NADP(+)-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Synthesis of pyrazole substituted methyl pheophorbide-a derivatives and their preliminary in vitro cell viabilities

The unique ability of chlorophyll derivatives to accumulate in tumorous tissues and to cause a photodynamic effect under laser radiation has been successively used in photodynamic therapy. In this connection, enormous research on modifications of the peripheral substituents in chlorophyll has shown that the presence and position of the substituents in the chlorin derivatives make a remarkable difference in biological activity. As a result, we directed our attention to synthesize methyl pheophorbide-a derivatives, chlorophyll-a derivatives, possessing heterocyclic aromatic substituents or pyrazole moieties on the peripheral positions and to examine preliminary in vitro effects of these new derivatives on A549 cancer cells for PDT. From our experimental results, it was observed that the biological effect induced by photosensitizers on cancer cells may also depend upon the chemical structure of the photosensitizers.