Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.     

Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae)

The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi.     

Oxytocin-induced prostaglandin E2 (PGE2) synthesis is regulated by progesterone via oxytocinase in Ishikawa cells.

Cell-surface oxytocinase inactivates oxytocin and regulates oxytocin stimulation. We reported that oxytocinase in human endometrial epithelial cells was secreted from the cell membrane in the mid-secretory phase and disappeared from the cell surface. On the other hand, the production in human endometrium of prostaglandins, which play important roles in the reproductive process, has been reported to be upregulated by oxytocin. We investigated whether progesterone affects cell-surface oxytocinase and oxytocin-induced prostaglandin E2 (PGE2) production in vitro. Progesterone induced secretion of oxytocinase into the culture medium, which resulted in a decrease in cell-surface oxytocinase. Production of PGE2 was increased slightly by oxytocin without progesterone, and significantly with progesterone. The inhibition of oxytocinase activity by amastatin had a similar effect to the loss of cell-surface oxytocinase caused by progesterone. It is therefore likely that the cell-surface oxytocinase of endometrial epithelial cells modified by progesterone plays an important role in the function of the human endometrium through PGE2.     

Hyperphagia and Obesity in Na,K‐ATPase α2 Subunit‐Defective Mice

Objective: The Na,K‐ATPase α2 subunit gene (Atp1a2) is expressed in the brain, skeletal muscles, heart, and adipocytes. Specific function of the α2 subunit, such as involvement in differentiation and function of adipocytes, has not been addressed. The aim of this study was to examine whether Atp1a2‐defective heterozygous mice show obesity and reveal the mechanisms underlying the obesity. Research Methods and Procedures: We measured the differentiation and glucose uptake function of in vitro‐differentiated adipocytes derived from embryonic fibroblasts of Atp1a2‐defective mice. Food intake, body temperature, metabolic rate, and spontaneous activity and mRNA levels of neuropeptide genes were compared between the heterozygous and wild‐type adult mice. Results: Atp1a2 heterozygous female mice developed obesity after middle age. The time course of in vitro adipocyte differentiation of embryonic fibroblasts isolated from wild type, heterozygous, and homozygous mice was not different, glucose and Rb uptake activities of the in vitro‐differentiated adipocytes were not altered, and the effects of insulin on glucose uptake and those of monensin and ouabain on Rb uptake were similar among the genotypes. However, food intake in the light phase was significantly greater in the heterozygous mice than the wild type in the 24‐hour dark‐light cycle, whereas it was similar under constant‐light condition. Body temperature, metabolic rate at rest, and spontaneous motor activity of the heterozygous mice were similar to those of the wild type. Orexin mRNA level was lower in heterozygous than wild‐type mice. Discussion: The Na,K‐ATPase α2 subunit is not involved in the differentiation or in glucose and Rb uptake function of in vitro‐differentiated adipocytes. Hyperphagia is the likely primary cause of obesity in Atp1a2 heterozygous mice.     

Comparative study of specific alpha-1,2-glucosidase activity toward glucosyl galactosyl hydroxylysine in various animal species

1. Assay of the activity of alpha-1,2-glucosidase was completed within 10 min using reversed-phase high performance liquid chromatography and purified dansylated glucosyl galactosyl hydroxylysine as the substrate. 2. A comparative study was made on the enzyme activity of liver homogenate from eight animal species, mouse, frog, chicken, rabbit, pig, rat, human, bovine and that of a spinach leaf homogenate. alpha-1,2-glucosidase activity in human and bovine liver was very low, and that of alpha-1,2-glucosidase could not be detected in the spinach homogenate as expected. 3. 1-deoxynojirimycin, a well known potent inhibitor of alpha-1,2-glucosidases which act on the N-glycosidic type carbohydrate chain, also inhibited alpha-1,2-glucosidase acting specificity on glucosyl galactosyl hydroxylysine derived from the collagen molecule.     

Evolution of Genomic Structures on Mammalian Sex Chromosomes

Throughout mammalian evolution, recombination between the two sex chromosomes was suppressed in a stepwise manner. It is thought that the suppression of recombination led to an accumulation of deleterious mutations and frequent genomic rearrangements on the Y chromosome. In this article, we review three evolutionary aspects related to genomic rearrangements and structures, such as inverted repeats (IRs) and palindromes (PDs), on the mammalian sex chromosomes. First, we describe the stepwise manner in which recombination between the X and Y chromosomes was suppressed in placental mammals and discuss a genomic rearrangement that might have led to the formation of present pseudoautosomal boundaries (PAB). Second, we describe ectopic gene conversion between the X and Y chromosomes, and propose possible molecular causes. Third, we focus on the evolutionary mode and timing of PD formation on the X and Y chromosomes. The sequence of the chimpanzee Y chromosome was recently published by two groups. Both groups suggest that rapid evolution of genomic structure occurred on the Y chromosome. Our re-analysis of the sequences confirmed the species-specific mode of human and chimpanzee Y chromosomal evolution. Finally, we present a general outlook regarding the rapid evolution of mammalian sex chromosomes.     

Specific arbuscular mycorrhizal fungi associated with non-photosynthetic Petrosavia sakuraii (Petrosaviaceae)

Mycorrhizal fungi in roots of the achlorophyllous Petrosavia sakuraii (Petrosaviaceae) were identified by molecular methods. Habitats examined were plantations of the Japanese cypress Chamaecyparis obtusa in Honshu, an evergreen broad-leaved forest in Amami Island in Japan and a mixed deciduous and evergreen forest in China. Aseptate hyphal coils were observed in root cortical cells of P. sakuraii, suggesting Paris-type arbuscular mycorrhiza (AM). Furthermore, hyphal coils that had degenerated to amorphous clumps were found in various layers of the root cortex. Despite extensive sampling of P. sakuraii from various sites in Japan and China, most of the obtained AM fungal sequences of the nuclear small subunit ribosomal RNA gene were nearly identical and phylogenetic analysis revealed that they formed a single clade in the Glomus group A lineage. This suggests that the symbiotic relationship is highly specific. AM fungi of P. sakuraii were phylogenetically different from those previously detected in the roots of some mycoheterotrophic plants. In a habitat in C. obtusa plantation, approximately half of the AM fungi detected in roots of C. obtusa surrounding P. sakuraii belonged to the same clade as that of P. sakuraii. This indicates that particular AM fungi are selected by P. sakuraii from diverse indigenous AM fungi. The same AM fungi can colonize both plant species, and photosynthates of C. obtusa may be supplied to P. sakuraii through a shared AM fungal mycelial network. Although C. obtusa plantations are widely distributed throughout Japan, P. petrosavia is a rare plant species, probably because of its high specificity towards particular AM fungi.