Studies on the anorectic effect of N-acylphosphatidylethanolamine and phosphatidylethanolamine in mice

N-acyl-phosphatidylethanolamine is a precursor phospholipid for anandamide, oleoylethanolamide, and other N-acylethanolamines, and it may in itself have biological functions in cell membranes. Recently, N-palmitoyl-phosphatidylethanolamine (NAPE) has been reported to function as an anorectic hormone secreted from the gut and acting on the brain (Gillum et al., [5]). In the current study, two of our laboratories independently investigated whether NAPE metabolites may be involved in mediating the anorectic action of NAPE i.p. injected in mice. Thus, the anorectic activity of a non-hydrolysable NAPE analogue, having ether bonds instead of ester bonds at sn1 and sn2 was compared with that of NAPE in molar equivalent doses. Furthermore, the anorectic effect of NAPE in NAPE-hydrolysing phospholipase D knockout animals was investigated. As negative controls, the NAPE precursor phosphatidylethanolamine and the related phospholipids phosphatidylcholine and phosphatidic acid were also tested. All compounds except one were found to inhibit food intake, raising the possibility that the effect of NAPE is non-specific.     

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D(3) derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1alpha,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1), whereas 1,25(OH)(2)D(3) was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.     

Retardation of the G2-M phase progression on gene disruption of RNA binding protein Sam68 in the DT40 cell line

Genes encoding the NarG and NarH subunits of the molybdo-iron-sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe-4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe-4S] cluster and three [4Fe-4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain.     

Establishment of X-linked Alport syndrome model mice with a Col4a5 R471X mutation

Alport syndrome (AS) is an inherited disorder characterized by glomerular basement membrane (GBM) abnormality and development of chronic kidney disease at an early age. The cause of AS is a genetic mutation in type IV collagen, and more than 80% of patients have X-linked AS (XLAS) with mutation in COL4A5. Although the causal gene has been identified, mechanisms of progression have not been elucidated, and no effective treatment has been developed. In this study, we generated a Col4a5 mutant mouse harboring a nonsense mutation (R471X) obtained from a patient with XLAS using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system. Col4a5 mRNA and protein expressions were not observed in the kidneys of hemizygous R471X male mice. R471X mice showed proteinuria and hematuria. Pathology revealed progression of glomerulosclerosis and interstitial fibrosis by age. Electron microscopy identified irregular thickening in GBM accompanied by irregular lamination. These observations were consistent with the clinical and pathological features of patients with AS and other established models. In addition, our mice models develop end-stage renal disease at the median age of 28 weeks, much later compared to previous models much more consistent with clinical course of human XLAS. Our models have advantages for future experiments in regard with treatment for human XLAS.     

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 μM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.     

Plasticity of flower longevity in Corydalis ambigua

We investigated the longevity of individual flowers of Corydalis ambigua Cham. et Schlecht. during different periods of pollinator activity and at different temperatures. To measure potential (unpollinated) flower longevity of C. ambigua, this study was conducted at forest islands where pollinator visits were scarce. The longevity of individual flowers of C. ambigua indicated high plasticity. The longevity of unpollinated flowers in natural pollination ranged from 2 to 25 days and continuously decreased with the date of flower opening. The temperature increased as the flowering season progressed. Furthermore, the greenhouse transplanting experiment showed that higher temperatures shorten the life span of flowers. The longevity of pollinated flowers subjected to hand pollination of newly opened flowers was shorter than that of unpollinated flowers in natural pollination regardless of the date of flower opening. These results showed that not only high temperature but also pollination shortens flower longevity. We discuss the role of plasticity in flower longevity for C. ambigua in relation to pollination success and reduction in the maintenance cost of the flowers.     

A Monoclonal Antibody Against the PSTAIR Sequence of p34cdc2, Catalytic Subunit of Maturation-Promoting Factor and Key Regulator of the Cell Cycle

A homolog of the serine/threonine protein kinase (p34^cdc2), encoded by the cdc2 ⁺ gene of the fission yeast ( Schizosaccharomyces pombe ), is a catalytic subunit of maturation-promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of p34^cdc2 This antibody recognizes 31–34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either p34^cdc2 itself or proteins highly homologous to p34^cdc2 in the given species, since, in all species studies to date, they are all precipitated with p13^suc1, the fission yeast suc 1⁺ gene product, which binds to p34^cdc2 with high specificity. The anti-PSTAIR immunoprecipitate had no histone H1 kinase activity and did not contain cyclin B, suggesting that the PSTAIR region is masked when p34^cdc2 forms a complex with cyclin B as an active kinase. Immunoblotting with the anti-PSTAIR antibody demonstrated that the fastest-migrating form of p34^cdc2 homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34^cdc2 The observed broad cross-reactivity of the anti-PSTAIR antibody against p34^cdc2 homologues in various species should permit us to examine the role of p34^cdc2 homologues in the regulation of the cell cycle in a variety of organisms.     

Prediction of birch airborne pollen counts by examining male catkin numbers in Hokkaido,northern Japan

Birch pollen is a very common cause of pollinosis in Hokkaido, northern Japan. Birch airborne pollen concentrations vary each year; hence, the development of a method for predicting annual airborne pollen concentration is very important in preventing widespread symptoms of pollinosis. In the current study, we investigated airborne pollen counts and male catkin numbers (male flower index) of birch in four cities of Hokkaido between 2002 and 2008. Airborne pollen surveys were conducted using Durham’s sampler, and male catkin numbers determined for three major birch species (Betula platyphylla var. japonica, B. emanii, and B. maximowicziana). We found an annual variation in male flower index for all the three birch species investigated. This variation worked in combination with the amount of precipitation during the pollen season to influence total birch pollen counts. In conclusion, the male catkin numbers of three major birch species reliably predict airborne pollen counts in Hokkaido, but only when the effect of precipitation during pollen season is considered.     

Physical and functional mapping of Tn2603, a transposon encoding ampicillin, streptomycin, sulfonamide, and mercury resistance

Summary A map of cleavage sites for restriction endonucleases EcoR1, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.:: Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.