• Journal of Genetic Medicine
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• v.5 no.1
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• pp.41-46
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• 2008

Purpose : Fragile X syndrome (FXS) is the most common heritable cause of cognitive impairment. FXS is caused by hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retadation-1(FMR1) gene. Combination of Southern blotting and simple polymerase chain reaction(PCR) amplification of the FMR1 repeat region is commonly used for diagnosis in females. To give a definite diagnosis in a female child suspected of having FXS, we carried out the molecular diagnostic test for FXS using the recently developed Abbott Molecular Fragile X PCR Kit. Methods : The PCR amplification of the FMR1 repeat region was performed using the Abbott Mdecular Fragile X PCR Kit. The amplified products were analyzed by size-separate analysis on 1.5% agarose gels and by DNA fragment analysis using Gene scan. Results : Agarose gel and Gene scan analyses of PCR products of the FMR1 repeat region showed that the patient had two heterozygous alleles with a normal 30 repeats and full mutation of >200 repeats whereas her mother had two heterozygous alleles with the normal 30 repeats and premutation of 108 repeats, suggesting that the premutation of 108 repeats in her mother may have led to the full mutation of >200 repeats in the patient. Conclusion : We diagnosed FXS in a female patient using a simplified molecular diagnostic test. This commercially available diagnostic test for FXS, based on PCR, may be a suitable alternative or complement method to Southern blot analysis and PCR analysis and/or methylation specific(MS)-PCR analysis for the molecular diagnosis of FXS in both males and females.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.12
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• pp.1695-1704
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• 2014

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

• Journal of Nutrition and Health
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• v.26 no.6
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• pp.661-671
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• 1993

The change in fatty acid composition in brain tissue of the second generation rats(Sprague-Dawley strain) was studied using four different fat diets(Corn oil=CO, Soybean oil=SO, Perilla oil=PO, Fish oil=FO, 10% by Wt). The experimental diets were started from pregnancy in four different groups, each consisting of 9 rats. The seound generation rats were fed the same diet as their mothers. Animals were anesthetized with ether at 0, 3, 9 & 16 weeks of age. Whole brains were dissected out, brain tissues were, then, homogenized and lipids were extracted from brain tissues. The fatty acid compositions were measured after methylation by gas-liquid chromatography at 0, 3, 9 and 16 weeks of age of offspring. The changes in the relative concentrations of polyunsaturated fatty acids(PUFA) or more specifically docosahexaenoic acid(22 : 6, $\omega$3, DHA), the major $\omega$3 fatty acid component in rat brain at different age were similar to changes in the amount of DNA in brain tissue showing the maximum value during the lactation. The changes in saturated fatty acid(SFA) content showed a contrasting patten to those of PUFA, while monounsaturated fatty acid(MUFA) increased steadily throughout the experimental period. At birth, the relative concentrations of $\omega$3 series fatty acids the relative concentrations of PUFA, MUFA and SFA converged to very similar values respectively regardless of the dietary fatty acid compositions. In brain tissue, it is of value to note that while changes in relative concentrations of linoleic acid (18 : 2, $\omega$6, LA) and arachidonic acid(20 : 4, $\omega$6, AA) showed a precursor-product-like relationship, $\alpha$-linolenic acid(18 : 3, $\omega$3, $\alpha$-LnA) and DHA showed a different pattern. Even when the $\omega$3 fatty acid content in very low in maternal diet(CO), the second generation rat brain tissues appeared to secure DHA content, suggesting an essential role of this fatty acid in the brain. The fact that a large amount of $\alpha$-LnA in the maternal diet did not have a significant effect on the second generation rat brain $\alpha$-LnA content, indicated that DHA seemed essential component for the brain development in our experimental condition. In all groups, the relative content of $\alpha$-LnA in the brain tissues remained relatively constant throughout the experimental period at the very low level. The study of the specific concentrations and essential role(s) of DHA in each parts of brain tissue is needed in more details.

