Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography

Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

O contrabaixo como instrumento solista na segunda metade do século XIX em Lisboa: perspectiva histórica e analítica, edição crítica

A presente dissertação de estrutura tripartida tem como objetivo primordial dar a conhecer três obras para contrabaixo solista escritas em Lisboa na segunda metade do século XIX, através da análise musical destas composições e de uma Edição Crítica, acompanhada pelo respetivo enquadramento histórico. Estas três obras foram escritas pelos contrabaixistas/compositores João Rodrigues Cordeiro (1826, Rio de Janeiro – 1881, Lisboa), Francisco de Freitas Gazul (1842 – 1925, Lisboa) e Júlio António Avelino Soares (1846 – 1888), aos quais são dedicados alguns capítulos de teor biográfico. Coloca-se o foco de investigação no ensino no Conservatório de Música de Lisboa, assim como na passagem por Lisboa, no Teatro de São Carlos (palco principal da Lisboa novecentista), dos virtuosos italianos Luigi Anglois, Giovanni Bottesini e Italo Caimmi; Abstract: The double bass as a soloist instrument in the second half of the nineteenth century in Lisbon: Historical and analytical perspective, critical edition. This dissertation of tripartite structure has the primary objective of bringing to light three works for double bass soloist written in Lisbon in the second half of the nineteenth century through the musical analysis of these compositions and a Critical Edition accompanied by an examination of their historical background. These three works were written by the double bassists and composers João Rodrigues Cordeiro (1826, Rio de Janeiro – 1881, Lisbon), Francisco de Freitas Gazul (1842, Lisbon – 1925, Lisbon) and Júlio António Avelino Soares (1846, Lisbon – 1888, Lisbon), to whom are dedicated some chapters with biographical information. The focus of this investigation is on the teaching practices at the Conservatório de Música de Lisboa as well as on the passage through Lisbon, at the Teatro de São Carlos (the principal stage in nineteenth century Lisbon) of the italian virtuosi Luigi Anglois, Giovanni Bottesini and Italo Caimmi. NOTA: Contém Anexos indisponiveis online. Consulta presencia

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de BiofísicaSciEL

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

Efeito de níveis de melão em substituição ao milho moído sobre o desempenho, o consumo e a digestibilidade dos nutrientes em ovinos Morada Nova.

Avaliaram-se o desempenho, o consumo e a digestibilidade aparente de componentes nutritivos em 32 ovinos Morada Nova em confinamento, 20 machos não-castrados e 12 fêmeas, recebendo dietas contendo 0, 30, 60 e 100% de melão em substituição ao milho moído. Os animais tinham em média 6 meses de idade, peso médio inicial de 15 kg e foram abatidos aos 25 kg. Utilizou-se delineamento experimental inteiramente casualizado, com quatro tratamentos e oito repetições. O consumo de MS apresentou tendência quadrática para ambos os sexos, variando de 769 a 837 g para os machos e de 722 g a 646 g para as fêmeas. Os consumos de matéria orgânica (MO), proteína bruta (PB), extrato etéreo (EE), fibra em detergente neutro (FDN), nutrientes digestíveis totais (NDT), carboidratos totais (CT) e carboidratos não-fibrosos (CNF) apresentaram comportamento quadrático, refletindo o mesmo comportamento da ingestão da MS. A digestibilidade aparente da MS apresentou tendência linear decrescente. Os coeficientes de digestibilidade aparente de MO, PB, EE, FDN e CT apresentaram comportamento linear crescente com a adição de melão em substituição ao milho moído. O ganho de peso diário com o aumento do nível de melão na dieta foi maior nos machos. A conversão e eficiência alimentar não foram influenciadas pelo aumento do nível de melão na dieta. Os dias de confinamento variaram de 79 a 90 dias. A substituição na dieta do milho moído por melão possibilitou obter desempenho satisfatório em ovinos Morada Nova em confinamento. O nível de 60% de substituição é o que permite melhor retorno financeiro, entretanto, em níveis superiores a 30%, aumentam os dias de confinamento, elevando a idade ao abate

Contribución para la caracterización florístico-ambiental del Norte de Portugal

En el presente trabajo se hace mención a diferentes taxones específicos e infraespecíficos con distribuciones diversas en el territorio portugués continental. En este sentido, la intensa variabilidad geomorfológica y la intrincada bioclimatología del Norte de Portugal constituyen, ambos, factores ambientales decisivos para comprender el comportamiento florístico de esta parte del país. Los taxones analizados en esta aportación son: Teucrium salviastrum Trigonella polyceratia subsp. amandiana, Ononis viscosa subsp. breviflora var. breviflora, Ononis viscosa subsp. pedroi, Digitalis amandiana, Myosotis welwistchii y Myosotis secunda. De modo a poder contribuir eficientemente en la gestión de la riqueza florística, se procede a la aplicación de una rutina programática multivariada, basada en un sistema de información geográfico (SIG), a partir del cual cada uno de los taxones estudiados es georeferenciado sobre cartografía ambiental (confeccionada y publicada por el Instituto para a Conservação da Naturaza, del Ministerio del Ambiente portugués). Los resultados obtenidos confirman el efecto gradual de transición entre el área de influencia bioclimática atlántica y la mediterránea, ambas determinantes para comprender el comportamiento florístico del Norte de Portugal.Several specific and infraspecific taxa with different occurrences in the continental portuguese area are referred in the present work. In the light of these behaviours, the climatic and geomorphological variability of the North of Portugal has been important environmental factors to explain the floristic diversity in this part of the country. In this case, the taxa analysed are Teucrium salviastrum Trigonella polyceratia subsp. amandiana, Ononis viscosa subsp. breviflora var. breviflora, Ononis viscosa subsp. pedroi, Digitalis amandiana, Myosotis welwistchii and Myosotis secunda. Because of the appropriate management of the floristic richness, a multivariate statistic routine is applied on a Geographical Informatic System (GIS) the chorological, geomorphological and climatic characterization for each one are described in this paper. The geological substrate and the altitudinal classes are focused for the geomorphological approach, as well as two biogeographic classifications are used to correlate the occurrence of the taxa and their bioclimatic preferences. In the light of this analytical scheme, every taxa is geo-referenced upon an environmental cartography (elaborated by the Instituto para a Conservação da Naturaza). The results obtained are pointing out the floristic progressive differentiation between the atlantic bioclimatic influence and the mediterranean one, both bioclimatic influences decisive to understand the floristic behaviour of the North of Portugal