The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice

Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes inDNAmethylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals

Associations between diabetes mellitus and pulmonary hypertension in chronic respiratory disease patients

博士（医学）福島県立医科大

Novel Methods to Study Angiogenesis Using Tissue Explants

Tissue explants of skeletal muscles, brain, kidney, liver and spleen from mice were cultured using collagen gel. Electron microscopic observation revealed that formation of capillary tubes with pericyte-like cells occurred only from the tissue explant of skeletal muscles. The capillary tubes formed in the collagen gel were positive for tomato lectin and platelet/endothelial cell adhesion molecule (PCAM)-1 antibody. Formation of capillary tubes in the rat was more predominant than in the mouse. Plasmalemmal vesicles were clearly observed in the capillary tubes from rat tissue explant. Muscle fiber-type differences were also observed. In the soleus muscle, the formation of capillary tubes was predominant than the tibialis anterior muscle. Using this culture model from the rat soleus muscle, effects of α-isoproterenol (β-adrenergic receptor agonist) and low-frequency electrical stimulation were examined on the formation of capillary tubes and fine structures of skeletal muscle explant. The formation of capillary tubes was promoted by α-isoproterenol administration. At low-frequency electrical stimulation, the formation of capillary tubes was inhibited. Both α-isoproterenol and electrical stimulation reduced the degeneration of skeletal muscles. This culture method of skeletal muscles may provide a useful model that can examine the effects of various drugs and physical stimulations

Current Status for Application of RNA Interference Technology as Nucleic Acid Drug

RNA interference (RNAi) is a convenient and useful gene suppression technology induced by small interfering RNA (siRNA) composed of 21-nucleotide long double-stranded RNA. The successful application of RNAi for clinical use is expected for a long time. Although siRNA drug is categorized into a nucleic acid drug, it has a prominent advantage that genetic function can be suppressed by destroying mRNA at the posttranscriptional level without wounding genomic DNA. Nevertheless, unfortunately there are no siRNA certified as pharmaceuticals passing through clinical trials, since there are several problems, such as gene suppression efficiency, stability in blood stream, or other undesirable effects. Here, we describe the current status and future prospects for clinical application of the siRNA nucleic acid drug

Calibration-free analysis of immersed brass alloys using long-ns-duration pulse laser-induced breakdown spectroscopy with and without correction for nonstoichiometric ablation

Long-ns-duration, single pulse laser-induced breakdown spectroscopy (LIBS) is known to be an effective method to observe well resolved spectra from samples immersed in water at high hydrostatic pressures. The aim of this study is to investigate whether the signals obtained using this method are suitable for quantitative analysis of chemical composition. Six certified brass alloys consisting of copper (Cu), zinc (Zn) and lead (Pb) were measured underwater using a laser pulse of duration 250 ns, and their compositions were determined using calibration-free LIBS (CF-LIBS) and corrected CF-LIBS (CCF-LIBS) methods. The mass fractions of Cu and Zn calculated using CF-LIBS showed better agreement with the certified values than those determined using CCF-LIBS, with relative errors of Cu 4.2 ± 3.3 % and Zn 7.2 ± 6.4 %. From the results, it can be said that the difference of preferential evaporation and ablation among elements does not need to be considered for underwater measurements with the long-pulse LIBS setup used in this work. While the results indicate that the CF-LIBS method can be applied for in situ quantitative analysis of major elements with concentrations > ~ 10 %, the mass fractions determined for Pb, with concentrations < 5 % had large relative errors, suggesting that an alternative method is required to quantify minor elements

Development of spontaneous neuropathy in NF-κBp50-deficient mice by calcineurin-signal involving impaired NF-κB activation

