Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Relationship between blood calcium level and ST peak interval of electrocardiographic variables in peripartum Holstein cows

The association between blood calcium levels and electrocardiographic variables was compared in 137 normal parturient and 36 peripartum recumbent Holstein cows to determine whether hypocalcemia in peripartum dairy cows can be rapidly diagnosed using electrocardiograph. Inverse of STc (ST peak interval/SS interval^0.5) and blood ionized calcium or serum calcium concentrations were strongly correlated, and both correlation coefficients were 0.81 (P<0.001). The 95% prediction interval indicated that cows with STc >0.385 ± 0.001 sec are very likely to be hypocalcemic (blood ionized or serum calcium concentrations of <0.9 mmol/l or <7.5 mg/dl, respectively). These findings indicate that hypocalcemia in parturient cows can be non-invasively estimated using the STc.     

First report of trunk rot caused by <Emphasis Type="Italic">Fomitiporia torreyae</Emphasis> in Kyoto prefecture on cultivars of Japanese cedar with no relatedness to ‘Sanbu-sugi’

Trunk rot, caused by Fomitiporia torreyae, is one of the most economically important sap rot diseases on Cryptomeria japonica, especially on a cutting cultivar ‘Sanbu-sugi’. This disease had been reported only from Chiba and Ibaraki prefectures in Japan; however, a similar trunk rot on Japanese cedars was found recently in Kyoto prefecture. We identified the causal agent of the trunk rot on Japanese cedars in Kyoto prefecture, clarified the genotype of ‘Sanbu-sugi’ (sensu stricto) using microsatellite markers, and then investigated the relatedness of these infected cedars with ‘Sanbu-sugi’. The pathogen was identified as F. torreyae by its DNA sequences and morphological characteristics of the fruiting body. The 25 ramets of ‘Sanbu-sugi’ had only one genotype based on eight microsatellite loci. Infected Japanese cedars in Kyoto were comprised of different genotypes and showed no close relatedness to ‘Sanbu-sugi’. This is the first report of trunk rot caused by F. torreyae on cultivars of Japanese cedar with no relatedness to ‘Sanbu-sugi’ outside of Chiba and Ibaraki prefectures and showed the potential for expansion of this disease to Japanese cedar plantations in other areas.     

Cross-sectional cutting of bamboo with a pair of shearing blades for bamboo cube production

As a new use of bamboo, bamboo cubes may be useful as a shot-blast material for surface treatments. However, a suitable processing technique for bamboo cubes has not been established. In this study, to obtain basic knowledge regarding the processing technology for bamboo cubes, we installed shearing blades (upper and lower blades) in a universal testing machine to test cross-sectional shearing of bamboo. The shearing force generated in this shearing was composed of a vertical component (F _v), a forward component (F _f), and a side component (F _s). This shearing force (F _v, F _f, and F _s) and the machining accuracy were investigated under various processing conditions. The shearing force became larger as the thickness of bamboo increased. In particular, F _v showed a tendency to increase rapidly. Changes in the shear angle of the upper blade had a remarkable influence on F _v. This result suggests that the shearing force could be greatly decreased by adjusting the shear angle. The shearing force within bamboo of 3mm in thickness was almost unaffected by the blade angle, and the change of shearing force with increasing clearance was almost indiscernible. It was clearly demonstrated that a large number of bamboo cubes could be made when the shear angle was large and cross-sectional shearing was performed from the bark side. However, a consequence of a large shear angle is that burr area increases. Part of this article was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, March 2003     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Production of trout offspring from triploid salmon parents

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.     

Cartilage acidic protein-1B (LOTUS), an endogenous Nogo receptor antagonist for axon tract formation

Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein-1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor-1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.