3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.     

The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.     

Can soy phytoestrogens decrease DNA methylation in BRCA1 and BRCA2 oncosuppressor genes in breast cancer?

Although soy phytoestrogens have been postulated to exert a protective effect against breast cancer, the attendant mechanisms, in particular epigenetics underpinnings, have remained elusive. We investigated the putative effects on DNA methylation by two naturally occurring isoflavones, genistein and daidzein, in a study of the BRCA1 and BRCA2 oncosuppressor genes in breast cancer cell lines (MCF-7, MDA-MB 231, and MCF10a). A demethylant agent, the 5-azacytidine, and a methylant, the budesonide, were used as treatment controls. DNA methylation of BRCA1 and BRCA2 was investigated with methylated DNA immunoprecipitation coupled with PCR. In parallel, protein expression was determined by Western blot, immunohistochemistry, and confocal microscopy. Our results suggest that treatment with 18.5?μM Genistein or 78.5?μM Daidzein might reverse DNA hypermethylation and restore the expression of the oncosuppressor genes BRCA1 and BRCA2. 5-Azacitydine also enhanced the reexpression of these genes while budesonide had an opposite effect. To the best of our knowledge, these observations, while requiring replication, provide new evidence on potential epigenetic mechanisms by which genistein and daidzein might contribute to regulation of the BRCA1 and BRCA2. Future studies are warranted on whether the demethylating effect of genistein and daidzein is global or focused on select candidate genes.