Hyperglycemia and HIV-1 Nef significantly enhanced the production of nitric oxide in the CNS in vitro and in vivo. (A) Hyperglycemia and HIV-1 Nef enhanced the production of nitric oxide in human primary astrocytes (in vitro) in dose dependent fashion. Primary human astrocytes were cultured and exposed to glucose solutions for12 hours as indicated earlier. Astrocytes with 5 mM glucose containing medium were used as control. After exposure to glucose, the astrocytes were transduced with HIV-1 Nef expressing virus. 48 hours later, the Nef-transduced astrocytes and cellular supernatants were collected and oxidative stress was determined by measuring the release of nitric acid in the astrocytes and in the supernatant with an ELISA kit (Stressgen, Victoria, BC, Canada). (B) Hyperglycemia and HIV-1 Nef significantly enhanced the production of nitric acid in mice brain: 1 × 107 viral particles generated through HIV-1 vectors or SNV vectors were injected into the brain of diabetes-induced mice via the cortex as described previously (Parveen et al 2003). Age-matched non-diabetic mice injected with an equal volume of citrate buffer served as control. After 8 weeks, the mice were sacrificed and the brain tissue lysates were subjected to ELISA to determine the release of total nitrate in the brain. The results depicted in this figure clearly indicate that hyperglycemia and Nef, either alone or in combination enhance oxidation reaction by increasing the release of total nitrates in CNS. The results are mean values of duplicate samples.