A New Cell Death Inhibitor, Bax-inhibiting-peptide (BIP) Derived from Ku70(Animal Biology, Animal Reproduction)

Programmed cell death (apoptosis) plays a pivotal role in the homeostasis of multicellular organisms. Bax is a key mediator of apoptosis. Apoptotic stress induces the translocation of Bax from the cytosol to mitochondria, and then Bax induces mitochondria-dependent cell death. Recently, we have found that Ku70 inhibits the translocation of Bax from the cytosol to mitochondria, suggesting that Ku70 inhibits mitochondria-dependent cell death (Sawada et al., 2003b). Moreover, we have designed a new type of cell death inhibitor, Bax-inhibiting-peptide (BIP) derived from Ku70 (Sawada et al., 2003b; Yoshida et al., 2004). We have demonstrated that BIPs inhibit the cell death induced by anti-cancer drugs, UVC irradiation, and tropic factor deprivation (Sawada et al., 2003b; Yoshida et al., 2004). BIP directly binds Bax and inhibits the cytotoxic activity of Bax. BIP may become a new tool to control degenerative diseases.ApoptosisKu70BaxBax-inhibiting-peptide (BIP)cell death inhibito

Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus–oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitromatured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozenthawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.</p

Vitrification of Germinal Vesicle Stage Oocytes

In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of nylon mesh, and plunged directly into liquid nitrogen for vitrification, the recovery rate was significantly higher compared with that in 15 ones on the electron microscope (EM) grid as a control, followed by obtaining the resultant cleavage and developmental rates after in vitro fertilization and culture (IVFC) without significant difference. Using bovine and murine oocytes, we found that a step-wise manner to expose them with the vitrification solution increased rates of in vitro maturation, subsequent development to blastocysts and hatching/hatched blastocysts after IVFC. Our results show that nylon mesh is an alternative material for cryopreserving large quantities of bovine GV oocytes, and that a step-wise exposure to cryoprotectants may have befit for decreasing disadvantage during vitrification

Gene expression profiles of novel caprine placental prolactin-related proteins similar to bovine placental prolactin-related proteins

BACKGROUND: This study reports the identification of a full-length cDNA sequence for two novel caprine prolactin-related proteins (cPRP1 and cPRP6), and their localization and quantitative expression in the placenta. Caprine PRPs are compared with known bovine PRPs. We examined their evolution and role in the ruminant placenta. RESULTS: Full-length cPRP1 and cPRP6 cDNA were cloned with a 717- and 720- nucleotide open-reading frame corresponding to proteins of 238 and 239 amino acids. The cPRP1 predicted amino acid sequence shares a 72% homology with bovine PRP1 (bPRP1). The cPRP6 predicted amino acid sequence shares a 74% homology with bovine PRP6 (bPRP6). The two cPRPs as well as bPRPs were detected only in the placentome by RT-PCR. Analysis by in situ hybridization revealed the presence of both cPRPs mRNA in the trophoblast binucleate cells. These mRNA were quantified by real-time RT-PCR analysis of the placentome at 30, 50, 90 and 140 days of pregnancy. Both new cPRP genes were able to translate a mature protein in a mammalian cell-expression system. Western blotting established the molecular sizes of 33 kDa for cPRP1 with FLAG-tag and 45 kDa for cPRP6 with FLAG-tag. The sequence properties and localized expression of cPRP1 and cPRP6 were similar to those of bovine. However, their expression profiles differed from those in bovine placenta. Although this study demonstrated possible roles of PRPs in caprine placenta, PRPs may regulate binucleate-cell functions like those in bovine, but their crucial roles are still unclear. CONCLUSION: We have identified the novel PRPs in caprine placenta. Localization and quantitative expression of caprine PRPs were compared with bovine PRPs. The data indicate that PRP genes in caprine placenta have coordination functions for gestation, as they do in bovine. This is the first study of PRPs function in caprine placenta

Relationship between Cytologic Results and the Extent of Intraductal Spread in Nonpalpable Breast Cancers with Nipple Discharge

Cytologic results of smears and ductal washings for 51 consecutive cases of nonpalpable breast cancers with nipple discharge were studied. Noninvasive can-cer was confirmed in 40 cases and microinvasive in 11 cases. The cytologic results were compared with the extent of cancerous lesions which was measured as the angle of cancerous spread (Q) on a map made from histological prepara-tions. Although the sensitivity of cytologic examinations was quite low (9.8% and 33.3%), the cytologic results correlated with the extent of cancerous spread in the breast (P<0.01) and with the distance from the nipple to the lesion (P<0. 01). The results in this study suggest that cytologic results can be affected by the extent of cancerous spread. The results of cytologic examination should be made use of in the process of assessing the presence of extensive cancerous lesions which cause nipple dis-charge with no palpable mass