Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo

The morphology and proportion of inner cell mass (ICM) of bovine blastocysts cultured in vitro or in vivo in rabbit oviducts after in-vitro fertilization of in-vitro matured follicular oocytes were compared with those of blastocysts fertilized in vivo by a differential fluorochrome staining technique. The delineation of each ICM cell was improved by the transfer of embryos derived from in-vitro fertilization to a rabbit oviduct although the cell-cell contacts of ICM cells were not as tight as those from in-vivo fertilization. The proportions (15.8 and 14.9%) of ICM in blastocysts cultured in vitro at early and expanded stages were significantly lower than those cultured in rabbit oviducts after in-vitro fertilization and fertilized in vivo. These results show that the transfer of bovine embryos derived from in-vitro fertilization to the rabbit oviduct increased the proliferation of ICM cells to the level of embryos fertilized in vivo although the cell-cell contact of ICM cell is not improved by the process.     

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG_2a-type, 47-mG_2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.     

Mapping of the Gene for Machado-Joseph Disease within a 3.6-cM Interval Flanked by D14S291/D14S280 and D14S81, on the Basis of Studies of Linkage and Linkage Disequilibrium in 24 Japanese Families

The gene locus of Machado-Joseph disease (MJD) has recently been mapped within a 29-cM subregion of 14q chromosome. We did a linkage study of 24 multigenerational MJD Japanese pedigrees, in an attempt to narrow the candidate region of this gene. Pairwise and multipoint linkage analysis, together with haplotype segregation analysis, led to the conclusion that the MJD gene is located at the 6.8-cM interval between D14S256 and D14S81 (Z_max = 24.78, multipoint linkage analysis). D14S291 and D14S280, located at the center of this interval, showed no obligate recombination with the MJD gene (Z_max = 5.93 for D14S291 and 9.99 for D14S280). A weak, but significant, linkage disequilibrium of MJD gene was noted with D14S81 (P < .05) but not with D14S291 or D14S280. These results suggest that a 3.6-cM interval flanked by D14S291/D14S280 and D14S81 is the most likely location of the MJD gene and that it is closest to D14S81.     

Nonenzymatic sequence-specific cleavage of duplex DNA via triple-helix formation by homopyrimidine phosphorothioate oligonucleotides

Phenanthroline was attached covalently to the 5′-terminus of the unmodified and modified (3′-terminal phosphorothioate) oligonucleotide sequences, TTTTTTCTTCTCTTTCC (OP-17 mer) and TTTTTTTCTTCTCTTTCsC (OPRp-17 mer or OPSp-17 mer) via a phosphoramidite bond. Simian virus 40 DNA contains a single target site for these oligonucleotides. In the presence of copper ions, the efficient double-stranded cleavage at 37 °C and pH 7.0 was observed by agarose gel electrophoresis. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove, even though the linker was located in the major groove. Of particular interest are the 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp), which were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes and antisense nucleic acid based anti-viral therapies, which could be used as tools in cellular biology.

The 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp) were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes, which could be used as tools in cellular biology.