• Korean Journal of Medicinal Crop Science
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• v.17 no.4
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• pp.266-272
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• 2009

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.95), catalyzes the reduction of hydroxycinnamaldehydes to give hydroxycinnamyl alcohols, or "monolignols," the monomeric precursors of lignin. Lignins are important components of cell walls and lignified secondary cell walls play crucial roles in long distance transport of water and nutrients during plant growth and development and in plant defense against biotic and abiotic stresses. Here a cDNA clone containing a CAD gene, named as PgCAD, was isolated from a commercial medicinal plant Panax ginseng. PgCAD is predicted to encode a precursor protein of 177 amino acid residues, and its sequence shares high homology with a number of other plant CADS. The expression of PgCAD in adventitious roots and hairy roots of P. ginseng was analyzed using reverse transcriptase (RT)-PCR under various abiotic stresses such as salt, salicylic acid, wounding and chilling treatment that triggered a significant induction of PgCAD at different time points within 2-48 h post-treatment. This study revealed that PgCAD may help the plants to survive against various abiotic stresses.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.606-612
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• 2012

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth, apoptosis and other IGF-stimulated signaling pathways. In order to explore the significance of IGFBP-5 in cells of the Inner Mongolian Cashmere goat (Capra hircus), IGFBP-5 gene complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the animal's fetal fibroblasts and tissue-specific expression analysis was performed by semi-quantitative RT-PCR. The gene is 816 base pairs (bp) in length and includes the complete open reading frame, encoding 271 amino acids (GenBank accession number JF720883). The full cDNA nucleotide sequence has a 99% identity with sheep, 98% with cattle and 95% with human. The amino acids sequence shares identity with 99%, 99% and 99%, respectively. The bioinformatics analysis showed that IGFBP-5 has an insulin growth factor-binding protein homologues (IB) domain and a thyroglobulin type-1 (TY) domain, four protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, three prenyl group binding sites (CaaX box). The IGFBP-5 gene was expressed in all the tested tissues including testis, brain, liver, lung, mammary gland, spleen, and kidney, suggesting that IGFBP-5 plays an important role in goat cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.3713-3716
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• 2012

Objective: Transmembrane protein 166 (TMEM166) expression in esophageal squamous cell carcinoma (ESCC) and remote normal esophageal tissues was examined to assess any role in tumour biology. Methods: TMEM166 mRNA expression in 36 cases with ESCC (36 tumour samples, 36 remote normal esophageal tissue samples) was detected by RT-PCR. TMEM166 protein expression was analysed in paraffin-embedded tissue samples from the same cases by immunohistochemistry. Results: Semi-quantitative analysis showed TMEM166 mRNA expression in ESCCs to be significantly lower than in remote normal esophageal tissues ($0.759{\pm}0.713$ vs. $2.622{\pm}1.690$, P=0.014). TMEM166 protein expression was also significantly reduced (69.4% vs. 94.4%, P<0.01). Conclusion: TMEM166 mRNA and protein expression demonstrated significant reduction in ESCCs compared with remote esophageal tissues, albeit with no correlation with tumour size, differentiation, stage, and lymph node metastasis, suggesting a role in regulating autophagic and apoptotic processes in the ESCC.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1635-1640
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• 2004

Two experiments were conducted to evaluate developmental gene expression of antimicrobial peptide PR-39 and effect of zinc oxide on gene regulation of PR-39 in piglets using semi-quantitative RT-PCR analysis. In experiment 1, fifteen female Tai-Hu pigs (a local breed in China) in five groups, each of three pigs at 1, 14, 28, 42 and 56 days of age were used to determine effect of age and weaning on mRNA expression of PR-39. In experiment 2, nine groups of pigs (total seventy-two female 36 days-age weanling Tai-Hu piglets) were assigned to three treatments (${ZnO}_0$, ${ZnO}_{100}$ and ${ZnO}_{3000}$). The feeding experimental period lasted 15 days. After feeding experiment, nine pigs with three animals in each treatment were chosen to determine the effect of ZnO on PR-39 mRNA expression of pigs. The results showed that PR-39 mRNA levels increased steadily in postnatal day 1-28 (preweaning), and weaning significantly decreased PR-39 mRNA expression of piglets (p<0.05). ${ZnO}_{3000}$ (3,000 mg zinc/kg diet) significantly increased PR-39 mRNA expression (p<0.05) when piglets were feed ${ZnO}_{3000}$ diet for 15 days. ${ZnO}_{100}$ (100 mg zinc/kg diet) also increased PR-39 gene expression, but the result was not statistically significant (p>0.05). The result was in accordance with the effect of ${ZnO}_{3000}$ and ${ZnO}_{100}$ on weight gain of piglets and prevention of diarrhea.

