Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Immunohistochemical localization of steroidogenic enzymes in the testis of Hokkaido Sika deer (Cervus nippon yesoensis)

The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.     

Measurement for breath concentration of hydrogen and methane in horses.

This study concerns the establishment of a simple testing method for breath concentration of hydrogen and methane in horses. Twenty-eight healthy thoroughbreds and 24 Arabians were used. Breath samples were collected using one-minute closed circulatory respiration through an aluminum bag filled with 10 liters of pure oxygen, which was mounted on the subjects by means of a face mask. Breath samples obtained, were analyzed by gas chromatography. A significant correlation in both hydrogen and methane levels was observed for samples collected at separate times. These findings confirmed the usefulness of our approach for testing breath concentrations of hydrogen and methane in horses.     

Isolation and species distribution of staphylococci from animal and human skin

From April 1999 to December 2000, a survey was made on the distribution of Staphylococcus species on the skin of 7 kinds of animals and humans. Staphylococci were isolated from 12 (100%) of 12 pigs, 17 (89.5%) of 19 horses, 30 (100%) of 30 cows, 73 (90.1%) of 81 chickens, 10 (40%) of 25 dogs, 23 (76.7%) of 30 laboratory mice, 20 (52.6%) of 38 pigeons, and 80 (88.9%) of 90 human beings. The predominant staphylococci isolated from a variety of animal species were novobiocin-resistant species, S. xylosus and S. sciuri regardless of the animal host species. The novobiocin-resistant species including S. xylosus and S. sciuri were only occasionally isolated from human skin. The predominant staphylococci found on human skin were novobiocin-sensitive species, S. epidermidis (63.8%), followed by S. warneri (28.8%) and S. hominis (13.8%). The results suggest that the staphylococcal flora inhabiting animal skin are different from those of human skin in regard to the predominant species isolated. In this study, we used pulsed-field gel electrophoresis to examine the chromosomal polymorphisms of S. epidermidis isolated most frequently from human skin. Strains of S. epidermidis showed the greatest genomic diversity in their fragment patterns.     

Immunohistochemical localization of inhibin subunits in the testis of the bull

The differential localization of the inhibin beta subunits betaA and betaB in the testis of adult bull was studied using specific monoclonal and polyclonal primary antibodies. Inhibin betaA- and betaB-subunits were localized only in the Sertoli cells. The inhibin betaA-subunit was observed in the cytoplasm while the betaB-subunit was localized in the nucleus. No specific findings depending on spermatogenic stages were observed among the seminiferous tubules. Moreover, the inhibin alpha-subunit was not detected in the testis of the bulls. In addition, no inhibin subunits were detected in the Leydig cells and spermatogenic cells. These findings indicate the presence of betaA- and betaB-subunits in the bull, which may suggest a possibility that activin is produced and/or stored in the Sertoli cells and regulates spermatogenesis in an autocrine/paracrine manner. Moreover, the inhibin betaB-subunit may be produced in the nucleus but the functional meaning of this is not yet clear.     

The relationship between the size of caudolateral curvilinear osteophyte of the canine femoral neck and the radiographic view

Caudolateral curvilinear osteophyte (CCO), an osteophyte at the site of joint capsule attachment on the caudal aspect of the femoral neck, has been advocated as a radiographic criterion for coxofemoral subluxation. The correlation between the presence of CCO on radiographs (radiographic-CCO), the size of the CCO (CCO index) on three-dimensional computed tomographic (CT) images, and hip evaluation using transverse CT images was assessed in 22 Border Collies. CCOs were detected on the radiographs and CT images of 32% and 100% femurs, respectively. The CCO index correlated significantly with radiographic-CCO, but a large CCO index did not necessarily imply that the CCO was visible on radiographs. Hence, radiographic-CCO findings should be used cautiously in hip evaluation of Border Collies.