Inhibition of acidic sphingomyelinase by xanthone compounds isolated from Garcinia speciosa

Sphingomyelinase is considered to be involved in the regulation of apoptosis and cell growth. In the course of our screening for acidic sphingomyelinase inhibitors we isolated three xanthone compounds, alpha-mangostin, cowanin, and cowanol, from the bark of Garcinia speciosa. These compounds competitively inhibited bovine brain-derived acidic sphingomyelinase with IC(50) values of 14.1, 19.2, and 10.9 microM, respectively and inhibited the acidic sphingomyelinase more effectively than the neutral sphingomyelinase of bovine brain. alpha-Mangostin inhibited the acidic sphingomyelinase in the most selective manner. alpha-Mangostin was chemically modified and its structure-activity relationships are discussed.     

A novel enzyme, L-tryptophan oxidase, from a basidiomycete, Coprinus sp. SF-1: purification and characterization

A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated "L-Trp oxidase (deaminating and decarboxylating)". The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50 degrees C. The enzyme was stable at pH 6.0-10.5 and below 50 degrees C, and at 4 degrees C for 1 year.     

Distribution of the cyanobacterium Richelia intracellularis as an epiphyte of the diatom Chaetoceros compressus in the western Pacific Ocean

The few available records on the association of the diazotrophheterocystous cyanobacterium Richelia intracellularis epiphyticallywith Chaetoceros compressus are restricted to the Indian andwestern Pacific Oceans, whereas the association of R. intracellularisas an endosymbiont in other diatoms is ubiquitous in warm oceans.From ten cruises in the western Pacific Ocean, the Richelia-Chaetocerosconsortia were exclusively observed in the periphery of thegeographic proliferations of C. compressus, coinciding withthe overlapping area of the populations of asymbiotic C. compressusand R. intracellularis as an endosymbiont in Rhizosolenia clevei.     

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents

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Palmitoyl lactic acid induces adipogenesis and a brown fat-like phenotype in 3T3-L1 preadipocytes

Brown adipose tissue is specialized to generate heat by dissipating chemical energy and may provide novel strategies for obesity treatment in humans. Recently, advances in understanding the pharmacological and dietary agents that contribute to the browning of white adipose tissue have been made to alleviate obesity by promoting energy expenditure. Krill oil is widely used as a health supplement in humans. In this study, the components from krill oil that promote adipogenesis of 3T3-L1 cells were screened to reveal palmitoyl lactic acid (PLA) as a promoter of adipogenesis. The PLA-induced adipocytes contained large number of small lipid droplets. Moreover, similar to the peroxisome proliferator-activated receptor (PPAR)γ agonists, pioglitazone and rosiglitazone, PLA significantly enhances adipogenesis in the presence of dexamethasone compared with PLA alone. Treatment with PLA causes a brown fat-like phenotype in 3T3-L1 cells by enhanced expression of various brown/beige cell-specific genes, such as PR domain containing 16 (Prdm16) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Pgc1a), as well as adiponectin gene. The expression profile of the brown/beige cell-specific genes induced by PLA was similar to that of the PPARγ agonist in 3T3-L1 cells. Our findings suggest that PLA induces a brown fat-like phenotype and, thus, likely has therapeutic potential in treating obesity.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.     

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.     

Transfection of Corynebacterium glutamicum with Temperate Phage ϕ CG1

Versatile plasmid vectors useful for gene cloning in Brevibacterium lactofermentum, a glutamic acid-producing bacterium, have been constructed. The trimethoprim (Tp)-resistant dihydrofolate reductase gene derived from chromosomal DNA of the Tp-resistant mutant of B. lactofermentum was introduced into pAM330, a cryptic plasmid in B. lactofermentum. The constructed cloning vector pAJ228 (7.6kb) exists in 10 to 20 copies in cells of B. lactofermentum and donated Tp-resistance, which is a useful selective marker of transformants. pAJ228 was further improved to a versatile plasmid vector pAJ224 having some profitable characteristics such as smaller size (3.7 kb), higher copy number (60 ~ 80 copies), and additional useful cloning sites (Bam HI, Pst I and Sal I) equipped with two different promoters arranged at both orientations for the expression of passenger DNA without promoter. These plasmids were stably retained in B. lactofermentum even in the absence of Tp over many generations. Thus, they have been found very powerful vectors for gene cloning in Brevibacterium and the related bacteria.     

Needle browning and death in the flagged crown of Abies mariesii in the timberline ecotone of the alpine region in central Japan

In the flagged crown, which is asymmetric growth formed by severe stresses during winter in alpine regions, needles of evergreen conifers often became brown and died in early spring, but did not in a cushion-shaped crown. Needle browning and death is thought to occur by increasing transpiration due to a thinner cuticle or mechanical damage to the cuticle by wind-born snow and ice particles. To confirm whether the needle browning and death in the flagged crown of Abies mariesii Mast., in the alpine region of Japan conform with this concept, we assessed mechanical damage of the needle cuticle in a timberline ecotone and evaluated the effect of cuticle thickness on cuticular resistance. Mechanical damage on needle cuticles of A. mariesii was not observed. In the cushion-shaped crown, epicuticular wax covered the cuticle and plugged stomatal antechambers. In the flagged crown, epicuticular wax was mostly absent. Cuticular resistance in the flagged crown was lower than that in the cushion-shaped crown. However, the cuticle in the flagged crown was thicker than that in the cushion-shaped crown. The needle browning and death in the flagged crown of A. mariesii occurred even though needle cuticles were not mechanically damaged. The thicker cuticle of the flagged crown may play a role in other stresses. To estimate desiccation stress in relation to the cuticle, we need to elucidate not only cuticular resistance and cuticle thickness, but also cuticle quality and structure.