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Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight
2004
Namiki, Y. (Hokkaido Univ., Sapporo (Japan)) | Kon, Y. | Sasaki, N. | Agui, T. | Endoh, D.
In MRJ/MpJ mice, there is a genetic mutation of exonuclease 1 (Exol), in which the exon 9 is sometimes deleted. In the present study, to check the gen-eration of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/ Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B , but was present little or very weakly in pCX/Ex/EIE/M . Next, the same spliced band was demonstrated in pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the del1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-l and tr-2 Exol).
Mostrar más [+] Menos [-]Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro
2004
Hosaka, Y. (Rakuno Gakuen Univ., Ebetsu, Hokkaido (Japan)) | Sakamoto, Y. | Kirisawa, R. | Watanabe, T. | Ueda, H. | Takehana, K. | Yamaguchi, M.
Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-Rassociated factor 2 (TRAF2) and nuclear factor-kappa B (NE-KappaB) , and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTS of thoroughbred horses. The tendinocytes were treated with 10ng/ ml equine TNFAlpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF-R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFAlpha-treated cells (inflamed condition). Intense TRAF2 and NF-KappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in uitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.
Mostrar más [+] Menos [-]Effects of cycloheximide treatment on in-vitro development of porcine parthenotes and somatic cell nuclear transfer embryos
2003
Diaz, M.A.M. (Hokkaido Univ., Sapporo (Japan)) | Suzuki, M. | Kagawa, M. | Ikeda, K. | Takahashi, Y.
Passive immunization with monoclonal antibodies: Effects on Haemaphysalis longicornis tick infestation of BALB/c mice
2003
Nakajima, M. (Hokkaido Univ., Sapporo (Japan)) | Yanase, H. | Iwanaga, T. | Kodama, M. | Ohashi, K. | Onuma, M.
Growth pattern and seasonal weight changes of the feral raccoon (Procyon lotor) in Hokkaido, Japan
2003
Asano, M. (Hokkaido Univ., Sapporo (Japan)) | Matoba, Y. | Ikeda, T. | Suzuki, M. | Asakawa, M. | Ohtaishi, N.
Polymerase chaine reaction-restriction fragment length polymorphism (PCR-RFLP) method for mtDNA typing in Hokkaido brown bear (Ursus arctos yesoensis) [Japan]
2003
Satoh, Y. (Hokkaido Univ., Sapporo (Japan)) | Mano, T. | Tsuruga, H | Masuda, R. | Matsuhashi, T. | Onuma, M. | Suzuki, M. | Ohtaishi, N.
Sustained hypomyelination and high serum thyroid hormone in aged black tremor hamster
2003
Kim, H.O. (Hokkaido Univ., Sapporo (Japan)) | Kimura, T. | Ochiai, K. | Yazawa, H. | Itakura, C. | Umemura, T.
Oligodendrocytes and myelin in the corpus callosum of black tremor and normal hamsters aged over 1.5 years were ultrastructurally examined to determine the myelination index (ratio of myelin thickness/diameter of axon), percentage of naked axons, and proportions of oligodendroglial subtypes (light, medium and dark). The mutant hamsters were remarkably hypomyelinated, with a low myelination index and a high proportion of naked axons, and high proportions of the dark subtypes. Serum concentrations of thyroid hormones (T sub(3) and T sub(4)) in 6-week-old mutant hamsters were 2-fold (T sub(3)) to 3-fold (T sub(4)) higher than those of age-matched normal animals. However, in the aged animals (over 1.5 years old) only T sub(4) levels of the mutant hamsters were higher in the mutant than normal hamsters. The black tremor hamsters were hypomyelinated throughout their life and high serum level of thyroid hormones might have played a role in the hypomyelination.
Mostrar más [+] Menos [-]Blood meal acquisition by ticks; molecular advances and implications for vaccine development
2002
Mulenga, A. (Hokkaido Univ., Sapporo (Japan)) | Tsuda, A. | Sugimoto, C. | Onuma, M.
