Expression of Bacillus sp. β-xylosidase gene (xylB) in Saccharomyces cerevisiae

-Xylosidase gene (xylB) from Bacillus sp. was amplified and inserted between GAL10 promoter and GAL7 terminator. For the secretory production of xylB in Saccharomyces cerevisiae, in-frame fusion of the exoinulinase signal sequence (INU1s) of Kluyveromyces marxianus to the upstream of xylB was conducted. When a transformant of S. cerevisiae harboring the resulting plasmid was grown on galactose-containing medium, most of -xylosidase activity was localized in the periplasmic space of yeast and a maximum total activity reached about 2.9 unit ml^–1 at 42 h cultivation. The recombinant -xylosidase was produced as an active dimer form.     

T-DNA insertional mutagenesis for functional genomics in rice

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.     

Structure of the terminal oxygenase component of angular dioxygenase, carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.     

A carbohydrate fraction, AIP1, from Artemisia iwayomogi down-regulates Fas gene expression and suppresses apoptotic death of the thymocytes induced by 2,3,7,8-tectrachlorodibenzo-p-dioxin

Apoptotic death of mouse thymocytes in vitro, as induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), involves the up-regulation of Fas gene expression, while a carbohydrate fraction, AIP1, from Artemisia iwayomogi suppresses the death of thymocytes in culture along with the down-regulation of Fas gene expression. We have now investigated whether the AIP1 fraction modulates TCDD-induced thymocyte death. When treated with TCDD and AIP1 fraction together, the thymocytes do not show apoptosis induced by the TCDD treatment. The AIP1 supplementation to the TCDD treatment also down-regulates the TCDD-induced Fas gene up-regulation. These findings indicate that the AIP1 fraction suppresses TCDD-induced thymocyte apoptosis through the modulation of Fas gene expression. Revisions requested 29 October 2004; Revisions received 10 December 2004     

Presence of a nucleoplasmic complex composed of the inositol 1,4,5-trisphosphate receptor/Ca2+ channel, chromogranin B, and phospholipids

Although the inositol 1,4,5-trisphosphate (IP(3)) induced nuclear Ca(2+) releases have been shown to play key roles in nuclear functions, the presence and operation of the IP(3)-dependent Ca(2+) control mechanism in the nucleoplasm have not been shown. Recently, we found the presence of a high-capacity, low-affinity Ca(2+)-storage protein chromogranin B (CGB) and all three IP(3) receptor (IP(3)R) isoforms in the nucleoplasm, localizing widely in both the heterochromatin and euchromatin regions. In view of the essential role of CGB-IP(3)R coupling in IP(3)-dependent Ca(2+) release in the endoplasmic reticulum, the potential coupling between CGB and the IP(3)Rs in the nucleoplasm was investigated. Hence, we found in the present study the presence of a nucleoplasmic complex, which is composed of the IP(3)R, CGB, and phospholipids, with an estimated molecular mass of approximately 2-3 x 10(7) Da, suggesting the possibility of the presence of an IP(3)-sensitive Ca(2+) store in the nucleoplasm. Moreover, double-labeling immunogold electron microscope studies showed the colocalization of all three IP(3)R isoforms with CGB to the extent that the majority of each IP(3)R isoform-labeling gold particles found in the nucleoplasm was literally next to the CGB-labeling gold particles. In line with the potential existence of an IP(3)-dependent vesicular nucleoplasmic Ca(2+) store, our preliminary results indeed showed a sudden release of Ca(2+) from a putative nucleoplasmic Ca(2+) store in response specifically to IP(3) but not to inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate.     

Hypochlorous acid-mediated mitochondrial dysfunction and apoptosis in human hepatoma HepG2 and human fetal liver cells: role of mitochondrial permeability transition

Liver cirrhosis is often preceded by overt signs of hepatitis, including parenchymal cell inflammation and infiltration of polymorphonuclear (PMN) leukocytes. Activated PMNs release both reactive oxygen species and reactive halogen species, including hypochlorous acid (HOCl), which are known to be significantly cytotoxic due to their oxidizing potential. Because the role of mitochondria in the hepatotoxicity attributed to HOCl has not been elucidated, we investigated the effects of HOCl on mitochondrial function in the human hepatoma HepG2 cell line, human fetal liver cells, and isolated rat liver mitochondria. We show here that HOCl induced mitochondrial dysfunction, and apoptosis was dependent on the induction of the mitochondrial permeability transition (MPT), because HOCl induced mitochondrial swelling and collapse of the mitochondrial membrane potential with the concomitant release of cytochrome c. These biochemical events were inhibited by the classical MPT inhibitor cyclosporin A (CSA). Cell death induced by HOCl exhibited several classical hallmarks of apoptosis, including annexin V labeling, caspase activation, chromatin condensation, and cell body shrinkage. The induction of apoptosis by HOCl was further supported by the finding that CSA and caspase inhibitors prevented cell death. For the first time, these results show that HOCl activates the MPT, which leads to the induction of apoptosis and provides a novel insight into the mechanisms of HOCl-mediated cell death at sites of chronic inflammation.     

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.     

Neuroprotective effects of naturally occurring biflavonoids

We examined neuroprotective effects of naturally occurring biflavonoids on oxidative stress-induced and amyloid beta peptide-induced cell death in neuronal cells. Among the nine biflavonoids tested, amentoflavone, ginkgetin, and isoginkgetin exhibited strong neuroprotection against cytotoxic insults induced by oxidative stress and amyloid beta, suggesting their therapeutic potential against neurodegenerative diseases, including ischemic stroke and Alzheimer's disease.