信州大学博士（医学）・学位論文・平成24年3月28日授与（乙第21144号）・中村朋子Purpose: The transcriptional regulator, nuclear factor-kappa B (NF-kappa B)/Rel family are involved in neuronal cell death and survival. Previously, we reported that NF-kappa Bp50-deficient (p50-deficient) mice exhibit many features resembling human normal tension glaucoma (NTG). The developmental mechanism of human NTG is not clearly understood, and a radical curative treatment has yet to be established. Our aim is to elucidate the signal cascade which mediates the spontaneous optic neuropathy in p50-deficient mice as a model of NTG. Methods: To demonstrate the expression and activation of pro-apoptotic factors, which mediate the death of retinal ganglion cells (RGCs) in p50-deficient mice, western blot (WB) and luciferase reporter assays with retinas from p50-deficient and wild type mice, and cultured RGC-5 cells were performed. Furthermore, we tested the neuroprotective effects of chemical reagents (memantine, lomerizine, and tacrolimus) against N-methyl-D-aspartate (NMDA)-susceptible RGC damage according to in vitro experiments with RGC-5 cells. To elucidate the NF-kappa B-mediated death signaling, the effects of chemical reagents on spontaneous optic neuropathy were examined by histopathological studies. Results: WB experiments and luciferase reporter assays showed that NF-kappa B-inducible BCL2-associated X protein (Bax) and a pro-apoptotic factor, activated caspase 3 were expressed in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Further, the constitutively active cleaved forms of calcineurin (CaN), which have been reported to lead to apoptosis, were detected in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Pre-treatment with tacrolimus markedly protected RGC-5 cells from NMDA-induced neurotoxicity, and then both spontaneous RGC death and degenerative changes to the optic nerve in p50-deficient mice were significantly reduced by the chronic administration of tacrolimus. The experiments with cultured RGC-5 cells supported the results of histological examinations with p50-deficient mice, suggesting that CaN activation leads to NF-kappa B-induced Bax activation and caspase 3 activation, and mediates spontaneous optic neuropathy in p50-deficient mice. Conclusions: Research findings show that the chronic administration of tacrolimus significantly reduces spontaneous optic neuropathy in p50-deficient mice. We demonstrated a potential CaN signal cascade, which spontaneously induces age-dependent RGC death and degenerative optic nerve changes in p50-deficient mice.ArticleMOLECULAR VISION. 17:2157-2170 (2011)journal articl

Development of spontaneous neuropathy in NF-kappa Bp50-deficient mice by calcineurin-signal involving impaired NF-kappa B activation

信州大学博士（医学）・学位論文・平成24年3月28日授与（乙第21144号）・中村朋子Purpose: The transcriptional regulator, nuclear factor-kappa B (NF-kappa B)/Rel family are involved in neuronal cell death and survival. Previously, we reported that NF-kappa Bp50-deficient (p50-deficient) mice exhibit many features resembling human normal tension glaucoma (NTG). The developmental mechanism of human NTG is not clearly understood, and a radical curative treatment has yet to be established. Our aim is to elucidate the signal cascade which mediates the spontaneous optic neuropathy in p50-deficient mice as a model of NTG. Methods: To demonstrate the expression and activation of pro-apoptotic factors, which mediate the death of retinal ganglion cells (RGCs) in p50-deficient mice, western blot (WB) and luciferase reporter assays with retinas from p50-deficient and wild type mice, and cultured RGC-5 cells were performed. Furthermore, we tested the neuroprotective effects of chemical reagents (memantine, lomerizine, and tacrolimus) against N-methyl-D-aspartate (NMDA)-susceptible RGC damage according to in vitro experiments with RGC-5 cells. To elucidate the NF-kappa B-mediated death signaling, the effects of chemical reagents on spontaneous optic neuropathy were examined by histopathological studies. Results: WB experiments and luciferase reporter assays showed that NF-kappa B-inducible BCL2-associated X protein (Bax) and a pro-apoptotic factor, activated caspase 3 were expressed in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Further, the constitutively active cleaved forms of calcineurin (CaN), which have been reported to lead to apoptosis, were detected in the retina of p50-deficient mice as well as NMDA-treated RGC-5 cells. Pre-treatment with tacrolimus markedly protected RGC-5 cells from NMDA-induced neurotoxicity, and then both spontaneous RGC death and degenerative changes to the optic nerve in p50-deficient mice were significantly reduced by the chronic administration of tacrolimus. The experiments with cultured RGC-5 cells supported the results of histological examinations with p50-deficient mice, suggesting that CaN activation leads to NF-kappa B-induced Bax activation and caspase 3 activation, and mediates spontaneous optic neuropathy in p50-deficient mice. Conclusions: Research findings show that the chronic administration of tacrolimus significantly reduces spontaneous optic neuropathy in p50-deficient mice. We demonstrated a potential CaN signal cascade, which spontaneously induces age-dependent RGC death and degenerative optic nerve changes in p50-deficient mice.ArticleMOLECULAR VISION. 17:2157-2170 (2011)journal articl

Obesity and the risk of diabetes mellitus in middle-aged Japanese men.

The morbidity of diabetes mellitus is increasing gradually in Japanese populations. It is important to clarify the risk factors of diabetes in Japanese populations in order to take adequate measures against the increasing morbidity of diabetes. In order to evaluate the link between past and concurrent obesity and diabetes in middle-aged Japanese men, we conducted a worksite-based historical cohort study in Okayama, Japan in 1999. Annual health examination data of middle-aged male workers in a worksite were collected. The relative risks of past and concurrent obesity for developing diabetes were calculated. Subjects with a past history of obesity at between 40 and 50 years of age had a significantly higher risk of developing diabetes by age 55 than did subjects in the normal weight group. These results suggest that, in order to prevent diabetes in middle-aged Japanese men, health guidance for normal weight maintenance should be provided not only for middle-aged men, but also for men under age 40.</p