• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.3
• /
• pp.1171-1176
• /
• 2014

Objective: The tumor suppressor gene, Ras-association domain family (RASSF)2A, is inactivated by promoter hypermethylation in many cancers. The current study was performed to evaluate the methylation status of RASSF2A in epithelial ovarian cancer (EOC) tissues and plasma, and correlations with gene expression and clinicopathologic characteristics. Method: We detected methylation of the RASSF2A gene in tissues and corresponding plasma samples from 47 EOC patients and 14 patients with benign ovarian tumors and 10 with normal ovarian tissues. The methylation status was determined by methylation-specific PCR while gene expression of mRNA was examined by RT-PCR. The EOC cell line, SKOV3, was treated with 5-aza-2'-deoxycytidine (5-azadC). Results: RASSF2A mRNA expression was significantly low in EOC tissues. The frequency of aberrant methylation of RASSF2A was 51.1% in EOC tissues and 36.2% in corresponding plasma samples, whereas such hypermethylation was not detected in the benign ovarial tumors and normal ovarian samples. The expression of RASSF2A mRNA was significantly down-regulated or lost in the methylated group compared to the unmethylated group (p<0.05). After treatment with 5-aza-dC, RASSF2A mRNA expression was significantly restored in the Skov3 cell line. Conclusion: Epigenetic inactivation of RASSF2A through aberrant promoter methylation may play an important role in the pathogenesis of EOC. Methylation of the RASSF2A gene in plasma may be a valuable molecular marker for the early detection of EOC.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.9
• /
• pp.1070-1076
• /
• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

• Toxicological Research
• /
• v.26 no.1
• /
• pp.47-52
• /
• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

• Journal of Korean Medicine for Obesity Research
• /
• v.8 no.1
• /
• pp.33-49
• /
• 2008

Objectives In order to investigate the effects of Sansayukbokhap-bang (SSYBHB) on the hematological and histological changes. Methods C57BL/6 mice were fed with high fat diet. C57BL/6 mice were divided into four groups and fed for 15weeks. Results 1. The body weight of SSYBHB intake mice was significantly lower than high fat diet group. 2. The final increase of body weight was decreased significantly. 3. The levels of ALT, AST, total cholesterol, LDL-Cholesterol, triglyceride, Leptin were decreased significantly. 4. The levels of creatinine were decreased but did not show significance. 5. The level of HDL-cholesterol and the expression of ${\beta}$3AR mRNA gene in 3T3-L1 Adipocytes were increased significantly. 6. Adipocytes' size was decreased significantly. 7. The expression of ${\beta}$3AR mRNA gene, Leptin mRNA gene and serotinin mRNA gene in Adipocytes tissue was decreased significantly. Conclusion Based on these results, it is proved that SSYBHB is effective on the therapy of obesity by referring to obese-gene and obese inhibitory. So, it is espected that the clinical application of SSYBHB can help the treatment of obesity.

• Korean Journal of Plant Tissue Culture
• /
• v.28 no.3
• /
• pp.165-171
• /
• 2001

New visible and fast assay system have been developed for tobacco transformant introduced with adenosine deaminase (ADA) marker gene, which converts cytotoxic adenosine analogues to non-toxic inosine analogues and ammonia. Ammonia was changed to blue color in the solution of phenol-nitoprusside and alkaline-hypochlorite. It was possible to detect activity of ADA visibly on the holes of 96 well plate using tiny explant of transgenic tobacco leaves within 1 hour incubation time. As substrates of ADA enzyme from transgenic plant on the plate, a number of adenosine analogues such as 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine were possible for detection of ADA activity. Optimal condition of substrate for ADA enzyme was each 10 mM and pH 7.5 in adenosine solution. Especially, transgenic plant did not convert adenosine to inosine and ammonia in the presence of ADA inhibitor deoxycoformycin, which means that ammonia produced from transgenic plant is due to expression of ADA gene. Now, we show that this detection system can be easily, sensitively, fast and cheaply as well as visibly assayed in vitro as GUS gene system with very small size of transformant explant.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.12
• /
• pp.1670-1674
• /
• 2001