In their quest for a blood meal, hematophagous arthropods must first defeat the host's hemostatic defense. Following injury as it occurs when hematophagous arthropods insert their proboscis into host skin to feed, the host will attempt to stop excessive blood loss through its hemostatic defense mechanism involving platelet aggregation, blood clotting and vasoconstriction. To acquire a full blood meal hematophagous arthropods inject an arsenal of bioactive enzymes which ultimately overpower the host's hemostatic defense. We have looked at a selected number of studies on the molecular biology of arthropod anti-hemostatic proteins and developed commentaries on the suitability of these molecules as target tick vaccine antigens.
Mostrar más [+] Menos [-]Taenia taeniaeformis larval product induces gastric mucosal hyperplasia in SCID mice
2002
Lagapa, J.T.G. (Hokkaido Univ., Sapporo (Japan)) | Oku, Y. | Nonaka, N. | Kamiya, M.
The effects of intraperitoneal implantation of Taenia taeniaeformis larvae and inoculation of in vitro larval products on gastric mucosa of SCID mice were investigated in this study. Mice surgically implanted with T, taeniaeformis larvae developed slight and moderate gastric hyperplasia. When in vitro cultured T. taeniaeformis larval excretory-secretory (TtLES) products containing 1 mg of protein were injected daily into mice, they caused gastropathy after 5-7 days. Mice injected daily with 0.5 mg of TtLES products also showed slight gastric hyperplasia after day 14 and 28. The gastropathy was characterized by reduction of both parietal and zymogenic cell number and increased number of alcian blue-periodic acid Schiff (AB-PAS)-positive cells and by two-fold extension of proliferative zone of gastric units. Larval implantation demonstrated a more potent effect in inducing gastropathy than did in vitro larval culture products. Significant decrease in number of parietal cells with con-comitant increase of tive zone and AB-PAS-positive cell number indicated their important roles in inducing the hyperplastic lesion. Similarities with other gastropathies indicated that there is a common fundamental regulatory mechanism involved, and that the host response may not be specific to parasites. Present study validated the induction of gastric mucosal hyperplasia by larval ES products of T. taeniaeformis. This proved the hypothesis of previous studies suggesting the role of larvae-derived products in inducing gastric mucosal hyperplasia in T. taeniaeformis-infected rats.
Mostrar más [+] Menos [-]Efficiency of fecal steroid hormone measurement for assessing reproductive function in the Hokkaido brown bear (Ursus arctos yesoensis)
2002
Ishikawa, A. (Hokkaido Univ., Sapporo (Japan)) | Kikuchi, S. | Katagiri, S. | Sakamoto, H. | Takahashi, Y.
The present study aimed to establish simple systems for measuring fecal steroid hormones in order to monitor the reproductive profiles of captive Hokkaido brown bears. The efficiency of fecal sample processing at the steps of dehydration and extraction and the correlation between steroid concentrations in matched fecal and blood samples were studied. Then, monthly changes in fecal estradiol-17beta and progesterone in female bears, and testosterone in male bears were examined. The procedure was finalized as follows. Fecal samples were dried at 100degC for 3 hr and extracted with diethyl ether. The diethyl ether in the extracts was evaporated and residues were reconstituted in ethanol for the assays. Hormone concentrations were quantified using enzyme immunoassays. Concentrations of progesterone and testosterone in fecal and plasma samples were correlated in the systems. The changes in fecal progesterone and testosterone concentrations were similar to those in serum concentrations of bears as reported previously. In contrast, fecal estradiol concentrations did not correlate with plasma levels probably because of the time lag in excretion. However, the changes in estradiol-17beta concentrations in feces in the present study were similar to those reported in serum. In conclusion, fecal progesterone and testosterone assay systems appear practical for monitoring ovarian and testicular activities without immobilization, though methodological improvements and further validation may be required. For the fecal estradiol-17beta assay, there is a need to solve the problem of excretion time lag before the system can be used in the study of reproductive physiology.
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