Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.     

Retardation of removal of radiation-induced apoptotic cells in developing neural tubes in macrophage galactose-type C-type lectin-1-deficient mouse embryos

MGL1/CD301a is a C-type lectin that recognizes galactose and N-acetylgalactosamine as monosaccharides and is expressed on limited populations of macrophages and dendritic cells at least in adult mice. In this study, pregnant mice with Mgl1+/- genotype were mated with Mgl1+/- or Mgl1-/- genotype males, and the embryos were used to assess a hypothesis that this molecule plays an important role in the clearance of apoptotic cells. After X-ray irradiation at 1 Gy of developing embryos at 10.5 days post coitus (d.p.c.), the number of Mgl1-/- pups was significantly reduced as compared with Mgl1+/+ pups. Distributions of MGL1-positive cells, MGL2-positive cells, and apoptotic cells were histologically examined in irradiated Mgl1+/+ embryos. MGL1-positive cells were detected in the neural tube in which many cells undergo apoptosis, whereas MGL2-positive cells were not observed. Biotinylated recombinant MGL1 bound a significant portion of the apoptotic cells. When Mgl1+/+ and Mgl1-/- embryos were examined for the presence of apoptotic cells, similar numbers of apoptotic cells gave rise, but the clearance of these cells was slower in Mgl1-/- embryos than in Mgl1+/+ embryos. These results strongly suggest that MGL1/CD301a is involved in the clearance of apoptotic cells. This process should be essential in the repair and normal development of X-ray-irradiated embryos.     

A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis

We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5′-phosphorylated methylation-specific and 5′-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn²⁺-Phos-tag), which selectively captures the 5′-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid.     

Substrate Inhibition Competes with Halide Inhibition in Polyphenol Oxidase

Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis?CMenten kinetics at catechol concentrations of up to 1?mM, it slowly decreased at catechol concentrations above 2?mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.     

Triphala,a formulation of traditional Ayurvedic medicine,shows protective effect against X-radiation in HeLa cells

Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.     

Beta2-microglobulin required for cell surface expression of blastocyst MHC

Blastocyst MHC is a mouse MHC class Ib gene that is selectively expressed in blastocysts and placenta like human HLA-G, which protect fetal trophoblasts and some tumor cells from NK cell attack, and in TAP-dependent expression on the cell surface. We expressed blastocyst MHC cDNA in beta2-deficient EL-4 S3 or beta2m-transfected EL-4 S3 cells. In parental EL-4 S3 cells, only 47-kDa blastocyst MHC protein was expressed and retained in the cytoplasm. However, additional 51-kDa blastocyst MHC protein was expressed on the surface of beta2m-transfected EL-4 S3 cells. The 51-kDa protein was resistant to Endo-H, whereas the 47-kDa protein was sensitive for Endo-H. The results suggested that beta2m as well as TAP was necessary for the transportation of blastocyst MHC from endoplasmic reticulum to cell surfaces through the Golgi apparatus, similar to other MHC class I molecules.