Tunicamycin inhibits prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F₂-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25–250 ng/ml) inhibited subsequent PGF₂ (1 M)-stimulated PI hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked [³⁵S]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF₂. These observations suggest that PGF₂ receptor is N-linked glycosylated.     

Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein, which harbors five or six PSD-95/Discs large/ZO-1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and beta-catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S-SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified beta-dystroglycan (beta-DG) as a binding partner for S-SCAM at inhibitory synapses. WW domains of S-SCAM bind to three sequences of beta-DG. We have also revealed that S-SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S-SCAM are involved in the interaction with neuroligin 2. Beta-DG, neuroligin 2 and S-SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti-beta-DG antibody from rat brain. S-SCAM, beta-DG and neuroligin 2 are partially co-localized in rat hippocampal neurons. These data suggest that S-SCAM is associated with beta-DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin-neuroligin complex.     

Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer,Streptomyces albulus

Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.     

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: Antioxidants, lipid peroxidation and DNA damage

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

Multi-scale analysis of the effect of nano-filler particle diameter on the physical properties of CAD/CAM composite resin blocks

The objective of this study was to assess the effect of silica nano-filler particle diameters in a computer-aided design/manufacturing (CAD/CAM) composite resin (CR) block on physical properties at the multi-scale in silico. CAD/CAM CR blocks were modeled, consisting of silica nano-filler particles (20, 40, 60, 80, and 100 nm) and matrix (Bis-GMA/TEGDMA), with filler volume contents of 55.161%. Calculation of Young’s moduli and Poisson’s ratios for the block at macro-scale were analyzed by homogenization. Macro-scale CAD/CAM CR blocks (3 × 3 × 3 mm) were modeled and compressive strengths were defined when the fracture loads exceeded 6075 N. MPS values of the nano-scale models were compared by localization analysis. As the filler size decreased, Young’s moduli and compressive strength increased, while Poisson’s ratios and MPS decreased. All parameters were significantly correlated with the diameters of the filler particles (Pearson’s correlation test, r = ?0.949, 0.943, ?0.951, 0.976, p < 0.05). The in silico multi-scale model established in this study demonstrates that the Young’s moduli, Poisson’s ratios, and compressive strengths of CAD/CAM CR blocks can be enhanced by loading silica nanofiller particles of smaller diameter. CAD/CAM CR blocks by using smaller silica nano-filler particles have a potential to increase fracture resistance.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.