Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Maternal proteins are rapidly degraded by the ubiquitin-proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficient gad. Furthermore, we assessed morphological features in gad mouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the 'maternal antigen that embryos require' (NLRP5 (MATER)) protein level increased significantly in gad mouse ova compared with that in wild-type mice. In an ultrastructural study, gad mouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.     

Developmental ability of cloned embryos from neural stem cells

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.     

Effects of siRNAs targeting the spacer sequence of plant RNAi vectors on the specificity and efficiency of RNAi

Small interfering RNAs (siRNAs) generated from the spacer region of hairpin RNAs were not detected in the RNA interference (RNAi) plants targeting the fatty acid desaturase gene. The expression of the desaturase gene was stably suppressed even when siRNAs targeting the spacer sequences were introduced into this plant.     

Molecular structures of gellan gum imaged with atomic force microscopy (AFM) in relation to the rheological behavior in aqueous systems in the presence of sodium chloride

Aqueous solutions of gellan gum with comparable molecular mass but with different acyl contents were investigated by atomic force microscopy and rheological measurements in the presence of sodium chloride (NaCl). Results obtained were discussed in relation to our previous report using potassium chloride (KCl) as an added salt. For a low-acyl sample, continuous fibrous network structures were identified microscopically as in the case of KCl. The network structures were more heterogeneous than those formed with KCl in terms of the height distribution of molecular assemblies. Rheological thermal hysteresis between sol–gel transitions was detected as in the case of KCl. The storage modulus (G′) of the gelled system was ca. 15% of the corresponding data with KCl at 20 °C. For a high-acyl sample, no continuous network structures were identified but branches with observable ends were identified as in the case of KCl. The hysteresis was less evident than the corresponding data with KCl and for the low-acyl sample with NaCl. Also, G′ values at 20 °C were ca. 30% and 20% of the corresponding data with KCl and for the low-acyl sample with NaCl, respectively. Continuousness and homogeneity of network structures related to the hysteresis and elasticity of the system, respectively.     

Suppression of inducible nitric oxide synthase expression and amelioration of lipopolysaccharide-induced liver injury by polyphenolic compounds in Eucalyptus globulus leaf extract

Eucalyptus leaf extract (ELE) is rich in hydrolyzable tannins. We examined the effects of ELE and its constituents on lipopolysaccharide (LPS)-induced liver injury in mice. Mice fed a diet supplemented with 1% ELE were intraperitoneally administered LPS. Six hours later, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly lower in the ELE-supplemented mice than in the controls; LPS-induced hepatic inducible nitric oxide synthase (iNOS) expression was also suppressed. ELE lowered LPS-stimulated iNOS expression in cultured RAW 264.7 macrophages. Furthermore, the aglycones of hydrolyzable tannins, gallic acid (GA) and ellagic acid (EA), inhibited iNOS induction to a greater extent than did ELE (15-fold higher). When mice were fed a 1% GA or EA diet, the increase in the serum ALT and AST activities and hepatic iNOS expression in response to the LPS challenge were significantly attenuated. Thus, hydrolyzable tannins in ELE ameliorate LPS-induced liver injury.     

Electron transfer reactions of glucose oxidase at Au111 electrodes modified with phenothiazine derivatives

The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x 10(5) dm(3) mol(-1) s(-1)) than for the PT-PEO1000 system (1.4 x 10(4) dm(3) mol(-1) s(-1)). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and PT(+) immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PEO1000 thickly modified on the electrode.     

Preparation of a crack-free rough titania coating on stainless steel mesh by electrophoretic deposition

TiO₂ (anatase) coating was prepared on stainless mesh by electrophoretic (EPD) process utilizing an isopropyl alcohol (IPA)-based suspension with submicron TiO₂ powder. When the deposition time was 30 s, a smooth thin coating was obtained. It remained crack-free even after sintering. Coating surface morphology was roughened by UV pre-illumination of the suspension. Photocatalytic decomposition of IPA to acetone and resultant electrochemical reaction at cathode during EPD provides heterogeneous deposition.     

Effects of dietary α-lipoic acid enantiomers on hepatic fatty acid metabolism in rats

Effects of dietary α-lipoic acid enantiomers on hepatic and serum lipid concentrations and the activity and mRNA levels of enzymes involved in hepatic fatty acid metabolism were examined in rats. Rats were fed experimental diets containing 0 or 2.5 g/kg of α-lipoic acid enantiomers (a racemic mixture of R/S-, S- or R-α-lipoic acid) for 21 days. Various α-lipoic acid preparations decreased serum and liver concentrations of triacylglycerol, and also lowered serum concentrations of cholesterol and phospholipid and the concentration of cholesterol in the liver. α-Lipoic acid decreased the activity and mRNA levels of various hepatic lipogenic enzymes and also decreased the activities of carnitine acyltransferase and 3-ketoacyl-CoA thiolase and mRNA levels of carnitine palmitoyltransferase 1a involved in fatty acid oxidation in the liver; however, R- and S-enantiomers of α-lipoic acid as well as well as a racemic mixture of R/S-α-lipoic acid were indistinguishable in altering these indices for lipid metabolism.