Comparison of the blood coagulation profiles of ferrets and rats

The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.     

Plasma Profiles of Glucose, Insulin and Lipids in the Male WBN/Kob-Lepr(fa) Rat, a New Model of Type 2 Diabetes with Obesity

Plasma profiles of glucose, insulin and lipids were examined in the male WBN/Kob-Lepr(fa) (fa/fa) rat, a new model of type 2 diabetes (T2D), in comparison with age-matched original male WBN/Kob (lean) rats. The fa/fa rats developed hypertriglycemia, obesity and hyperglycemia from 5, 7, and 9 weeks of age, respectively. Plasma insulin levels in fa/fa rats were significantly higher than those in lean rats at 5 weeks of age, but after 11 weeks of age gradually declined to the levels in lean rats. HOMA-IR, a measure of insulin resistance status, showed that fa/fa rats had insulin resistance. The fa/fa rat has the potential to become an important animal model of T2D with obesity.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

Changes in bedload transport rate associated with episodic sediment supply in a Japanese headwater channel

We conducted field monitoring of bedload transport rate associated with experimental sediment release in a natural channel to clarify behavior of the supplied sediment on mixed size bed. Observation of bedload rate at two sites along the 30 m channel reach revealed that downstream migration of finer particles delay compared with coarser particles. Ratio of the bedload sediment that deposited during the migration was higher for finer sediments. These behaviors of the mixed size particles were clear during passage of the sediment wave without changes in water discharge. Flashing peak of discharge that caused artificially by opening of the dam gate did not destroy channel bed structure including steps and pools formed by coarser sediments, and only small amount of bedload was mobilized. Both reach scale channel features including steps, pools, and riffles as well as fine scale features (i.e., armour coat) likely increased critical shear stress of particles and decreased bedload rate during our experiment. Extreme sediment supply induced two types of sediment deposition; (1) filling the pools in reach sales and (2) the intrusion of fine particles into the coarser sediment that formed an armour layer. The all grain size fractions can deposit as type (1) when shear stress of stream water is not enough to entrain bedload particles, while deposition type (2) occurs when finer sediment pass over channel bed on which armour coat is well-developed. Deposition of finer sediment into coarser sediment that forms armour coat is affected by grain size distribution of bed surface sediment. Thus, impact of the sediment supply on downstream channel depends on both bedforms and grain size distribution of bed surface sediment over which the supplied sediment pass.     

Mammalian brain morphogenesis and midline axon guidance require heparan sulfate

Heparan sulfate (HS) is required for morphogen signaling during Drosophila pattern formation, but little is known about its physiological importance in mammalian development. To define the developmental role of HS in mammalian species, we conditionally disrupted the HS-polymerizing enzyme EXT1 in the embryonic mouse brain. The EXT1-null brain exhibited patterning defects that are composites of those caused by mutations of multiple HS-binding morphogens. Furthermore, the EXT1-null brain displayed severe guidance errors in major commissural tracts, revealing a pivotal role of HS in midline axon guidance. These findings demonstrate that HS is essential for mammalian brain development.     

A novel member of the genus Nepovirus isolated from Cucumis melo in Japan

An unusual virus was isolated from a Japanese Cucumis melo cv. Prince melon plant showing mild mottling of the leaves. The virus had a broad experimental host range including at least 19 plant species in five families, with most infected plants showing no symptoms on inoculated and uninoculated systemically infected leaves. The virus particles were spherical, approximately 28 nm in diameter, and the coat protein (CP) had an apparent molecular mass of about 55 kDa. The virus possessed a bi-partite genome with two RNA species, of approximately 8,000 and 4,000 nucleotides. Both genome components for the new virus were sequenced. Amino acid sequence identities in CP between the new virus and previously characterized nepoviruses were found to be low (less than 27%); however, in phylogenetic reconstructions the closest relationship was revealed between the new virus and subgroup A nepoviruses. These results suggest that the new virus represents a novel member of the genus Nepovirus. A new name, Melon mild mottle virus, has been proposed for this new virus.     

Diversity of fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.