• IMMUNE NETWORK
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• v.7 no.2
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• pp.57-65
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• 2007

Background: Cordyceps militaris have been reported to modify the immune and inflammatory responses both in vivo and in vitro. Macrophages play important roles in the innate immunity through the phagocytosis of antigens. This study examined the effects of Cordyceps militaris on the activation of murine macrophage RAW 264.7 cells and primary macrophages. Methods: The components contained in culture broth of Cordyceps militaris were purified by propyl alcohol extraction and HP 20 column chromatography to CMDB, CMDBW, CMDB5P, and CMDB25P. The amounts of nitric oxide (NO) were determined by using ELISA, Griess reagent respectively. The amounts of some cytokines were determined by using ELISA, western blot, and RT-PCR The expression levels of cell surface molecules (ICAM-1, B7-1 and B7-2) were measured by flow cytometric analysis. Results: All the components of Cordyceps militaris produced significant amounts of NO. In particular, CMDB produced much more NO in RAW 264.7 cells and primary macrophages than other fractions of Cordyceps militaris. CMDB increased significantly the production of tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-1${\beta}$, and IL-6 dose-dependently in RAW 264.7 cells. Examination of the gene expression level also showed that the enhanced production of cytokines was correlated with the up-regulation of i-NOS expression, cycloxygenase (COX)-2 expression, IL-1${\beta}$ and IL-6 expression, and TNF-${\alpha}$ expression on the expression of mRNAs by semi-quantitative RT-PCR Western blot analysis also confirmed that CMDB enhances the expression level of these cytokines. Conclusion: These results show that CMDB stimulates the production of NO and pro-inflammatory cytokines and can also up-regulate the gene expression levels in macrophages.

• IMMUNE NETWORK
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• v.11 no.4
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• pp.203-209
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• 2011

Background: T cell immunoglobulin mucin containing molecule (TIM)-3 is expressed in differentiated Th1 cells and is involved in the suppression of the cytokine production by these cells. However, the regulation of the expression of other T cell genes by TIM-3 is unclear. Herein, we attempted to identify differentially expressed genes in cells abundantly expressing TIM-3 compared to cells with low expression of TIM-3. Methods: TIM-3 overexpressing cell clones were established by transfection of Jurkat T cells with TIM-3 expression vector. For screening of differentially expressed genes, gene fishing technology based on reverse transcription-polymerase chain reaction (RT-PCR) using an annealing control primer system was used. The selected candidate genes were validated by semi quantitative and real-time RT-PCR. Results: The transcription of TIMP-1, IFITM1, PAR3 and CCL1 was different between TIM-3 overexpressing cells and control cells. However, only CCL1 transcription was significantly different in cells transiently transfected with TIM3 expression vector compared with control cells. CCL1 transcription was increased in primary human $CD4^+$ T cells abundantly expressing TIM-3 but not in cells with low expression of TIM-3. Conclusion: CCL1 was identified as a differentially transcribed gene in TIM-3-expressing $CD4^+$ T cells.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.383-387
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• 1999

Objective: There is increasing evidence for the expression of rat in gene in several extrapituitary sites including testis and ovary. We also have demonstrated that the local LH expression in the rat epididymis and uterus, the major accessory sex organs in male and female reproductive system, respectively. Design: The present study was undertaken to elucidate whether the gene for LH receptor is expressed in rat uterus and whether the expressions of uterine LH and its receptor are differentially regulated during estrous cycle. Presence of the transcripts for rat LH receptor in the rat uterine tissue were confirmed by touchdown reverse transcription-polymerase chain reaction (RT-PCR). Results: In $LH{\beta}$ semi-quantitative RT-PCR, the highest expression level was shown in estrus stage. The level of ill receptor transcripts was also fluctuated during estrous cycle. In ovariectomized rats (OVX + Oil), the expressions of both uterine LH and LH-R were markedly reduced when compared to those from normal rats. Supplement with estradiol $17{\beta}$ to the ovariectomized rats (OVX + $E_2$) restored the expression levels of LH and its receptor to the levels in uteri from normal rats. Conclusion: Our findings indicated that 1) LH and its receptor gene are expressed in the rat uterus from cycling rats, 2) the expression of uterine LH and its receptor is mainly, if not all, under the control of ovarian sex steroid(s). These results suggested that the uterine LH may act as a local regulator with auto and/or paracrine manner, though the posibility that the pituitary LH may act directly on the regulation of uterine functions could not be discarded.

• Genomics & Informatics
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• v.2 no.1
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• pp.30-35
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• 2004

To investigate the XIST gene expression and its effect in a Klinefelter's patient, we used Klinefelter's syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter's syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.