6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P . patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'-and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Succharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS-and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. putulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.
The ability of glyoxal to induce apoptosis was confirmed by our findings demonstrating the time- and dose-dependent acidification of retinal cells. The onset of acidification is a hallmark because acidification is a measurable cytosolic event that follows the mitochondrial change but precedes caspase activation. Therefore, monitoring of pHi can allow one to assess cell stresses such as hypoxia, and metabolic stress (AGEs), and to test whether these stresses have a cumulative effect on apoptosis induction. Our study showed that intracellular pH and mitochondrial potential in living retinal cells are useful parameters for monitoring metabolic status in retinal tissue. These findings may be relevant in the study of retinal cell death mechanisms associated with age-related diabetic retinopathy and macular degeneration.
The glyoxal-induced rapid formation of CML shows the ability of our retina model to simulate AGE-related effects in vitro. The dose-dependent expression of apoptosis-promotor molecules indicates that the apoptosis-inducing machinery starts in most retinal cells within 9 h. The neurotoxicity of glyoxal-induced AGE formation was shown by the significantly increased rate of cell death in the retina. The significant decrease of apoptotic events (P<0.01) indicates that antioxidants and AGE formation blocker can exert a differentiated cytoprotection for each of the retinal cell layers.
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