Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning-related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.     

Noise reduction from genotyping microarrays using probe level information

Genomic copy number change is one of the important phenomenon observed in cancer and other genetic disorders. Recently oligonucleotide microarrays have been used to analyze changes in the copy number. Although high density microarrays provide genome wide useful data on copy number, they are often associated with substantial amount of experimental noise that could affect the performance of the analyses. We used the high density oligonucleotide genotyping microarrays in our experiments that uses redundant probe tiling approach for individual SNPs. We found that the noise in the genotyping microarray data is associated with several experimental steps during target preparation and devised an algorithm that takes into account those experimental parameters. Additionally, defective probes that do not hybridize well to the target and therefore could not be modified inherently were detected and omitted automatically by using the algorithm. When we applied the algorithm to actual datasets, we could reduce the noise substantially without compressing the dynamic range. Additionally, combinatorial use of our noise reduction algorithm and conventional breakpoint detection algorithm successfully detected a microamplification of c-myc which was overlooked in the raw data. The algorithm described here is freely available with the software upon request to all non-profit researchers.     

The Identification of Fucosterol in the Marine Brown Algae Hizikia fusiformis

The enzyme uridine diphosphate N-acetylglucosamine pyrophosphorylase was purified about 330-fold from an extract of baker’s yeast by the treatment with protamine sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G–150. The purified enzyme was proved to be homogeneous by disc gel electrophoresis. The molecular weight was determined to be approximately 37,000 by gel filtration. The enzyme had an optimum reactivity in the pH range of 7.5-8.5 and was stable at 4°C in potassium phosphate buffer, pH 7.5, containing 0.1 mm dithiothreitol, but was unstable when stored at ?20°C. The addition of dithiothreitol also increased the thermal stability of enzyme. The enzyme was specific for UDP-N-acetylglucosamine as substrate, and none of the other sugar nucleotides could serve as nucleotide substrate. The estimated values of Km were 6.1 × 10^?3 m for UDP-N-acetylglucosamine and 5.0 × 10^?3 m for inorganic pyrophosphate. The enzyme required some divalent cations for activity. Magnesium ion was the most effective among the cations tested. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol.     

Adsorption of Hemoglobin and Casein onto Air Bubbles

Effects of pH, sodium chloride, temperature, and particle size were studied on the adsorption of proteins onto air bubbles. The results suggested that the froth flotation could be applied to the recovery of dissolved proteins with a comparable efficiency to the active sludge method. Such a strategy was proposed as to strip the dissolved hemoglobin under alkaline conditions or in the presence of sodium chloride, and to concentrate it under acidic conditions. Temperature was of little effect. Flotation of the precipitated proteins was much more efficient than that of the dissolved proteins. Particle size greatly affected the efficiency of flotation of the precipitated proteins.     

Chlorophycean parasite on a marine fish,Sillago japonica (Japanese sillago)

A green spotted Japanese sillago (Sillago japonica) was caught by a fisherman and brought to the laboratory for pathological inspection. The green spots were abundant on the lateral line and more extensively so within the mouth cavity. In both sites, green spots were embedded within the fish flesh and formed 2–3 mm dome-shaped colonies. SEM revealed these colonies to harbor numerous unknown cells with small, surface warts (ornamentations). Molecular analysis showed the cells were Desmodesmus (D. komarekii), a common freshwater coccoid green alga found in ponds and rivers worldwide. It is uncertain how the host fish came to be infected with the alga which was not merely attached externally but embedded within the flesh and inside the mouth cavity. This is the first case of parasitic form of coccoid green algae in marine fish and provides new insights into the variable nature of green algae.     

Generation of hydroxyl radical from linoleic acid hydroperoxide in the presence of epinephrine and iron.

When linoleic acid hydroperoxide was reacted with ferrous iron, the electron spin resonance signals characteristic of the spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide and the hydroxyl radical were detected. Although the signals were not detected with the hydroperoxide and ferric iron, they were actually found if epinephrine was added, indicating that the hydroxyl radical could be generated from the hydroperoxide upon reduction of the latter with ferrous iron formed by epinephrine. The possible generation of the hydroxyl radical from lipid peroxides in vivo was discussed from the viewpoint of pathogenesis of lipid peroxide-related diseases.     

Involvement of nectin in inactivation of integrin alpha(v)beta(3) after the establishment of cell-cell adhesion

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.     

Absence of Ku70 gene obliterates X-ray-induced lacZ mutagenesis of small deletions in mouse tissues

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.     

Intercomparison study on 152Eu gamma ray and 36Cl AMS measurements for development of the new Hiroshima–Nagasaki Atomic Bomb Dosimetry System 2002 (DS02)

In the process of developing a new dosimetry system for atomic bomb survivors in Hiroshima and Nagasaki (DS02), an intercomparison study between (152)Eu and (36)Cl measurements was proposed, to reconcile the discrepancy previously observed in the Hiroshima data between measurements and calculations of thermal neutron activation products. Nine granite samples, exposed to the atomic-bomb radiation in Hiroshima within 1,200 m of the hypocenter, as well as mixed standard solutions containing known amounts of europium and chlorine that were neutron-activated by a (252)Cf source, were used for the intercomparison. Gamma-ray spectrometry for (152)Eu was carried out with ultra low-background Ge detectors at the Ogoya Underground Laboratory, Kanazawa University, while three laboratories participated in the (36)Cl measurement using accelerator mass spectrometry (AMS): The Technical University of Munich, Germany, the Lawrence Livermore National Laboratory, USA and the University of Tsukuba, Japan. Measured values for the mixed standard solutions showed good agreement among the participant laboratories. They also agreed well with activation calculations, using the neutron fluences monitored during the (252)Cf irradiation, and the corresponding activation cross-sections taken from the JENDL-3.3 library. The measured-to-calculated ratios obtained were 1.02 for (152)Eu and 0.91-1.02 for (36)Cl, respectively. Similarly, the results of the granite intercomparison indicated good agreement with the DS02 calculation for these samples. An average measured-to-calculated ratio of 0.98 was obtained for all granite intercomparison measurements. The so-called neutron discrepancy that was previously observed and that which included increasing measured-to-calculated ratios for thermal neutron activation products for increasing distances beyond 1,000 m from the hypocenter was not seen in the results of the intercomparison study. The previously claimed discrepancy could be explained by insufficient understanding of the measured data.