The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.     

Molecular diversity of bamboo-associated fungi isolated from Japan

Bamboos are common and useful plants in Japan but little information is available about their endophytes. In this study, 257 fungal strains were isolated from bamboo tissues, and 71 representative strains were characterized by 18S rRNA gene and internal transcriber spacer region sequence analysis. Phylogenetic analysis showed that the fungal isolates were located in Sordariomycetes and Dothideomycetes. Xylariales was the dominant group within bamboos. Several rRNA gene sequences were not similar to any current sequence in the database and might be a novel species or genera that could represent sources of novel biological compounds. These findings reveal that bamboos are a huge reservoir of microorganisms that should be extensively investigated.     

Expression and distribution of very late antigen-5 in mouse peritoneal macrophages upon ingestion of fibronectin-bound Staphylococcus aureus

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.     

Capsaicin induces the production of IL-6 in human upper respiratory epithelial cells

Capsaicin, a type of alkaloid and the pungent component of chili peppers, is used as a therapeutic drug against allergic rhinitis and also as an index of bronchial hypersensitivity. Capsaicin receptor (TRPV1) expression has been identified in non-neuronal cells as well as neuronal cells. In our previous study, both TRPV1 protein and its gene expression on nasal epithelial cells were confirmed by immunohistochemistry and RT-PCR, respectively. In order to clarify whether or not TRPV1 acts as a functional receptor, we examined the effects of capsaicin on the production of IL-6 from primary cultured human airway epithelial cells at both protein and mRNA levels. Human nasal epithelial cells (HNECs) and normal human bronchial/tracheal epithelial cells (NHBE cells) were stimulated with increasing concentrations of capsaicin and/or pretreatment with capsazepine (TRPV1 antagonist) at 37 degrees C. The supernatant and total RNA were collected at 0, 4, 12, 24 and 48 h after treatment. IL-6 concentration and the IL-6 mRNA level were evaluated by ELISA and real-time PCR, respectively. Capsaicin (10 nM-10 muM) induced production of IL-6 from HNECs and NHBE cells and this effect was inhibited by pretreatment with capsazepine. Our findings suggest that topical application of capsaicin to the airway induces IL-6 production from respiratory epithelial cells via activation of TRPV1.     

Successful superovulation of cattle by a single administration of FSH in aluminum hydroxide gel

We investigated whether Al-gel could adsorb and release FSH effectively in vitro and in vivo, and whether a single administration of FSH in Al-gel could successfully induce superovulation (SOV) in cattle. Porcine FSH (pFSH; 30 mg) was mixed with 5 mL of Al-gel; 99.98+/-0.01% of pFSH was adsorbed by the gel and 71.6+/-1.1% of the adsorbed pFSH was subsequently released in the presence of BSA. In cattle given a single i.m. treatment of 30 mg of pFSH in 5 mL of Al-gel, the numbers of CL, total ova recovered, and transferable embryos per cow were not significantly different from conventional (twice daily for 4 d) pFSH treatment (12.3+/-1.7 versus 11.7+/-1.8, 10.0+/-2.5 versus 9.3+/-1.7, and 8.6+/-2.3 versus 8.0+/-1.8, respectively, mean+/-S.E.M.); plasma pFSH concentrations were increased for 4 d, indicating sustained release from the Al-gel. Five cows were given 30 mg pFSH in 5 mL of Al-gel i.m. on five occasions (once every 2-3 months); there was no significant difference among treatments for the number of CL (12.4+/-3.8, 13.8+/-4.8, 9.0+/-1.9, 9.8+/-3.0, 12.0+/-2.1), total ova recovered (12.0+/-3.8, 12.6+/-5.1, 6.8+/-1.9, 7.6+/-1.8, 11.4+/-2.5), and transferable embryos (11.4+/-3.9, 10.4+/-5.8, 6.6+/-2.1, 4.8+/-1.4, 10.4+/-2.6). In conclusion, a single i.m. treatment of 30 mg pFSH in 5 mL Al-gel effectively induced SOV in cattle.     

Knockdown of Golgi Stress-Responsive Caspase-2 Ameliorates HLD17-Associated AIMP2 Mutant-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation

Hypomyelinating leukodystrophy 17 is an autosomal recessive disease affecting myelin-forming oligodendroglial cells in the central nervous system. The gene responsible for HLD17 encodes aminoacyl-tRNA synthase complex-interacting multifunctional protein 2, whose product proteins form a scaffold that supports aminoacyl-tRNA synthetases throughout the cell body. Here we show that the HLD17-associated nonsense mutation (Tyr35-to-Ter [Y35X]) of AIMP2 localizes AIMP2 proteins as aggregates into the Golgi bodies in mouse oligodendroglial FBD-102b cells. Wild type AIMP2 proteins, in contrast, are distributed throughout the cell body. Expression of the Y35X mutant proteins, but not the wild type proteins, in cells upregulates Golgi stress signaling involving caspase-2 activation. Cells expressing the wild type proteins exhibit differentiated phenotypes with web-like structures bearing many processes following the induction of differentiation, whereas cells expressing the Y35X mutant proteins fail to differentiate. Furthermore, CASP2 knockdown but not control knockdown reverses the phenotypes of cells expressing the mutant proteins. These results suggest that HLD17-associated AIMP2 mutant proteins are localized in the Golgi bodies where their proteins stimulate Golgi stress-responsive CASP2 to inhibit differentiation; this effect is ameliorated by knockdown of CASP2. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD17 and possible approaches to ameliorating the disease’s effects.