• Journal of Chest Surgery
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• v.37 no.12
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• pp.967-977
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• 2004

Background: DNA methylation is one of the important gene expression mechanisms of the cell. When cytosine of CpG dinucleotide in promotor is hypomethylated, expression of some genes that is controlled by this promoter is altered. In this study, the author investigated the effect of DNA demethylating agent, 5-aza-2'-deoxycytidine (ADC), on the expressions of cancer antigen genes, MHC and B7 in 4 lung cancer cell lines, NCIH1703, NCIH522, MRC-5, and A549. Material and Method: After treatment of cell lines, NCIH1703, NCIH522, MRC-5 and A549 with ADC (1 uM) for 48 hours, RT-PCR was performed by using the primers of MAGE, GAGE, NY-ESO-1, PSMA, CEA, and SCC antigen gene. In order to find the optimal ADC treatment condition for induction of cancer antigen, we studied the effect of ADC treatment time and dose on the cancer antigen gene expression. To know the effect of ADC on the expression of MHC or B7 and cell growth, cells were treated with 1 uM of ADC for 72 hours for FACS analysis or cells were treated with 0.2, 1 or 5 uM of ADC for 96 hours for cell counting. Result: After treatment of ADC (1 uM) for 48 hours, the expressions of MAGE, GAGE, NY-ESO-1, and PSMA genes increased in some cell lines. Among 6 MAGE isotypes tested, and gene expression of MAGE-1, -2, -3, -4 and -6 could be induced by ADC treatment. However, CEA gene expression did not change and SCC gene expression was decreased by ADC treatment. Gene expression was generally induced 24 - 28 hours after ADC treatment and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC ADC teatment, and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC teatment in ADC-Free medium. Most gene expression could be induced at 0.2 uM of ADC, but gene expression increased dependently on ADC treatment dose. The expression of MHC and B7 was not increased by ADC treatment in all four cell lines, and the growth rate of 4 cell lines decreased significantly with the increase of ADC concentrations. Conclusion: Treatment of lung cancer cell lines with ADC increases the gene expression MAGE, GAGE and NY-ESO-1 that are capable of induction of cytotoxic T lymphocyte response. We suggest that treatment with 1 uM of ADC for 48 hours and then culturing in ADC-free medium is optimal condition for induction of cancer antigen. However, ADC has no effect on MHC and B7 induction, additional modification for increase of expression of MHC, B7 and cytokine will be needed for production of efficient cancer cell vaccine.

• Journal of Nutrition and Health
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• v.42 no.5
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• pp.423-433
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• 2009

Folate and vitamin $B_{12}$ are essential cofactors for homocysteine (Hcy) metabolism. Homocysteinemia has been related with cardiovascular and neurodegenerative disease. We examined the effect of folate and/or vitamin $B_{12}$ deficiency on biomarkers of one carbon metabolism in blood, liver and brain, and analyzed the correlation between vitamin biomarkers in mild and moderate homocysteinemia. In this study, Sprague-Dawley male rats (5 groups, n = 10) were fed folatesufficient diet (FS), folate-deficient diet (FD) with 0 or 3 g homocystine (FSH and FDH), and folate-/vitamin $B_{12}$-deficient diet with 3 g homocystine (FDHCD) for 8 weeks. The FDH diet induced mild homocysteinemia (plasma Hcy 17.41 ${\pm}$ 1.94 nmol/mL) and the FDHCD diet induced moderate homocysteinemia (plasma Hcy 44.13 ${\pm}$ 2.65 nmol/mL), respectively. Although liver and brain folate levels were significantly lower compared with those values of rats fed FS or FSH (p < 0.001, p < 0.01 respectively), there were no significant differences in folate levels in liver and brain among the rats fed FD, FDH and FDHCD diet. However, rats fed FDHCD showed higher plasma folate levels (126.5 ${\pm}$ 9.6 nmol/L) compared with rats fed FD and FDH (21.1 ${\pm}$ 1.4 nmol/L, 22.0 ${\pm}$ 2.2 nmol/L)(p < 0.001), which is the feature of "ethyl-folate trap"by vitamin $B_{12}$ deficiency. Plasma Hcy was correlated with hepatic folate (r = -0.641, p < 0.01) but not with plasma folate or brain folate in this experimental condition. However, as we eliminated FDHCD group during correlation test, plasma Hcy was correlated with plasma folate (r = -0.581, p < 0.01), hepatic folate (r = -0.684, p < 0.01) and brain folate (r = -0.321, p < 0.05). Hepatic S-adenosylmethionine (SAM) level was lower in rats fed FD, FDH and FDHCD than in rats fed FS and FSH (p < 0.001, p < 0.001 respectively) and hepatic S-adenosylhomocysteine (SAH) level was significantly higher in those groups. The SAH level in brain was also significantly increased in rats fed FDHCD (p < 0.05). However, brain SAM level was not affected by folate and/or vitamin $B_{12}$ deficiency. This result suggests that dietary folate- and vitamin B12-deficiency may inhibit methylation in brain by increasing SAH rather than decreasing SAM level, which may be closely associated with impaired cognitive function in nutritional homocysteinemia.