Na+ / H+ exchanger-3 is involved in mouse blastocyst formation

The mouse blastocyst consists of the trophectoderm, the inner cell mass, and a fluid-filled cavity, the blastocoel. Formation and subsequent expansion of this cavity is important for further differentiation of the inner cell mass and successful implantation. Previous work provided evidence that vectorial transport of Na+ and CL- ions through the trophectoderm into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. As the activity of the Na+ / H+ exchanger (NHE) has been implicated as the exchanger responsible for facilitating the transtrophectodermal Na+ flux, the functional role of NHE in mouse blastocoel development was determined. Embryos were cultured in the presence of subtype-specific NHE inhibitors to examine the role of NHEs in blastocoel development. When 2-cell stage embryos were treated continuously with a specific inhibitor of NHE-1, cariporide, the embryos passed beyond the 8-cell stage and became blastocysts. However, in the presence of a specific inhibitor of NHE-3, S3226, the 2-cell stage embryos developed to the morula stage but formation of the blastocyst were inhibited in a dose-dependent manner. Cariporide did not inhibit the formation of the blastocoel cavity from the morula stage whereas S3226 did inhibit that process. S3226 also reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. An immunofluorescence study showed that NHE-3 was detected in the vicinity of the cell membrane of the trophectoderm, especially in the apical cell margins of the trophectoderm. These results suggest that NHE-3 is likely involved in blastocyst formation.     

Substrate specificity of beta-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing

We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified beta-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed beta-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified beta-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the beta-primeverosyl (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as beta-primeveroside, beta-vicianoside, beta-acuminoside, beta-gentiobioside and 6-O-alpha-L-arabinofuranosyl-beta-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl beta-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the beta-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.     

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.     

Analysis of mushroom diversity in successional young forests and equilibrium evergreen broad-leaved forests

Eight examination stations were constructed in young red pine forests and mature evergreen broad-leaved woods. A total of 32 quadrats of 10 × 10 m each were examined for vegetation and mushroom distribution and diversity once a week for one year. A total of 9707 individual mushrooms, amounting to 1470.23 g in dry weight, were analyzed for fungal communities. The results were arranged in a sequence of succession. The mushroom diversities, expressed as Shannon's , increased constantly to the equilibrium at around 3.0 in the climax forests. The distribution of the proportion of saprophytic fungi depicted a similar curve and reached equilibrium at around 0.3. The seasonal segregation in mushroom fruiting also developed and reached equilibrium. Ecological distances between the forests were estimated from the mushroom results and were compared with those estimated from the woody plant diversities. Fungal succession took place more rapidly or was more exaggerated than that of woody plants. From these results, the fungal succession and diversity in the climax forest was discussed.     

Immunocytochemical study of activin type IB receptor (XALK4) in Xenopus oocytes

Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.     

Reactivation of hyperglycemia-induced hypocretin (HCRT) gene silencing by N-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-O-GlcNAcylation on the HCRT gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the HCRT gene, which was characterized by H3/H4 hypoacetylation and hyper-O-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the HCRT-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the HCRT silencing. Thus, the epigenetic status of the HCRT gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated HCRT gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the HCRT gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.     

Disaccharide analysis of glycosaminoglycans synthesized by cardiac myxoma cells in tumor tissues and in cell culture

We analyzed the main disaccharide units of glycosaminoglycans synthesized by cardiac myxoma cells in vivo and in cell culture using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling. Cardiac myxoma tissues contained large amounts of chondroitin-6-sulfate (46%) and hyaluronic acid (32%), along with some chondroitin-4-sulfate (13%), chondroitin (6%), and much less dermatan sulfate (3%). Cultured cardiac myxoma cells synthesized mainly chondroitin-6-sulfate. The abundant glycosaminoglycans in myxoma tissues may make up the characteristic friable gelatinous matrix which is favorable for embolism and tumor cell growth.     

<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb. α-mannosidase belongs to the glycosyl hydrolase family 38

α-Mannosidase (EC 3.2.1.24) was purified from ‘Iseimo’, a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting α-mannosidase activity. We purified α-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65°C. The K _m value for p-nitrophenyl-α-d-mannopyranoside was 0.35 ± 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved α-1,2 linkages preferentially to α-1,6 and α-1,3 linkages. The M _r of purified α-mannosidase was estimated to be 250–260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme’s properties indicated that Iseimo α-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.