EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (Coll-G and Coll-H). In a well defined composition Coll-G/Coll-H together enzymes working on hydrophobic amminoacid, the neutral protease (Dispase) or the thermolysin (Thermostable Neutral Protease), are used in Langerhans islets purification. By electrophoresis and gelatin zymography approaches, in combination to densitometry quantitative valuation we have compared in composition, stability and autodigestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from two different producers, Roche and Serva.…

Formulation of Different Chitosan Hydrogels for Cartilage Tissue Repair

Different formulations of Chitosan/sulphate and Chitosan/PEGDE were produced by physical and chemical reticulation to obtain hydrogels with better physiochemical properties. The hydrogels were analyzed – in terms of their non-toxicity, proliferative, differentiative, inflammatory and immunology responses. Commercial grade Chitosan (Sigma) was solubilized and purified by progressive filtrations. Then, the polymer was freeze-dried in a water soluble cationic form. A physical hydrogel was prepared by mixing a 3 % w/w water solution of the a.m. polymer with different stoichiometric ratios of (SO4=) 1/0.5;1/0.75;1/1 respectively. The hydrogels were cross-linked in multiwells. The chemical hydrog…

A new method to valued efficiency of enzyme blend for pancreas tissue digestion.

One of the best successful example of cell therapy is represented by islet transplantation since the '90. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas condition, and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion are purified from Clostridium histolyticum; these enzymes has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. However, a major obstacle in human islet isolation successful is due to the variability in composition and concentration of the collagenases used…

Vesicles mediated cell-cell and cell-matrix interactions

Comparative study on enzymatic activity and molecules stability of some commercial proteolytic enzymes used in pancreatic islet isolation.

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (COLL G and COLL H). They are used in a defined tissue dissociation enzyme (TDE) mixtures together neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). The TDE mixtures were in part responsible for the success of the Edmonton protocol; however, just to now, people working in islets purification found discrepancy in an application to another one. This variability in application see in the enzymatic blend composition the higher accused, such as the contamination from endotoxines due to extractive production…

Proteolytic enzymes in membrane domains of endothelial cells cultured in type-1 collagen 3D gels

Novel protese from marine organisms with potential interest in restoration procedures

In the last decades molecular biology allowed the development of innovative protocols in the field of conservation/restoration of cultural assets. In this work we presents novel hydrolyses isolated from marine invertebrate organisms, whose protease activity was previously tested in laboratory by zymography on polyacrilamide gel (in presence of 0.1 of gelatin) Very interesting these enzymes are activated/work in a range of temperatures between 4 ° C and 37 ° C. In this study two sets of proteases were applied, to bio-clean works of art surfaces, at the environments temperature (19° to 25.5° C). Before remove, the water-soluble components of the layers have been analyzed by high-pressure size…

The nucleic acid binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii

Cellular nucleic acid binding proteins (CNBPs) represent a highly conserved protein family among vertebrates; they harbour seven tandem zinc finger repeats CCHC type and have been described as transcriptional and translational regulators. To date, there is little characterization of CNBP in invertebrates since its structure and function have been analysed solely in Drosophila melanogaster. However, no CNBP has been investigated in other arthropod systems. In an effort to isolate immune-related genes in Procambarus clarkii, a partial mRNA coding a zinc finger containing protein was found to be up-regulated during the response to white spot syndrome virus (WSSV) infection. The red swamp crayf…

A new method to valued efficiency of enzyme blends for pancreas tissue digestion

One of the best successful examples of cell therapy is represented by islet transplantation since 1990. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas conditions and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion include collagenases and neutral proteases: their composition and activity are largely variable between different batches. We set up a new in vitro method to better in vitro characterize enzymatic blend before its use in human pancreas. In our experimental approach human immortalized cells (ECV-304) or human is…