Transcriptional regulation of nuclear orphan receptor,NOR-1, by Ca(2+)/calmodulin-dependent protein kinase cascade

It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.     

Geraniol-inducible glutathione S-transferase in cultured soybean cells

When the cultured cells of Glycine max (soybean) were treated with 5 mM geraniol as a chemical stress, an mRNA level was elevated in a rapid but transient increase. The mRNA was cloned and sequenced, and found to correspond to the mRNA encoding glutathione S-transferase (GST). The GST mRNA level and GST activity were elevated to maxima at 4-6 h and 8 h, respectively, after treatment of the cultures with geraniol. These indicate that GST is one of the geraniol-responsive factors in soybean cells.     

Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Synthesis of DNA conjugates by solid phase fragment condensation

Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.     

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.     

Tissue distribution,molecular cloning,and gene expression of cytosolic glutathione peroxidase in Japanese monkey

Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.     

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.     

Excision repair of uracil-containing DNA in a mitochondrial extract of Xenopus oocytes

A mitochondrial extract of Xenopus oocytes was prepared to characterize DNA repair reactions operating in mitochondria. We asked whether and how uracils, spontaneously formed in DNA as a result of cytosine deamination, would be repaired in the extract. When a 40-mer oligonucleotide duplex with a single deoxyuridine at position 21 was incubated in the extract, incision took place at the 5' side of the lesion, and the uracil-containing strand was mostly cleaved within 15 min. Subsequent repair DNA synthesis produced DNA fragments of various sizes. Analysis of these repair intermediates indicated that the 3' border of the repair patches was at position 22. Completion of the repair reaction however was rare in this system, due in part to insufficient ligation activity of the extract.