Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.     

Temporal changes in NCAM immunoreactivity during taste cell differentiation and cell lineage relationships in taste buds

Neural cell adhesion molecule (NCAM) is a type III cell marker in the taste buds. In order to clarify the cell type of Mash1-expressing cells in taste buds, expression of NCAM was examined in Mash1-expressing taste cells of adult mice in comparison with gustducin- and T1r3-expressing cells, using a combination of NCAM immunohistochemistry and in situ hybridization. About 98% of Mash1-expressing cells were NCAM immunopositive (IP), suggesting that Mash1-expressing cells should be categorized as type III cells. Unexpectedly, small subsets of gustducin- and T1r3-expressing cells were also found to be NCAM-IP, contradicting previous immunohistochemical studies in rats, in which gustducin-IP cells were observed specifically in type II cells, which do not have NCAM immunoreactivity. Examinations of developing taste buds showed temporal changes in the ratio of NCAM-IP cells in gustducin- and T1r3-expressing cells; the ratio of NCAM-IP cells in these gene-expressing cells were approximately 90% at 0.5 days after birth and decreased markedly during development. In contrast, the majority of Mash1-expressing cells showed constant NCAM immunoreactivity throughout development. In addition, BrdU-labeling experiments showed that the differentiation of Mash1-expressing cells precedes those of gustducin- and T1r3-expressing cells in taste buds of adult mice. These results suggest that T1r3- and gustducin-expressing cells are NCAM-IP at the beginning of cell differentiation, and that NCAM immunoreactivity in gustducin- and T1r3-expressing cells might remain from the previous developmental stage expressing Mash1.     

Structural and functional analysis of essential pre-mRNA splicing factor Prp19p

U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.     

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin–biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Use of 11alpha-deuterium labeled cortisol as a tracer for assessing reduced 11beta-HSD2 activity in vivo following glycyrrhetinic acid ingestion in a human subject

This study describes an oral administration of 5 mg of [1,2,4,19-13C4,11alpha-2H]cortisol (cortisol-13C4,2H1) to a human subject performed on two separate occasions, one with cortisol-13C4,2H1 alone and the other with cortisol-13C4,2H1 plus 130 mg per day of glycyrrhetinic acid for 6 days. The stable isotope methodology employed allowed for the evaluation of the individual in vivo activities of the two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD1 and 11beta-HSD2, and to demonstrate the sensitivity of changes in cortisol elimination half-life for detecting inhibition of 11beta-HSD2 activity induced with glycyrrhetinic acid. The kinetic analysis associated with the loss of 11alpha-2H during the conversion of cortisol-13C4,2H1 to cortisone-13C4 by 11beta-HSD2 clearly indicated reduced 11beta-HSD2 activity with glycyrrhetinic acid ingestion, as observed by an increase in the elimination half-life of cortisol-13C4,2H1. The elimination half-life of cortisol-13C4,2H1 provided sensitive in vivo measures of 11beta-HSD2 activity and was more sensitive for detecting changes in renal 11beta-HSD2 activity than the measurement of the urinary ratio of free cortisol and free cortisone (UFF/UFE). The 2H-labeling in the 11alpha-position of cortisol served as an appropriate tracer for assessing the reduced 11beta-HSD2 activity in vivo induced by glycyrrhetinic acid.