Genetic activity of bleomycin in Escherichia coli

The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated λ-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies. (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes. (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain. (3) The plating efficiency of UV-irradiated λ-phage increased by Blm treatment of the host cell. This increase was not observed in the umuC mutant. The plating efficiency of UV-irradiated λ-phage was drastically reduced in the lexA and recA strains treated with Blm. (4) No significant increase of the reversion of nonsense mutation (his-4 to His⁺) in AB1157 by the treatment of Blm was observed. Possible implications of these results are discussed.     

PHYLOGEOGRAPHY OF CUTLERIA CYLINDRICA (CUTLERIALES,PHAEOPHYCEAE) IN NORTHEASTERN ASIA,AND THE IDENTITY OF AN INTRODUCED POPULATION IN CALIFORNIA1

Cutleria cylindrica Okamura was described from Japan in 1902 and has been reported only from northwestern Asia until its relatively recent discovery in California, USA, and Baja California, Mexico. To clarify the genetic relationships within and among the disjunct populations, we carried out a molecular phylogenetic study, as well as the examination of sex ratio and the life‐history patterns, of populations in Japan, Korea, and California. Based on the DNA sequences of mitochondrial genes cox2, cox3, the open reading frame (ORF) region, and the spacer between cox3 and ORF, a total of 23 haplotypes were detected in the 85 individuals from 20 localities in Japan, Korea, and California. All localities in Japan and Korea included multiple haplotypes, but only a single haplotype was found in California. There was a positive relationship between distance and genetic divergence in Japan and Korea. The single haplotype found in California was the same as one occurring in Japan (Aomori Pref. and Fukuoka Pref.) and Korea (Daedaepo, Pusan). Both male and female gametophytes were distributed in most northeastern Asian populations. Only female gametophytes, developing parthenogenetically from female gametes, were found in California and Aomori Pref., Japan. On the basis of these results, we conclude that the disjunct population of C. cylindrica in California originated from a relatively recent introduction from Japan and shares its origin with the parthenogenetic population in the Tsugaru Strait.     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

B cell receptor-mediated Syk-independent activation of phosphatidylinositol 3-kinase,Ras, and mitogen-activated protein kinase pathways

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.     

Fluorescent indicators for imaging protein phosphorylation in single living cells

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded fluorescent indicators, named phocuses. Two different color mutants of green fluorescent protein (GFP) were joined by a tandem fusion domain composed of a substrate domain for the protein kinase of interest, a flexible linker sequence, and a phosphorylation recognition domain that binds with the phosphorylated substrate domain. Intramolecular interaction of the substrate domain and the adjacent phosphorylation recognition domain within a phocus was dependent upon phosphorylation of the substrate domain by protein kinase, which influenced the efficiency of fluorescence resonance energy transfer (FRET) between the GFPs within a phocus. In the present study, we employed phocuses composed of insulin signaling proteins to visualize protein phosphorylation by the insulin receptor. This method may provide a general approach for studying the dynamics of protein phosphorylation-based signal transduction in living cells.     

Possible involvement of hydroxyl radical on the stimulation of tetrahydrobiopterin synthesis by hydrogen peroxide and peroxynitrite in vascular endothelial cells

We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.