Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes.

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.

Overexpression of catalase provides partial protection to transgenic mouse beta cells.

Pancreatic beta cells are sensitive to reactive oxygen species and this may play an important role in type 1 diabetes and during transplantation. Beta cells contain low levels of enzyme systems that protect against reactive oxygen species. The weakest link in their protection system is a deficiency in the ability to detoxify hydrogen peroxide by the enzymes glutathione peroxidase and catalase. We hypothesize that the deficit in the ability to dispose of reactive oxygen species is responsible for the unusual sensitivity of beta cells and that increasing protection will result in more resistant beta cells. To test these hypotheses we have produced transgenic mice with increased beta cell levels of catalase. Seven lines of catalase transgenic mice were produced using the insulin promoter to direct pancreatic beta cell specific expression. Catalase activity in islets from these mice was increased by as much as 50-fold. Northern blot analysis of several tissues indicated that overexpression was specific to the pancreatic islet. Catalase overexpression had no detrimental effects on islet function. To test whether increased catalase activity could protect the transgenic islets we exposed them to hydrogen peroxide, streptozocin, and interleukin-1beta. Fifty-fold overexpression of catalase produced marked protection of islet insulin secretion against hydrogen peroxide and significantly reduced the diabetogenic effect of streptozocin in vivo. However, catalase overexpression did not provide protection against interleukin-1beta toxicity and did not alter the effects of syngeneic and allogenic transplantation on islet insulin content. Our results indicate that in the pancreatic beta cell overexpression of catalase is protective against some beta cell toxins and is compatible with normal function.

Semi-synthesis of polymyxin-B conjugated ovalbumin: evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-alpha synthesis.

A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo-SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.

Inhibition of lipopolysaccharide-induced TNF-alpha production by semisynthetic polymyxin-B conjugated dextran.

During episode of severe endotoxemia, concentrations of both lipopolysaccharide and its lipid A-core subfraction are liberated from gram-negative bacteria and become elevated within the systemic circulation. Lipid-A core is the most homogeneous and physiologically toxic segment of the lipopolysaccharide molecule. Polymyxin-B has profound binding avidity for the lipid A-core subfraction of lipopolysaccharide. The mechanism of this binding avidity involves the development of attractive forces between the cationic groups of polymyxin-B and the anionic groups of the lipid A-core moiety of lipopolysaccharide. Complementary attractive forces include hydrophobic interactions which additionally become established between the octylheptanoyl group of polymyxin-B and the saturated carbon chains of the lipid A-core moiety. This paper describes a method for the semisynthetic production of polymyxin-B conjugated dextran in the form of polymyxin-B.ABH.dextran applying the photoreactive crosslinking reagent azidobenzoyl hydrazide (ABH). Molecular design and development of a semisynthetic technique for the conjugation of polymyxin-B to purified dextran fractions was motivated by the pronounced nephrotoxicity associated with this cationic polypeptide antibiotic. Conjugation of polymyxin-B to a relatively large molecular weight carrier compound would increase the overall size of the complex to a degree sufficient to theoretically reduce clearance through glomerular filtration mechanisms. Attributes of such a large molecular weight polymyxin-B conjugated biopharmaceutical would include diminished levels of nephrotoxicity due to a reduction of renal tubular concentrations and a simultaneous prolongation of its intravascular half-life (t (1/2)) and pharmacokinetic profile. Lipopolysaccharide (LPS) binding avidity of polymyxin-B.ABH.dextran was verified by Dot-Blot analysis in conjunction with the application of fluorescein isothiocyanate conjugated E. coli (0.55:B5) LPS (FITC-LPS). Capacity of polymyxin-B.ABH.dextran conjugates to inhibit in vitro LIP-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed by the application of a tissue culture based biological assay system capable of detecting cytotoxicity mediated by this potent monokine. Semisynthetic conjugates of polymyxin-B.ABH.dextran conjugates (0.6 microns/mL), thereby providing cytoprotectivity (95%; p < or - 0.001. to WEHI 164 clone 13 cell populations relative to untreated reference controls. Since TNF-alpha is currently believed to be the principal endogenous mediator involved in the host's inflammatory response during episodes of endotoxemia, results from these investigations provide a scientific foundation for warranting the elevation of the in vivo efficacy of large molecular weight semisynthetic polymyxin-B conjugates. Results from these investigations may ultimately lead to the application of semisynthetic polymyxin-B.ABH.dextran as a model for the molecular design of semisynthetic production of alternative biopharmaceutical or pharmaceutical agents possessing prophylactic and/or therapeutic efficacy for the management of severe endotoxemia conditions.