Gene suppression of mouse testis in vivo using small interfering RNA derived from plasmid vectors

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules.     

Duodenal perforation due to compression necrosis by the tip of percutaneous endoscopic gastrostomy tube

Percutaneous endoscopic gastrostomy (PEG) is a common and safe procedure for enteral nutrition. There are few reports concerning its complications. We managed a 31-y-old bedridden case with punched out duodenal perforation without inflammation, from which the tip of the PEG tube protruded. Simple x-ray and computed tomography showed incarceration of the balloon in the duodenal bulb and extravasation of the tip of the tube. We performed simple closure with omental patching for duodenal perforation. Postoperative gastrointestinal fiberscopy on the 11th day revealed scar phase. Some PEG tubes have a balloon, which can prevent the removal of the tube, fix the position of the tube, and prevent the leakage of gastric contents from fistula. However, in our case, the inflated balloon was transferred into the duodenal bulb according to gastric strong peristalsis. This pathophysiologic mechanism is the same as ball bulb syndrome, which is known as gastroduodenal obstruction by incarceration of the gastric submucosal tumor. There is a risk of wedging of the inflated balloon of the PEG tube and perforation of the duodenum. We must not insert the tube too deeply, must not continue to inflate the balloon for a long time, and must check its position using a stethoscope, simple x-ray examination, or ultrasound.     

Functional characterization of the novel BRAF complex mutation,BRAFV600delinsYM,identified in papillary thyroid carcinoma

An activating mutation in the BRAF gene is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The mutation in PTCs is almost a c.1799T>A transversion, resulting in a p.V600E amino acid substitution (BRAF^V600E). Here, we report a novel complex BRAF mutation identified in 4/492 Japanese PTC cases (0.81%). The mutation was comprised of one nucleotide substitution at position 1798, followed by an in‐frame insertion of three nucleotides, c.1798delinsTACA in Exon 15, resulting in p.V600delinsYM. In silico three‐dimensional protein structure prediction implied altered kinase activity of this mutant. In vitro kinase assay and western blotting revealed that this mutation conferred high kinase activity on the BRAF protein, leading to constitutive activation of the MAPK signaling pathway. The mutation also showed high transforming ability in focus formation assay using NIH3T3 cells. The degree of all the functional characteristics was comparable to that of BRAF^V600E, and treatment with a BRAF inhibitor Sorafenib was also equally effective in this mutant. These findings suggest that the novel BRAF mutation, BRAF^V600delinsYM, is a gain‐of‐function mutation and plays an important role in PTC development.     

Species differences of organic anion transporters involved in the renal uptake of 4‐amino‐3‐chlorophenyl hydrogen sulfate,a metabolite of resatorvid,between rats and dogs

Previous studies on the metabolic fate of resatorvid (TAK‐242) have shown that species differences in the pharmacokinetics of 4‐amino‐3‐chlorophenyl hydrogen sulfate (M‐III), a metabolite of TAK‐242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M‐III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p‐aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M‐III inhibited the uptake. The initial uptake clearance of M‐III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie‐Hofstee plot of M‐III uptake at 37 °C revealed two‐component transport processes with K_m values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M‐III was 8.70 × 10^‐3 and 9.00 × 10^‐3 ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1‐ and rOat3‐expressing oocytes mediated M‐III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M‐III in rats. Moreover, it is speculated that Oat(s) is unable to transport M‐III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M‐III between rats and dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability

Mdm2 and Mdm4 are homologous RING domain-containing proteins that negatively regulate the tumor suppressor p53 under physiological and stress conditions. The RING domain of Mdm2 encodes an E3-ubiquitin ligase that promotes p53 degradation. In addition, Mdm2 and Mdm4 interact through their respective RING domains. The in vivo significance of Mdm2-Mdm4 heterodimerization in regulation of p53 function is unknown. In this study, we generated an Mdm4 conditional allele lacking the RING domain to investigate its role in Mdm2 and p53 regulation. Our results demonstrate that homozygous deletion of the Mdm4 RING domain results in prenatal lethality. Mechanistically, Mdm2-Mdm4 heterodimerization is critical for inhibiting lethal p53 activation during early embryogenesis. However, Mdm2-Mdm4 interaction is dispensable for regulating p53 activity as well as the stability of Mdm2 and p53 at later stages of development. We propose that Mdm4 is a key cofactor of Mdm2 that inhibits p53 activity primarily during early embryogenesis but is dispensable for regulating p53 and Mdm2 stability in the adult mouse.     

Species differences in toxicokinetic parameters of glycidol after a single dose of glycidol or glycidol linoleate in rats and monkeys

Glycidol fatty acid esters (GEs) have been identified as contaminants in refined edible oils. Although the possible release of glycidol (G) from GEs is a concern, little is known about the conversion of GEs to G in the human body. This study addressed the toxicokinetics of glycidol linoleate (GL) and G in male Crl:CD(SD) rats and cynomolgus monkeys. Equimolar amounts of GL (341 mg/kg) or G (75 mg/kg) were administered by gavage to each animal. G was found in both species after the G and GL administration, while plasma GL concentrations were below the lower limit of quantification (5 ng/ml) in both species. In rats, the administration of GL or G produced similar concentration-time profiles for G. In monkeys, the Cmax and AUC values after GL administration were significantly lower than those after G administration. The oral bioavailability of G in monkeys (34.3%) was remarkably lower than that in rats (68.8%) at 75 mg/kg G administration. In addition, plasma G concentrations after oral administration at three lower doses of GL or G were measured in both species. In monkeys, G was detected only at the highest dose of G. In contrast, the rats exhibited similar plasma G concentration-time profiles after GL or G administration with significantly higher G levels than those in monkeys. In conclusion, these results indicate that there are remarkable species differences in the toxicokinetics of GEs and G between rodents and primates, findings that should be considered when assessing the human risk of GEs.