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The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8·1012dm3mol-1s-1, and 1.6·1012dm3mol-1s-1, respectively.
Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the αv promoter region and are directly involved in the regulation of transcriptional activity of the αv gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates αv expression during differentiation of pluripotent K562 cells in­duced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the «v gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was pro­tected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nu­clear extract of K562 cells treated with BA revealed an increase in GATA binding ac­tivity, which was associated with reduced αv mRNA and αv protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of αv. We conclude that the GATA-1 tran­scription factor specifically binds to the GATA element in the αv gene promoter and negatively regulates αv gene expression.
Vaccinia virus is able to replicate in many cell types and is known to modulate apoptosis in infected cells. In this study, expression of apoptosis-related genes was screened in human adherent monocytes after vaccinia infection using a DNA array. A marked increase of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was found. Increased expression and nuclear translocation of GAPDH have recently been reported to participate in apoptosis of many cell types. To confirm the array results, levels of GAPDH mRNA were estimated by RT-PCR, showing an increase at 4 h p.i. followed by a slight decrease, which corre­lated with the viral anti-apoptotic E3L gene transcript levels. Subcellular localization of the enzyme in human monocytes was examined by Western blot and immunostaining of the infected cells. Both experiments revealed accumulation of GAPDH in the nucleus at 14 h p.i., which was completely suppressed at 24 h p.i. This might indicate GAPDH as a novel target for vaccinia anti-apoptotic modulation.
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