Functional expression of Acetabularia acetabulum vacuolar H(+)-pyrophosphatase in a yeast VMA3-deficient strain

The function of the translation product of cDNA for Acetabularia vacuolar H(+)-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H(+)-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.     

Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and the increase of their expression by heat shock and exposure to a hazardous chemical

We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Production of mitochondrial DNA transgenic mice using zygotes

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.     

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.     

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.     

Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.     

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by ¹H and ¹³C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.     

Down-regulation of endodermal Shh is required for gland formation in chicken stomach

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.