Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

The Caenorhabditis elegans eukaryotic initiation factor 5A homologue, IFF-1, is required for germ cell proliferation, gametogenesis and localization of the P-granule component PGL-1

Eukaryotic initiation factor 5A (eIF-5A) was originally isolated as a translation initiation factor. However, this function has since been reconsidered, with recent studies pointing to roles for eIF-5A in mRNA metabolism and trafficking [Microbiol. Mol. Biol. Rev. 66 (2002) 460; Eur. Mol. Biol. Org. J. 17 (1998) 2914]. The Caenorhabditis elegans genome contains two eIF-5A homologues, iff-1 and iff-2, whose functions in vivo were examined in this study. The iff-2 mutation causes somatic defects that include slow larval growth and disorganized somatic gonadal structures in hermaphrodites. iff-2 males show disorganized tail sensory rays and spicules. On the other hand, iff-1 mRNA is expressed in the gonad, and the lack of iff-1 activity causes sterility with an underproliferated germline resulting from impaired mitotic proliferation in both hermaphrodites and males. In spite of underproliferation, meiotic nuclei are observed, as revealed by presence of immunoreactivity to the anti-HIM-3 antibody; however, no gametogenesis occurs in the iff-1 gonads. These phenotypes are in part similar to the mutants affected in the components of P granules, which are the C. elegans counterparts of germ granules [Curr. Top Dev. Biol. 50 (2000) 155]. We found that localization of the P-granule component PGL-1 to P granules is disrupted in the iff-1 mutant. In summary, the two C. elegans homologues of eIF-5A act in different tissues: IFF-2 is required in the soma, and IFF-1 is required in the germline for germ cell proliferation, for gametogenesis after entry into meiosis, and for proper PGL-1 localization on P granules.     

Antibacterial activity of hydrolyzable tannins derived from medicinal plants against Helicobacter pylori

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time- and dose-dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN-28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose-dependent membrane-damaging activity. However, it remains to be elucidated whether their membrane-damaging activity directly contributes to their antibacterial action.     

Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.