Heparanase upregulation in high glucose-treated endothelial cells is prevented by insulin and heparin.

Heparan sulfate proteoglycans on the endothelial cell surface and extracellular matrix play an important role in vascular homeostasis. Previous studies have shown that the quantity of heparan sulfate is reduced in kidney and other organs in diabetes. The objectives of this study were to determine if heparanase is induced by high glucose in endothelial cells and if heparin and/or insulin or basic fibroblast growth factor (bFGF) affect this upregulation. Cultured porcine aortic endothelial cells in M199 medium were treated with high glucose (30 mM) and/or bFGF (1 or 10 ng/ml) or high glucose plus insulin (1 U/ml) and/or heparin (0.5 microg/ml) for 7 days. To help define the mechanism of endothelial damage, cells were also exposed to H(2)O(2) (0.1 mM) for 1 day or mannitol (30 mM) for 7 days. Heparanase mRNA was detected by reverse transcription polymerase chain reaction. Heparanase activity was measured by incubating cell lysates with [(35)S]labeled extracellular matrix of bovine corneal endothelial cells and analyzing released radioactive products by gel filtration and beta-scintillation. Heparanase mRNA was found in high-glucose- and H(2)O(2)-treated cells; however, it was not found in control cells, mannitol- or high glucose plus insulin- and/or heparin-treated cells, or fresh porcine tissue. Heparanase activity was only found in high-glucose- and H(2)O(2)-treated cells. As well, bFGF did not prevent heparanase mRNA upregulation by high glucose. From these observations, we concluded that heparanase upregulation by high glucose is prevented by insulin and/or heparin but not bFGF. Reactive oxygen species, but not changes in osmolarity, may be involved in the upregulation of heparanase.

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA.

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.