New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.     

A sex-specific size–number tradeoff in clonal broods

Polyembryonic parasitoids producing single-sex broods of clonal offspring provide an unusually clear window into the classic tradeoff between the number and size of offspring. We conducted a laboratory study of the encyrtid parasitoid Copidosoma bakeri parasitizing the noctuid Agrotis ipsilon to examine the way that size and number of offspring tradeoff in broods of each sex and to determine how the fit between host and parasitoid brood is achieved. We found that brood mass (wasp body mass ×brood size) was proportional to host mass, independent of brood sex, indicating a tight fit between brood and host and ensuring a size–number tradeoff. By correcting brood size and body mass of each brood for host mass, we demonstrated the expected inverse relationship between wasp variables. We postulated that the wasp brood might achieve the fit to the host by (1) adjusting brood size based on information available early in host development before and during division of the embryo, (2) manipulating host size late in host development after completion of embryo division, or (3) simply adjusting individual wasp mass to fill the host. We evaluated host responses to parasitism – and correlations between brood size and host growth early and late in development – for broods of each sex. The data are consistent with adjustment of brood size to the amount of host growth early in host development and with manipulation of host mass late in host development. The tight link between host mass and brood mass also suggests a final adjustment by parasitoid growth to achieve complete filling. Within the tight fit, female broods were smaller but contained larger individuals than male broods. The sex-specific balance point of the tradeoff and sex differences in balancing mechanisms and responses to host size suggest different selection pressures on each sex requiring future investigation.     

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

In the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.     

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts

What is it that defines a bone marrow‐derived chondrocyte? We attempted to identify marrow‐derived cells with chondrogenic nature and immortality without transformation, defining “immortality” simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte‐specific cis‐regulatory element of the collagen α2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony‐stimulating factor‐deficient osteopetrotic mice and also known to be niche‐constituting cells for hematopoietic stem cells expressed chondrocyte‐specific or ‐associated genes such as type II collagen α1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF‐β3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen‐positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell‐type‐specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of “chondroblast,” that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo. J. Cell. Biochem. 100: 1240–1254, 2007. © 2006 Wiley‐Liss, Inc.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.