAMP); Universidade de São Paulo (USP)During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis, in late stages, this ligament undergoes relaxation to allow proper delivery, which is expected on the 19th day, Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracelfular matrix in these tissues. Gl cosarilinogl cans and protcogl cans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy.At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well Lis in D15, D17 and D18 pubic ligaments. the only sulfated glycosaminoglycan in these filaments was chondroitin sulfate. as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation. corroborated this inference, the ratio of chondroitin Sulfate dry weight of symphysis showed two phases of increase: between D12 and D15. and between D17 and D18 WC Suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during relaxation. Versican and hyaluronic acid. working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. the presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. the role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed. (c) 2005 International Federation for Cell Biology. Published by Elsevier B.V. All rights reserved.