Pharmacologic vitreolysis

The vitreoretinal interface is involved in a wide range of vitreoretinal disorders and separation of the posterior vitreous face from the retinal surface is an essential part of vitrectomy surgeries. A diverse range of enzymatic and non-enzymatic agents are being studied as an adjunct before or during vitrectomy to facilitate the induction of posterior vitreous detachment. There is a significant body of knowledge in the literature about different vitreolytic agents under investigation for a variety of pathologies involving the vitreoretinal interface which will be summarized in this review

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil) / Caracterização enzimática de cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Veneza (Itália) e Baía de Guanabara (Brasil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1 percent of sodium chloride (NaCl) and APW plus 3 percent NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4 percent), V. harveyi (19 percent) and V. parahaemolyticus (7.6 percent), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

O ecossistema aquático é o habitat natural de microrganismos incluindo aqueles dos gêneros Vibrio e Aeromonas os quais são patogênicos para o homem e animais. Na presente investigação foi avaliada a freqüência destas bactérias e a característica enzimática de 34 cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Venice (Itália) e Baía de Guanabara (Brasil) durante o período de Novembro-2003 a Fevereiro-2004. As amostras de mexilhões foram submetidas a enriquecimento em Água Peptonada Alcalina (APA) adicionada de 1 por cento de Cloreto de Sódio (NaCl) e APA com 3 por cento de NaCl (37 ºC/18-24h). Em seguida as amostras foram semeadas em Agar TCBS (Agar Tiossulfato Citrato Bile Sacarose) e as colônias suspeitas foram submetidas à caracterização bioquímica. As cepas de Vibrio alginolyticus foram avaliadas quanto à produção das enzimas colagenase, elastase e condroitinase. Os resultados demonstraram o isolamento de 127 microrganismos assim distribuídos: 105 cepas de Vibrio das quais V. alginolyticus (32,4 por cento), V. harveyi (19 por cento) e V. parahaemolyticus (7,6 por cento), 20 cepas de Aeromonas e 2 Plesiomonas shigelloides foram os principais patógenos isolados. Observou-se a produção das três enzimas a partir de V. alginolyticus, consideradas principais fatores de virulência da bactéria, em especial em casos de infecção dermatológica humana.

Purification and immobilization of chondroitinase from Aeromonas sobria YH 311 / 生物工程学报

Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose.

Purificação e utilização das condroitinases B e AC de Flavobacterium heparinum no estudo dos proteoglicanos de matriz extracelular / Purification and utilization of chondroitinases AC, B and C from Flavobacterium in the study of proteoglycans the extracellular matrix

As condroitinases AC, B e C de Flavobacferium heparinum são importantes instrumentos para a identificação e a análise da estrutura de condroitim sulfatos e dermatam sulfatos, bem como de seus proteoglicanos, de diferentes origens. Entretanto, para que sejam úteis, preparações de enzimas puras e estáveis devem ser obtidas. No presente trabalho, descrevemos um procedimento simples, reprodutível e com alto rendimento para preparo dessas enzimas, baseado em cromatografia de troca iônica em Q-Sepharose FF e cromatografia de interação hidrofóbica em Pheny-Sepharose HP. Para análise quantitativa da purificação e do rendimento das enzimas purificadas, estabelecemos uma metodologia que se baseia na queda em metacromasia que acompanha a despolimerização dos glicosaminoglicanos. Este método, que utiliza a interação dos glicosaminoglicanos com azul de dimetilmetileno, permitiu a dosagem das condroitìnases presentes em extratos brutos induzidos de F. heparinum e nas preparações purificadas. A vantagem deste procedimento sobre outros métodos comumente empregados, que quantificam os produtos insaturados formados, é que a. presença de glicuronidases e sulfatares não interfere na determinação das atividades enzimáticas. As condroitinases AC e B assim preparadas foram empregadas na identificação e na análise estrutural de proteoglicanos e glicosaminoglicanos extraídos de diversos tecidos e fluidos biológicos, como córnea humana, urina, soro e diversos tecidos de gato, miométrio e leiomioma humanos. Além disso, uma nova metodologia foi desenvolvida para imunolocalizar proteoglicanos de condroitim sulfato ou dermatam sulfato em cortes de tecidos. Essa metodologia baseia-se na incubação de cortes de tecidos com condroitinase AC (para condroitim sulfato) ou condroitinase 8 (para dermatam sulfato). Em seguida, os resíduos insaturados gerados pelas enzimas são reconhecidos por anticorpos monoclonais específicos. Estudos de dupla marcação para as cadeias de condroitim sulfato ou dermatam sulfato e o esqueleto protéico (decorim ou versicam) ou queratam sulfato ou, ainda, filamentos de actina foram também realizados. As imagens de dupla marcação foram submetidas a análise morfométrica, utilizando a ferramenta MATLAB, para determinação do grau de co-localização...

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