• Asian-Australasian Journal of Animal Sciences
• /
• v.7 no.3
• /
• pp.427-434
• /
• 1994

The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.4
• /
• pp.1307-1313
• /
• 2015

Colangiocarcinoma (CCA) is a progressively fatal disease which generally occurs due to malignant transformation of hepatic biliary cholangiocytes. The incidence of CCA has been increasing worldwide and there is an urgent requirement for effective diagnosis and treatment strategies against this devastating disease. Different factors including liver-fluke infestation, viral hepatitis, exogenous nitrosamine-mediated DNA damage, and chronic inflammation have been linked to CCA genesis. However, the risk factors and underlying complex mechanisms leading to development of CCA are not sufficiently understood to devise an effective targeted treatment therapy. In this review, we summarize currently known epidemiological and pathological aspects of the disease and briefly describe various potential biomarkers and experimental anticancer phytochemicals related to CCA research. In addition, we also sum up recent findings that link chronic inflammation of hepatic biliary cholangiocytes with CCA. The collective information concisely presented in this article would provide useful insights into the current understanding of this cancer.

• BMB Reports
• /
• v.33 no.6
• /
• pp.440-447
• /
• 2000

In this study HepG2 cells were used to express and purify HBV pol proteins. In order to facilitate purification of HBV pol proteins, HBV pol and its deletion mutants were fused to MBP (Maltose Binding Protein). As a result we successfully expressed and partially purified both wild type and mutant recombinant HBV pol proteins by using an amylose resin and anti-MBP antibody. In the case of wild type, the anti-MBP antibody detected three bands. One was full-length and the others were generated by proteolysis of the terminal domain region. The expressed MBP/POL proteins were localized both in the cytoplasm and in the perinuclear region. The purified proteins had polymerase activity toward an exogenous homo-polymer template. The MBP/POL protein also had DNA synthesis activity in vivo, since the MBP/POL expression construct was able to complement a HBV polymerase mutant in trans.

• BMB Reports
• /
• v.44 no.6
• /
• pp.393-398
• /
• 2011

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.77.1-77
• /
• 2003

We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

• Parasites, Hosts and Diseases
• /
• v.56 no.1
• /
• pp.1-9
• /
• 2018

Giardia lamblia, an anaerobic, amitochondriate protozoan parasite causes parasitic infection giardiasis in children and young adults. It produces pyruvate, a major metabolic product for its fermentative metabolism. The current study was undertaken to explore the effects of pyruvate as a physiological antioxidant during oxidative stress in Giardia by cysteine-ascorbate deprivation and further investigation upon the hypothesis that oxidative stress due to metabolism was the reason behind the cytotoxicity. We have estimated intracellular reactive oxygen species generation due to cysteine-ascorbate deprivation in Giardia. In the present study, we have examined the effects of extracellular addition of pyruvate, during oxidative stress generated from cysteine-ascorbate deprivation in culture media on DNA damage in Giardia. The intracellular pyruvate concentrations at several time points were measured in the trophozoites during stress. Trophozoites viability under cysteine-ascorbate deprived (CAD) medium in presence and absence of extracellular pyruvate has also been measured. The exogenous addition of a physiologically relevant concentration of pyruvate to trophozoites suspension was shown to attenuate the rate of ROS generation. We have demonstrated that Giardia protects itself from destructive consequences of ROS by maintaining the intracellular pyruvate concentration. Pyruvate recovers Giardia trophozoites from oxidative stress by decreasing the number of DNA breaks that might favor DNA repair.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.2
• /
• pp.643-646
• /
• 2014

Objective: This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2 (PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence on invasiveness. Methods: Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established. TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasiveness was evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. Results: TF protein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expression was significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR-2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TF ShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TF and MMP-9 expression was positively correlated with the invasiveness of tumor cells. Conclusion: TF, PAR-2, and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.

• Journal of Ginseng Research
• /
• v.36 no.4
• /
• pp.449-460
• /
• 2012

Plants have versatile detoxification systems to encounter the phytotoxicity of the wide range of natural and synthetic compounds present in the environment. Glutathione S-transferase (GST) is an enzyme that detoxifies natural and exogenous toxic compounds by conjugation with glutathione (GSH). Recently, several roles of GST giving stress tolerance in plants have demonstrated, but little is known about the role of ginseng GSTs. Therefore, this work aimed to provide further information on the GST gene present in Panax ginseng genome as well as its expression and function. A GST cDNA (PgGST) was isolated from P. ginseng cDNA library, and it showed the amino acid sequence similarity with theta type of GSTs. PgGST in ginseng plant was induced by exposure to metals, plant hormone, heavy metals, and high light irradiance. To improve the resistance against environmental stresses, full-length cDNA of PgGST was introduced into Nicotiana tabacum. Overexpression of PgGST led to twofold increase in GST-specific activity compared to the non-transgenic plants, and the GST overexpressed plant showed resistance against herbicide phosphinothricin. The results suggested that the PgGST isolated from ginseng might have a role in the protection mechanism against toxic materials such as heavy metals and herbicides.

• Reproductive and Developmental Biology
• /
• v.30 no.2
• /
• pp.87-92
• /
• 2006

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.

• Journal of Korean Neurosurgical Society
• /
• v.29 no.4
• /
• pp.471-476
• /
• 2000

Objective : p16/INK4a, a kind of tumor suppressor genes, encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. This prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumor suppressor protein(pRb), thus preventing exit from the G1 phase. According to previous reports, over 50% of glioma tissue and 80% of glioma cell lines have been demonstrated inactivation of p16/INK4a gene. The purpose of this study was to determine whether recombinant adenovirus-p16 virus is a suitable candidate for gene replacement therapy in cases of glioma. Methods : Three human glioma cell lines(U251MG, U87MG and U373MG) that express mutant p16 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p16 cDNA into the cells ; control cells were infected with the Ad-${\beta}$-gal expressing ${\beta}$-galactosidase. To monitor gene transfer and the expression of exogenous genes, we used Western Blotting analysis. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. Results : We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16 induced growth suppression in vitro. Flow cytometric study revealed that Ad-CMV-p16 infected U87MG cells were arrested during the G0-G1 phase of the cell cycle. Expression of p16 transferred by Ad-CMV-p16 in glioma cells was highly efficient and maintained for more than seven days. Conclusions : Our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.