To understand molecular mechanisms that regulate deposition and release of intramuscular fat, a fasting-induced clone was identified by differential screening from cDNA library of adipose tissues of Korean cattle. The clone had a total length of 1,319 nucleotides coding for 334 amino acids. It was identified as one encoding L-lactate dehydrogenase H chain (LDH-B). Comparison of the deduced amino acid sequences of bovine LDH-B with those of pig, human, rat, and mouse showed 98%, 98%, 97%, and 96% identity, respectively. Food deprivation for 48 h increased mRNA levels of LDH-B gene in adipose tissues of Korean cattle compared to fed- and 6 h refed- tissues. The expression of obese mRNA was examined for individual adipose tissue from several fat depots. Fasting induced expression of LDH-B gene in subcutaneous adipose tissues, but it did not affect expression levels in abdominal, perirenal and intramuscular tissues. Results demonstrate that induction of LDH-B gene during fasting may represent a metabolic shift from anaerobic state to aerobic predominance in fasted adipose tissues and that its responses to fasting are different among several adipose tissues.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.9
• /
• pp.5489-5494
• /
• 2013

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.

• The Journal of Korean Academy of Prosthodontics
• /
• v.45 no.6
• /
• pp.787-794
• /
• 2007

Statement of problem. Surface microgrooves on Ti substrata have been shown to alter the expression of genes responsible for various biological activities of cultured fibroblasts. However, their effect on enhancing cell proliferation is not yet clear. Purpose. The purpose of this study was to determine the dimension of surface microgrooves on Ti substrata that enhances proliferation and alters gene expression of cultured human gingival fibroblasts. Material and methods. Commercially pure Ti discs with surface microgrooves of monotonous $3.5{\mu}m$ in depth and respective 15 and $30{\mu}m$ in width were fabricated using photolithography and used as the culture substrata in the two experimental groups in this study (TiD15 and TiD30), whereas the smooth Ti was used as the control substrata (smooth Ti group). Human gingival fibroblasts were cultured on the three groups of titanium substrata and the proliferation, DNA synthesis, and gene expression of theses cells were analyzed and compared between all groups using XTT assay, BrdU assay, and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Results. From the XTT assay at 48 h incubation, the proliferation of human gingival fibroblasts in TiD30 was significantly enhanced compared to that in smooth Ti and TiD15. The results from the BrdU assay showed that, at 24 h incubation, the DNA synthesis was significantly enhanced in TiD30 compared to that in smooth Ti. In RT-PCR, increase in the expression of PCR transcripts of fibronectin, CDK6, $p21^{cip1}$ genes was noted at 48h incubation. Conclusion. Surface microgrooves $30{\mu}m$ in width and $3.5{\mu}m$ in depth on Ti substrata enhance proliferation and alter gene expression of cultured human gingival fibroblasts.

• Korean Journal of Plant Tissue Culture
• /
• v.22 no.4
• /
• pp.235-240
• /
• 1995

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plant. We established a new selectable marker system for tobacco transformation using chimeric adenosine deaminase (ADA) gene, which confers resistance to cytotoxic adenosine analogues, 9-$\beta$-D-arabinofuranosyl adenine(Ara-A) and cordycepin. The transformants with the chimeric ADA gene in tobacco grew in the presence of normally lethal level of cytotoxic adenosine analogues, 100 $\mu$M Ara-A and 50 $\mu$M cordycepin. We successfully distinguished transformed shoot from non-transformed shoot on the same selectable media with cytotoxic adenosine analogues. In this selectable media, we were able to select seeds with/ without ADA gene from transgenic tobacco seeds. Theses results show that the mammalian ADA gene may serve as a new selectable marker for tobacco transformation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.18 no.12
• /
• pp.1684-1690
• /
• 2005

Using mRNA differential display technique, we investigated differential gene expression in hybrids relative to their parents in a diallel cross involving four chicken breeds in order to provide an insight into the molecular basis of heterosis in chicken. The results indicated that there was extensive differential gene expression between chicken F1 hybrids and their parents which was classified into four kinds of patterns as following: (1) bands only detected in hybrid F1; (2) bands only absent in hybrid F1; (3) bands only detected in parent P1 or P2; (4) bands absent in parent P1 or P2. Forty-two differentially expressed cDNAs were cloned and sequenced, and their expression patterns were confirmed by Reverse-Northern dot blot. Sequence analysis and database searches revealed that genes showed differential expression between hybrid and parents were regulatory and functional genes involved in metabolism, mRNA splicing, transcriptional regulation, cell cycles and protein modification. These results indicated that hybridization between two parents can cause changes in expression of a variety of genes. In conclusion, that the altered pattern of gene expression in hybrids may be responsible for heterosis in chickens.