Establishing the Japan-Store house of animal radiobiology experiments (J-SHARE), a large-scale necropsy and histopathology archive providing international access to important radiobiology data.

Purpose: Projects evaluating the effects of radiation, within the National Institutes of Quantum and Radiological Science and Technology (QST), National Institute of Radiological Sciences (NIRS), have focused on risk analyses for life shortening and cancer prevalence using laboratory animals. Genetic and epigenetic alterations in radiation-induced tumors have been also analyzed with the aim of better understanding mechanisms of radiation carcinogenesis. As well as the economic and practical limitations of repeating such large-scale experiments, ethical considerations make it vital that we store and share the pathological data and samples of the animal experiments for future use. We are now constructing such an archive called the Japan-Storehouse of Animal Radiobiology Experiments (J-SHARE). Methods: J-SHARE records include information such as detailed experimental protocols, necropsy records and photographs of organs at necropsy. For each animal organs and tumor tissues are dissected, and parts are stored as frozen samples at -80 °C. Samples fixed with formalin are also embedded in paraffin blocks for histopathological analyses. Digital copies of stained tissues are being systematically saved using a virtual slide system linked to original records by barcodes. Embedded and frozen tissues are available for molecular analysis. Conclusion: Similar archive systems for radiation biology have also been under construction in the USA and Europe, the Northwestern University Radiation Archive (NURA), and STORE at the BfS, respectively. The J-SHARE will be linked with the sister-archives and made available for collaborative research to institutions and universities all over the world.

Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary.

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.

Adrenomedullin: a possible regulator of germinal vesicle breakdown.

Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.

New water-soluble prodrugs of HIV protease inhibitors based on O-->N intramolecular acyl migration.

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns-Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction at the alpha-hydroxy-beta-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37 degrees C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t(1/2) value of 32h that may not be suitable for practical use.