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Proteasomes have been shown to be involved in the regulation of melanin biosynthesis in melanoma cells. Here we report on the correlation between proteasome subunits and Tyrosinase (Tyr) activity in different cell phenotypes, and thereby regulation of melanin biosynthesis in B16F10 mouse melanoma cells. Our results indicated that the quantity of proteasome subunit p27 is higher and that of the enzyme Tyr and its activity are lower in amelanotic melanoma cells, while the reverse is true in melanotic melanoma cells. Proteasome subunit p27, compared to another subunit p31, shows increased co-localization with Tyr and Tyrosinase related protein 1 (Trp1) in amelanotic cells to a greater extent than that in melanotic cells. On exposure to cycloheximide, increased Tyr degradation was seen in amelanotic cells, as indicated by increased co-localization of p27 and Tyr. Further, exposure of amelanotic melanoma cells with proteasome-specific inhibitor MG132 resulted in an increased Tyr activity, increased levels of Tyr and Trp1, leading to increased melanin synthesis. These results therefore suggest that proteasomes, particularly p27 subunit, are directly involved in the regulation of melanin biosynthesis in mouse melanoma cells.

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The human immunodeficiency virus (HIV-1) Nef protein is now regarded as a regulatory protein responsible not only for establishment of infection and increased pathogenesis but also for enhancement of viral replication. However, the mechanism of Nef-induced activation of viral replication remains to be clearly understood. Using transient transfection assay, co-immunoprecipitation and pull-down analysis, we demonstrate in this report that the HIV-1 Nef protein physically interacts with Tat, the principal transactivating protein of HIV-1. Our observations with single cycle replication experiments further indicate that this interaction results not only in enhancement of Tat-induced HIV-1 long terminal repeat-mediated gene expression but also in virus production.

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Metabolism of glutathione by gamma-glutamyl transpeptidase (gamma-GT) at the level of cell membrane has been shown to generate hydrogen peroxide in many cell types including human melanomas. gamma-GT does not appear to be involved in cysteine uptake for pheomelanin production in melanoma cells and does not contribute significantly to the pheomelanin synthesized in B16 melanoma cells. We have therefore examined the possibility of gamma-GT mediated production of prooxidant reactions and its effect, if any, on pigmentation using B16 melanoma cells. Our results indicate that in B16 melanoma cells, gamma-GT activity leads to the production of hydrogen peroxide. We further show that the nuclear levels of the redox sensitive transcription factor NF-kappa B is regulated by H2O2 formed by the action of gamma-GT: stimulation and inhibition of gamma-GT affect the levels of NF-kappa B. Tumor necrosis factor alpha, a hypopigmenting cytokine, known to activate NF-kappa B also up-regulates the gamma-GT messenger RNA and activity. Stimulation of gamma-GT generated prooxidant reactions led to a decrease in tyrosinase activity. We therefore propose that prooxidant reactions mediated by gamma-GT in turn regulate the levels of tyrosinase in pigment cells. Our findings thus introduce a new aspect in the regulation of pigmentation and ascribe a novel role for gamma-GT in pigment cells.

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Gamma-glutamyl transpeptidase (gamma-GT), an ectoenzyme involved mainly in glutathione metabolism, is expressed in B16 melanoma cells. B16 melanoma cells under continuous culture conditions show a phenotypic drift from melanotic to amelanotic and re-melanotic stages. We have investigated the regulation of gamma-GT in B16 melanoma cells under such different pigmentary conditions. High levels of gamma-GT messenger RNA (mRNA) and activity were detected in pigmented B16 melanoma cells, whereas in amelanotic B16 melanoma cells the levels were very low. Treatment with lactic acid, a known inhibitor of tyrosinase gene expression, also led to the down-regulation of gamma-GT mRNA and activity. Thus our results indicate that gamma-GT regulation depends on the pigmentation status in pigment cells. We have also assessed the levels of gamma-GT in normal murine melanocytes (melan-a cells). It was seen that melan-a cells express very low levels of gamma-GT. As gamma-GT is known to be regulated in a tissue-specific manner, and is expressed from as many as six promoters giving rise to six different types of mRNAs each having unique 5' ends, we have further investigated the type of gamma-GT mRNA expressed in B16 melanoma and melan-a cells. In this study, we have conclusively demonstrated that type I mRNA transcript of gamma-GT is expressed in B16 melanoma and melan-a cells.

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Vitiligo among the community Somavashan Sahasajuna Kashatriya(SSK) of Baugalore surveyed by us during February-March, 1987, was found to be characterised by an earlier age of onset and a very high (90.38%) familial incidence. This community at present is small and is believed to have migrated'from Gujarat-Maharashtra border where the incidence of vitiligo in a related community, the Khatris is already high (3.6%). The results indicate that continuous inbreeding among the SSK community in Bangalore may have enhanced their genetic predisposition to this disease.