Effect of epithelial debridement on human cornea proteoglycans

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.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de OftalmologiaUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de OftalmologiaSciEL

Caracterização e imunolocalização de proteoglicanos de condroitim sulfato e/ou dermatam sulfato de matriz extracelular

-Proteoglicanos são macromoléculas complexas formadas por um esqueleto protéico ao qual está covalentemente ligada pelo menos uma cadeia de glicosaminoglicano. Glicosaminoglicanos (GAGs) são heteropolissacarídeos lineares formados por unidades dissacarídicas repetitivas em que um dos açúcares é uma hexosamina e o outro um açúcar não nitrogenado. Os PGs são componentes essenciais da matriz extracelular (MEC) e possuem papéis altamente diversificados agindo como organizadores celulares e influenciando o crescimento da célula e a maturação de tecidos especializados. Atuam também no armazenamento e modulação da atividade de fatores de crescimento. Alterações significativas no conteúdo, estrutura e concentração de PGs foram descritas em diferentes condições fisiopatológicas, como em artrose, diversos tipos de tumorais, além de células transformadas e mantidas em cultura. Nos tumores, estas modificações podem facilitar o crescimento, a progressão e a invasão tumoral. Estudos para a determinação das causas dessas modificações têm sido intensificados, mas questões relevantes permanecem ainda sem respostas. Por exemplo, que alterações ocorrem nas doenças que possam explicar tais modificações e como elas afetam a estrutura da matriz extracelular? Qual é o papel dos proteoglicanos nesse contexto? Com base nessas questões, o objetivo do presente trabalho foi caracterizar e analisar a distribuição de PGs de condroitim e/ou dermatam sulfato de MEC em 15 diferentes tecidos de coelhos da linhagem New Zealand. Proteoglicanos obtidos por extrações sucessivas com GuaHCl 4 M em tampão acetato 0,05 M, pH 6,4, foram submetidos à cromatografia de troca-iônica em Q-Sepharose para eliminação de outras moléculas de matriz, tais como, colágenos e proteínas não fibrosas. PGs purificados foram submetidos às análises por eletroforese em gel de agarose e SDS-PAGE e degradação proteolítica. Foram observadas bandas com migração característica de PGs em todos os tecidos. Por imunoblotting foi detectado decorim em aorta, intestino grosso músculo, pâncreas, traquéia e estômago e PGs de queratam sulfato em coração, fígado. Em aorta, cérebro, intestino delgado, córnea, útero e bexiga foram detectados ambos PGs. Análise histológica por imunofluorescência em aorta, útero revelou distribuição diferencial de PGs de condrotim e de dermatam sulfato na matriz desses tecidos

Induction and purification of Flavobacterium heparinum chondroitinases

O presente trabalho estabeleceu as melhores condicoes para o preparo das condroitinases de Flavobacterium heparinum. Foram realizados estudos sobre o crescimento bacteriano e a inducao enzimatica em diferentes meios de cultura, sobre as melhores condicoes para preparo de extrato bruto e foram estabelecidos metodos para a purificacao das condroitinases. O desenvolvimento de uma metodologia rapida para ensaio das enzimas, que se baseia na queda da metacromasia dos glicosaminoglicanos quando sao degradados, foi de grande importancia para a realizacao desses estudos. Para as analises do crescimento bacteriano e da inducao enzimatica, foram utilizados dois meios complexos (trypticase sem giicose e peptona) e um meio definido. Em todos os meios de cultura, a fase de crescimento exponencial foi seguida de uma fase estacionaria, que ocorreu mais tardiamente no meio definido que nos meios complexos. Embora as celulas de F. heparinum crescam bem em todos os meios utilizados, densidades celulares mais altas foram obtidas em trypticase sem glicose que nos demais meios. Em todos os meios de cultura, as atividades das enzimas que degradam condroitim sulfato e dermatam sulfato aumentaram muito quando a bacteria foi cultivada em presenca de condroitim 6-sulfato como indutor, embora a cinetica de crescimento bacteriano nao tenha sido afetada. Atividades enzimaticas maximas foram observadas proximo ao final da fase de crescimento exponencial. Em peptona, outro pico de atividade enzimatica apareceu, nas primeiras horas de crescimento bacteriano, tanto em presenca como em ausencia do indutor. Alem disso, nesse meio houve rapida queda na atividade das condroitinases durante a fase estacionaria. A concentracao minima de condroitim 6-sulfato capaz de induzir a expressao das condroitinases foi de 50 mg/L. Acima de 100 mg/L, pequeno aumento nas atividades enzimaticas foi observado. Dentre todos os glicosaminoglicanos utilizados como indutores, condroitim 6-sulfato foi o mais eficiente. Alem disso, oligossacarideos produzidos por quebra enzimatica de condroitim 4-sulfato tambem sao capazes de induzir as enzimas. Para o preparo de extrato bruto de F heparinum, a exposicao da suspensao bacteriana ao ultrassom (20 kHz) por periodos curtos de tempo (2 pulsos de 30 segundos) foi mais eficiente para solubilizacao das condroitinases que periodos mais longos. Para o fracionamento das condroitinases, dois metodos foram utilizados: cromatografia de adsorcao ...(au)BV UNIFESP: Teses e dissertaçõe

Purification and utilization of chondroitinases AC, B and C from Flavobacterium in the study of proteoglycans the extracellular matrix

BV UNIFESP: Teses e dissertaçõe

Reduced urinary excretion of sulfated polysaccharides in diabetic rats

The aim of the present study was to further understand the changes in renal filtration that occur in the early stages of diabetes mellitus. Diabetes was induced in male Wistar rats by a single injection of streptozotocin. Glycemia, body weight, 24-h urine volume and urinary excretion of creatinine, protein and glycosaminoglycans were measured 10 and 30 days after diabetes induction. All the diabetic animals used in the present study were hyperglycemic, did not gain weight, and presented proteinuria and creatinine hyperfiltration. in contrast, the glycosaminoglycan excretion decreased. Dextrain sulfates of different molecular weights (6.0 to 11.5 kDa) were administered to the diabetic rats, and to age-matched, sham-treated controls. Most of the dextran sulfate was excreted during the first 24 h, and the amounts excreted in the urine were inversely proportional to the dextran sulfate molecular weight for all groups. Nevertheless, diabetic rats excreted less and accumulated more dextran sulfate in kidney and liver, as compared to controls. These differences, which were observed only for the dextran sulfates of higher molecular weights (> 7 kDa), increased with the duration of diabetes. Our findings suggest differential renal processing mechanisms for proteins and sulfated polysaccharides, with the possible involvement of kidney cells. (c) 2004 Elsevier B.V. All rights reserved

INCREASE OF GLYCOSAMINOGLYCANS AND METALLOPROTEINASES 2 AND 9 IN LIVER EXTRACELLULAR MATRIX ON EARLY STAGES OF EXTRAHEPATIC CHOLESTASIS

Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Methods Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats&#8217; liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content

Cecropia pachystachya Leaves Present Potential to Be Used as New Ingredient for Antiaging Dermocosmetics

Several biological activities have been reported for leaf extracts of Cecropia pachystachya species, including antioxidant and wound healing activities. This study aims to report, for the first time, the antiaging potential of the hydroethanolic (HE) and the ethanolic (EE) extracts obtained from the leaves of C. pachystachya using different in vitro assays. Both HE and EE presented relevant antioxidant capacity in different models, including phosphomolybdenum, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), carotene/linoleic acid bleaching, and thiobarbituric acid reactive substances (TBARS) assays. Their ability to prevent the production of advanced glycation end products (AGEs) was also evaluated, and both extracts showed important activity, especially HE. The extracts also stimulated the fibroblasts proliferation in vitro, specialized cells that produce several mediators which maintain the skin integrity and youthfulness. Cytotoxicity of the extracts was not observed for this lineage or HEK-293, human embryonic kidney cells widely used to evaluate cytotoxicity of chemical compounds. HE also exhibited the ability to inhibit the collagenase (metalloproteinase MMP-2) and elastase activities. The total phenolic and flavonoids contents were also determined. HPLC analysis revealed the presence of the flavonoids orientin and iso-orientin, which were quantified to be used as chemical markers. The results suggested that the extracts of C. pachystachya leaves present the potential to be used in dermocosmetic formulations to prevent the skin aging process, which attracts the attention of pharmaceutical companies and researchers interested in the development of novel ingredients likely to be used as active principles in antiaging products