Differential regulation of two types of monogalactosyldiacylglycerol synthase in membrane lipid remodeling under phosphate-limited conditions in sesame plants.

Phosphate (Pi) limitation causes drastic lipid remodeling in plant membranes. Glycolipids substitute for the phospholipids that are degraded, thereby supplying Pi needed for essential biological processes. Two major types of remodeling of membrane lipids occur in higher plants: whereas one involves an increase in the concentration of sulfoquinovosyldiacylglycerol in plastids to compensate for a decreased concentration of phosphatidylglycerol, the other involves digalactosyldiacylglycerol (DGDG) synthesis in plastids and the export of DGDG to extraplastidial membranes to compensate for reduced abundances of phospholipids. Lipid remodeling depends on an adequate supply of monogalactosyldiacylglycerol (MGDG), which is a substrate that supports the elevated rate of DGDG synthesis that is induced by low Pi availability. Regulation of MGDG synthesis has been analyzed most extensively using the model plant Arabidopsis thaliana, although orthologous genes that encode putative MGDG synthases exist in photosynthetic organisms from bacteria to higher plants. We recently hypothesized that two types of MGDG synthase diverged after the appearance of seed plants. This divergence might have both enabled plants to adapt to a wide range of Pi availability in soils and contributed to the diversity of seed plants. In the work presented here, we found that membrane lipid remodeling also takes place in sesame, which is one of the most common traditional crops grown in Asia. We identified two types of MGDG synthase from sesame (encoded by SeMGD1 and SeMGD2) and analyzed their enzymatic properties. Our results show that both genes correspond to the Arabidopsis type-A and -B isoforms of MGDG synthase. Notably, whereas Pi limitation up-regulates only the gene encoding the type-B isoform of Arabidopsis, low Pi availability up-regulates the expression of both SeMGD1 and SeMGD2. We discuss the significance of the different responses to low Pi availability in sesame and Arabidopsis.

DNA damage caused by bisphenol A and estradiol through estrogenic activity.

Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induced chromosomal aberration. Estrogen receptors (ER) participate in some of these genotoxic effects by estrogen. In this study, we showed the effects of bisphenol A (BPA), an endocrine-disrupting chemical eliciting weak estrogenic activity, and of 17beta-estradiol (E2), on DNA damage in ER-positive MCF-7 cells by Comet assay. Higher concentrations of BPA, more than 1000 times of E2, were needed to induce the same levels of effects by E2. Immunofluorescence microscopy showed that gammaH2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in MCF-7 cells. gammaH2AX foci colocalized with Bloom helicase, which is considered to be responsible for the repair of DNA damage after treatment with E2 or BPA. Interestingly, DNA damage was not as severe in ER-negative MDA-MB-231 cells as in MCF-7 cells. The ER antagonist ICI182780 blocked E2 and BPA genotoxic effects on MCF-7 cells. These results together suggest that BPA causes genotoxicity ER dependently in the same way as E2.

Piperazine propanol derivative as a novel antifungal targeting 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan synthase, which synthesizes a main component of fungal cell wall, is one of the promising targets for antifungal agents. In order to identify novel chemical classes of 1,3-beta-D-glucan synthase inhibitors, we screened a chemical library monitoring inhibition of the Candida albicans 1,3-beta-D-glucan synthase activity. The piperazine propanol derivative GSI578 [(2,6-difluoro-phenyl)-carbamic acid 3-(4-benzothiazol-2-yl-piperazine-1-yl)-propyl ester] was identified as a potent inhibitor against 1,3-beta-D-glucan synthase with an IC50 value of 0.16 microM. GSI578 exhibited in vitro antifungal activity against pathogenic fungi including C. albicans and Aspergillus fumigatus. Temperature-sensitive mutations of the FKS1 gene in the Deltafks2 background of Saccharomyces cerevisiae, where FKS1 and FKS2 encode putative catalytic subunits of 1,3-beta-D-glucan synthase, altered sensitivity to GSI578. This suggests that the antifungal activity of the piperazine propanol derivative has an effect on 1,3-beta-D-glucan synthase inhibition. Results of our initial evaluation suggest that the piperazine propanol derivative is a novel chemical structure of the class of antifungals which inhibit fungal cell growth by inhibiting fungal 1,3-beta-D-glucan synthase.

Quantitative analysis of Werner helicase activity using the single-molecule fluorescence detection system MF10S.

We developed a system that uses the single-molecule fluorescence detection system MF10S to assess quantitatively the activity of WRN helicase, the product of the causative gene of Werner syndrome that includes premature ageing. Double-strand DNA substrates labeled with the fluorescence dye TAMRA at the 5' end and with a quencher at the 3' end of the counter strand were incubated with a single trapper oligonucleotide and Werner helicase, and the resultant single DNA fragments labeled with TAMRA produced by the unwinding of WRN helicase were detected using the MF10S. The results using this system and those using polyacrylamide gel electrophoresis were well correlated. The MF10S system provides a quantitative analysis that is much faster, simpler, and more economical than systems using polyacrylamide gel electrophoresis and radioisotopes, and could be used as a quantitative analysis system for Werner helicase and other DNA helicase activities.

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase, in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.

Differential sensitivity between Fks1p and Fks2p against a novel beta -1,3-glucan synthase inhibitor, aerothricin3 [corrected].

Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity. Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-glucan synthase inhibitor, aerothricin3 [corrected]. To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 [corrected]. As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 [corrected] sensitivity when the region was replaced with a corresponding region of Fks1p. In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis. Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 [corrected]. On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 [corrected]. These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 [corrected] sensitivity.

A novel copper-binding protein with characteristics of a metallothionein from a clinical isolate of Candida albicans.

It is known that clinical isolates of Candida albicans exhibit a high level of resistance to copper salts, although the molecular basis of this resistance is not clear. To investigate this, a novel copper-binding protein was purified from a clinical isolate of C. albicans. The protein was extracted from yeast cells after an induction period of 10 h in a copper-containing suspension medium. It was further purified by size-exclusion chromatography, ultrafiltration and reverse-phase HPLC. All protein fractions were analysed for their protein and copper contents. The copper/protein ratio increased steadily throughout the purification process; the most highly purified fraction showed a 210-fold increase compared to the whole-cell extract, with a recovery of 0.03%. The molecular mass of the protein was 10,000 Da and a reconstitution study using the purified apoprotein suggested that the equivalent extent of Cu(I) binding was approximately 14 mol eq. The amino-terminal segment of the copper-binding protein revealed three Cys-Xaa-Cys motifs, which is typical of a metallothionein (MT), and showed significant homology with mammalian MTs with respect to the positions of the cysteine residues. This is the first report of the isolation of a copper-binding protein from C. albicans.