Novel Mutations in the TMPRSS3 Gene May Contribute to Taiwanese Patients with Nonsyndromic Hearing Loss

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene’s association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

DPPA2 and DPPA4 are necessary to establish a totipotent state in mouse embryonic stem cells

After fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through so-called zygotic genome activation (ZGA), occurring in the mouse at the 2-cell stage. Amongst the first elements to be transcribed are the Dux gene, the product of which secondarily induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons, which regulate chromatin accessibility in the early embryo. The maternally-inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Here we identify the paralog proteins DPPA2 and DPPA4 as responsible for this process.

Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period

Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare’s serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus–oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15 : Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

Expression patterns of Nlrp9a, Nlrp9b and Nlrp9c during mouse development

The Nlrp gene family contains 20 members in the mouse. Recent studies have demonstrated that these genes play key roles in reproduction. In this study, we investigated the expression patterns of Nlrp9a, Nlrp9b and Nlrp9c in the mouse. In 2-week-old mouse tissues, Nlrp9a, Nlrp9b and Nlrp9c were all strikingly expressed in the ovary, while the transcripts of Nlrp9b and Nlrp9c were also detected in other tissues. The transcripts of Nlrp9a, Nlrp9b and Nlrp9c were restricted to the oocytes and declined with oocyte aging within the ovary. Furthermore, Nlrp9a, Nlrp9b and Nlrp9c transcripts presented evidence for the exclusive maternal origin, which were presented in oocytes and zygotes, immediately downregulated and not detected after the 2-cell stage during preimplantation development. In addition, Nlrp9a and Nlrp9c transcripts were not detected in other cells except for oocytes. Nevertheless, Nlrp9b expression was detected in oocytes, as well as in D3 ES and F9 ES. These results indicate that Nlrp9a, Nlrp9b and Nlrp9c display specific or preferential oocyte expression patterns and may play critical role in oogenesis and/or preimplantation embryo development in the mouse.

Raltegravir Is a Substrate for SLC22A6: a Putative Mechanism for the Interaction between Raltegravir and Tenofovir

ABSTRACTThe identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEMVBL100, and Caco-2 cells. Transport by uptake transporters was assessed by using aXenopus laevisoocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEMVBL100, and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. TheKmandVmaxfor SLC22A6 transport were 150 μM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 μM tenofovir with a 50% inhibitory concentration (IC50) of 14.0 μM, and tenofovir inhibited 1 μM raltegravir with an IC50of 27.3 μM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.