Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase

The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.     

Variation in metal concentrations in the brown alga Undaria pinnatifida in Osaka Bay, Japan

In order to evaluate the usefulness of a biomonitoring system using seaweeds for assessing the geographic distribution of metal ions in coastal seawaters, the metal concentrations in the sporophytes of an annual kelp, Undaria pinnatifida (Harvey) Suringar, were collected at 15 localities in Osaka Bay, Japan and compared. About 160 cm² of the blade was cut out from the central part of clean sporophytes, rinsed in filtered seawater using an ultrasonic cleaning bath, and freeze‐dried. After digestion with 12% HNO₃ in a microwave apparatus, metal concentrations in the samples were determined by inductively coupled plasma mass spectrometry. The concentrations (dry weight basis) of most examined elements were in the parts per million range; Cr, 0.48–3.18; Ni, 0.77–5.94; Cu, 3.20–43.8; Zn, 11.3–86.8; Pb, 0.14–3.53. Comparisons of metal compositions of the U. pinnatifida samples from the northeastern area of the bay, which has a large urban population and highly developed industries inland, showed high concentrations of Cd, Pb and Cu compared with the samples from the southwestern area of the bay where the population and industries are much smaller. This suggests that U. pinnatifida metal loads can be used as a marker to track the geographic distributions of the metal concentrations in coastal seawaters, reflecting inland human activities such as shipbuilding and repairing in port areas, and can be used as a useful biomonitoring system of coastal environments for long‐term trend.     

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.     

Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography

We developed a novel approach to enlarge phosphoproteome coverage using successive elution of phosphopeptides with various buffers in series from a single microcolumn packed with hydroxy acid-modified metal oxides, such as titania and zirconia. Elution conditions were investigated to maximize the recovery of phosphopeptides from three standard phosphoproteins. Secondary amines, such as piperidine and pyrrolidine, provided better efficiency than the conventional conditions using ammonium hydroxide and phosphate buffers. Furthermore, elution with these secondary amines provided unique phosphopeptides that were not eluted under the conventional conditions in the analysis of HeLa cell lysates. On the basis of these results, we fractionated phosphopeptides captured by a single metal oxide microcolumn using successive elution with 5% ammonium hydroxide solution, 5% piperidine solution and 5% pyrrolidine solution in series. We identified 1,803 nonredundant phosphopeptides from 100 microg of HeLa cells, which represented a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions. Since this approach is applicable to any single tip-based protocol without coupling with other enrichment methods, this simple strategy can be easily incorporated as an option into existing protocols for phosphopeptide enrichment, and would be suitable for high-throughput analysis in a parallel format.     

Nδ-benzoyl-l-ornithine and Nδ-benzoyl-l-γ-hydroxyornithine from Vicia pseudo-orobus

Two new non-protein amino acids, N^δ-benzoyl-l-ornithine and N^δ-benzoyl-l-γ-hydroxyornithine have been characterized from the seeds of Vicia pseudo-orobus.     

Chloroplast assemblage by mechanical stimulation and its intercellular transmission in diatom cells

Summary By touching a cell ofPleurosira laevis, a centric diatom, the chloroplasts in the cortical cytoplasm immediately migrate to the nuclear cytoplasm which is located at the center of the cell. The time required for migration, is about 10 s and that for restoration is about 15 min. The touch could be replaced by an electric stimulation. A 7 V pulse for 1 ms was required for the induction of chloroplast assemblage. To obtain chloroplast assemblage in 50% of the cells, the amount of time and voltage required were inversely proportional. Higher concentrations of KCl also induced chloroplast assemblage. Neither contact nor electric stimulation in Ca²⁺ free medium induced chloroplast assemblage. Verapamil, a Ca²⁺ channel blocker, in medium suppressed the chloroplast assemblage. Ca²⁺ ionophore caused the chloroplast assemblage. These results suggested that the depolarization of the plasma membrane was a trigger of the induction of chloroplast assemblage and the influx of Ca²⁺ to the cytoplasm through the channel was essential for this. The chloroplast assemblage was transmitted successively to the other cells of the filamentous colony, though these cells were linked only by a mucilaginous substance. Furthermore, the transmission occurred in cells which were placed separately. The maximum distance possible for this jumping transmission was 200 m in the culture medium. To search for the release of any chemical transmitter, contact stimulation was applied to one cell at the middle of a filamentous colony which was placed in water streaming along the axis of the filament. Chloroplast assemblage was induced more in downstream cells than in upstream ones. This result suggested the existence of some substance which mediated the transmission of the stimulation.     

Decrease of bone mineral density and muscle and/or strength in the leg during 20 days horizontal bed rest

Ten individuals underwent 20 days of horizontal bed rest for this study of the influence of muscle mass and strength on bone mineral density. Muscle mass volume and cross sectional area were measured using magnetic resonance imaging and bone mineral density was determined by dual energy X-ray absorptiometry before and after bed rest. Measurements were made at various parts of the leg, including the knee. Gender differences were also determined. Results are presented and discussed.