A naturally occurring substitution in human immunodeficiency virus Tat increases expression of the viral genome.

A natural amino acid substitution in the human immunodeficiency virus type 1 (HIV-1) transcriptional activator Tat increases its activity and compensates for deleterious mutations elsewhere in the Tat protein. Substitution of asparagine for threonine 23 increases Tat transactivation of the HIV-1 promoter and the binding of Tat to the cellular kinase positive transcription elongation factor b (P-TEFb). Of nine other position 23 mutations tested, only the serine substitution retained wild-type activity. Correspondingly, asparagine is the most frequent amino acid at this position in HIV-1 isolates, followed by threonine and serine. Asparagine is prevalent in Tat proteins of viruses in clades A, C, and D, which are major etiologic agents of AIDS. We suggest that selection for asparagine in position 23 confers an advantage to the virus, since it can compensate for deleterious mutations in Tat. It may also support the replication of otherwise less fit drug-resistant viruses and permit the emergence of virulent strains.

Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription.

To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 microg/ml ethidium bromide (EtBr) to the murine pancreatic beta cell line betaHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998) J. Biol. Chem. 273, 20300-20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in betaHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p < 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in beta cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca(2+)](i) increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion.