• Journal of Microbiology and Biotechnology
• /
• v.19 no.10
• /
• pp.1103-1108
• /
• 2009

The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.

• Molecules and Cells
• /
• v.19 no.2
• /
• pp.289-293
• /
• 2005

BAF53 is an actin-related protein that shuttles between nucleus and cytoplasm. In the nucleus, it constitutes an integral component of many chromatin-modifying complexes such as the SWI/SNF, TIP60, TRRAP, and TIP48/49 complexes. BAF53 is essential for growth, but its function remains elusive. BAF53 homologues from yeast to humans have a conserved N-terminal motif, MS_(G/A)(G/A)__(V/L)YGG, which is unique to these proteins. Previously we showed that over-expression of an N-terminal deletion mutant of BAF53 ($BAF53_-{\Delta}N$) reduced the viability of HEK293 and HeLa cells. When we replaced the serine 2 and tyrosine 6 of this N-terminal motif with alanine, over-expression of the alanine-replaced BAF53 strongly impaired the growth of HEK293 cells whereas replacement with aspartate/glutamate had no effect. The alanine-replaced BAF53 mutants also stimulated p53-dependent transcription, in which the SWI/SNF and TRRAP complexes are involved. Our results demonstrate that serine 2 and tyrosine 6 play important roles in BAF53 activity.

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.324.1-324.1
• /
• 2002

Human 5-reductase type II(5AR2) is an important target for the treatment of benign prostatic hyperplasia. In this study we describe the establishment of cell line which stably expressed 3X FLAG tagged human 5AR2. We used this cell line as a cell based assay tool and source for 5AR2 enzyme. First a plasmid (3XFLAGpCMVl0-5AR2) for the expression of 5AR2 was constructed by the use of the vector 3XFLAGpCMV10 and transfected into the HEK 293. By selection with G418 sulfate. ten HEK 293 single cell clones were obtained of which three stably exhibited high 5AR2 activity. (omitted)

• International Journal of Oral Biology
• /
• v.33 no.1
• /
• pp.1-5
• /
• 2008

Adenosine 5'-triphosphate (ATP) is an important extracellular signaling molecule which is involved in a variety of physiological responses in many different tissues and cell types, by acting at P2 receptors, either ionotropic (P2X) or G protein-coupled metabotropic receptors (P2Y). P2X receptors have seven isoforms designated as $P2X_{1^-}P2X_7$. In this study, we investigated the electrophysiological and pharmacological properties of rat $P2X_{1^-}P2X_4$ currents by using whole-cell patch clamp technique in a heterologous expression system. When ATP-induced currents were analyzed in human embryonic kidney (HEK293) cells following transient transfection of rat $P2X_{1^-}P2X_4$, the currents showed different pharmacological and electrophysiological properties. ATP evoked inward currents with fast activation and fast desensitization in $P2X_{^1-}$ or $P2X_{3^-}$ expressing HEK293 cells, but in $P2X_{2^-}$ or $P2X_{4^-}$ expressing HEK293 cells, ATP evoked inward currents with slow activation and slow desensitization. While PPADS and suramin inhibited $P2X_2$ or $P2X_3$ receptor-mediated currents, they had little effects on $P2X_4$ receptor-mediated currents. Ivermectin potentiated and prolonged $P2X_4$ receptor-mediated currents, but did not affect $P2X_2$ or $P2X_3$ receptor-mediated currents. We suggest that distinct pharmacological and electrophysiological properties among P2X receptor subtypes would be a useful tool to determine expression patterns of P2X receptors in the nervous system including trigeminal sensory neurons and microglia.

• Biomolecules & Therapeutics
• /
• v.18 no.4
• /
• pp.448-453
• /
• 2010

Drug-induced long QT syndrome is known to be associated with the onset of torsades de pointes (TdP), resulting in a fatal ventricular arrhythmia. QT interval prolongation can result from blocking the human ether-a-go-go-related gene (hERG) channel, which is important for the repolarization of cardiac action potential. Nicardipine, a Ca-channel blocker and antihypertensive agent, has been reported to increase the risk of occasional serious ventricular arrhythmias. We studied the effects of nicardipine on hERG $K^+$ channels expressed in HEK293 cells and Xenopus oocytes. The cardiac electrophysiological effect of nicardipine was also investigated in this study. Our results revealed that nicardipine dose-dependently decreased the tail current of the hERG channel expressed in HEK293 cells with an $IC_{50}$ of 0.43 ${\mu}M$. On the other hand, nicardipine did not affect hERG channel trafficking. Taken together, nicardipine inhibits the hERG channel by the mechanism of short-term channel blocking. Two S6 domain mutations, Y652A and F656A, partially attenuated (Y652A) or abolished (F656A) the hERG current blockade, suggesting that nicardipine blocks the hERG channel at the pore of the channel.

• Biomolecules & Therapeutics
• /
• v.28 no.6
• /
• pp.569-575
• /
• 2020

Crotamiton is an anti-scabies drug, but it was recently found that crotamiton also suppresses non-scabietic itching in mice. However, the underlying mechanism is largely unclear. Therefore, aim of the study is to investigate mechanisms of the anti-pruritic effect of crotamiton for non-scabietic itching. Histamine and chloroquine are used as non-scabietic pruritogens. The effect of crotamiton was identified using fluorometric intracellular calcium assays in HEK293T cells and primary cultured dorsal root ganglion (DRG) neurons. Further in vivo effect was evaluated by scratching behavior tests. Crotamiton strongly inhibited histamine-induced calcium influx in HEK293T cells, expressing both histamine receptor 1 (H1R) and transient receptor potential vanilloid 1 (TRPV1), as a model of histamine-induced itching. Similarly, it also blocked chloroquine-induced calcium influx in HEK293T cells, expressing both Mas-related G-protein-coupled receptor A3 (MRGPRA3) and transient receptor potential A1 (TRPA1), as a model of histamine-independent itching. Furthermore, crotamiton also suppressed both histamine- and chloroquine-induced calcium influx in primary cultures of mouse DRG. Additionally, crotamiton strongly suppressed histamine- and chloroquine-induced scratching in mice. Overall, it was found that crotamiton has an anti-pruritic effect against non-scabietic itching by histamine and chloroquine. Therefore, crotamiton may be used as a general anti-pruritic agent, irrespective of the presence of scabies.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.1
• /
• pp.154-161
• /
• 2007

Human immunodeficiency virus type 1 (HIV-1) infections are responsible for a substantial number of deaths annually and represent a significant threat to public health. According to the latest study, the Tat (Transactivator of transcription) protein is essential in transcription and replication of viral genes, and is among the early expression genes involved in the life cycle of HIV. The virion NC (nucleocapsid) plays an important role in early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection. Therefore, we attempted to elucidate the relationship between the Tat protein and nucleocapsid protein. In a comparison of two independently prepared and hybridized samples, flag NC overexpressed HEK 293T cells and pTat overexpressed HEK 293T cells, and hybridization showed the differences in expression in each case. Among the microarray results confirmed with real-time reverse transcriptase assay, twelve genes were identified to be involved according to their gene expression profiles. Of approximately 8,208 human genes that were analyzed, we monitored candidate genes that might have been related to NC and Tat genes from gene expression profiles. Additionally, the pathways could be viewed and analyzed through the use of Pathway Studio software. The pathways from the gene list were built and paths were found among the molecules/cell objects/processes by the curation method.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.28 no.2
• /
• pp.162-168
• /
• 2014

Fruits of Schisandra chinensis (SC) Baill are considered a traditional herbal medicine for the treatment and alleviation of various diseases. The purpose of this study was to investigate the anti-cancer effects of SC extract in human breast adenocarcinoma cells (MCF-7). We used human breast adenocarcinoma cell line, MCF-7 cells. We examined cell death by MTT assay and caspase 3 and 9 assay with SC extract. To examine the inhibitory effects of SC extract, cell cycle (sub G1) analysis and mitochondrial membrane depolarization was done the MCF-7 cells after one day with SC extract. In addition, to investigate the transient receptor potential melastatin 7 (TRPM7) currents, we used the whole cell patch clamp techniques. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in MCF-7 cell growth and survival. SC extract inhibited the growth of MCF-7 cells in a dose-dependent fashion. Also we showed that SC extract induced apoptosis in MCF-7 cells by MTT assay, caspase 3 and 9 assay, sub-G1 analysis and mitochondrial membrane depolarization. SC extract inhibited the TRPM7 currents in MCF-7 cells and in TRPM7 overexpressed HEK 293 cells. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SC extract-induced cell death. Our findings provide insight into unraveling the effects of SC extract in human breast adenocarcinoma cells and developing therapeutic agents against breast cancer.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.18
• /
• pp.7785-7792
• /
• 2014

Background: Valproic acid (VPA) is a potent anticancer and antiangiogenic agent. However, design and synthesis of chemical derivatives with improved antiangiogenic and anticancer activities are still necessary. In this study a library of novel derivatives of VPA was synthesized and tested. Methods: A human liver cancer cell line (HepG2) and a human normal embryonic kidney cell line (HEK 293) were exposed to various concentrations of VPA derivatives for 24 hours and cell viability was checked by MTT colorimetric assay. Anti-angiogenic properties were evaluated in transgenic zebrafish embryos. Results: N-valproylglycine derivatives suppressed survival almost 70% (p value 0.001) in HepG2 cells but only 10-12% in HEK 293 cells (p value 0.133). They also suppressed angiogenic blood vessel formation by 80% when used between $2-20{\mu}M$ in zebrafish embryos. Valproic acid hydrazides showed moderate level of anticancer activity by affecting 30-50% (p value 0.001) of cell viability in HepG2 cells and 8-10% in HEK293 cells (p value 0.034). Conclusion: The majority of compounds in this study showed potent and stronger antiangiogenic and anticancer activity than VPA. They proved selectively toxic to cancer cells and safer for normal cells. Moreover, these compounds inhibited developmental angiogenesis in zebrafish embryos. Based on the fact that liver is a highly vascularized organ, in case of liver carcinoma these compounds have the potential to target the pathological angiogenesis and could be an effective strategy to treat hepatocellular carcinoma.

• The Transactions of The Korean Institute of Electrical Engineers
• /
• v.56 no.12
• /
• pp.2208-2213
• /
• 2007

In this study we have developed a hyperspectrum imaging system for highly sensitive and effective imaging analysis. An optical setup was designed using acoustic optical tunable filter (AOTF) for high sensitive hyperspectrum imaging. Light emitted by mercury lamp gets split in to diffracted and undiffracted beams while passing though AOTF. GFP transfected HEK-293 cell line was used as a model for in vitro imaging analysis. Cells were first, analyzed by fluorescence microscope followed by flow cytometric analysis. Flow cytometric analysis showed 66.31% transfection yield in GFP transfected HEK-293 cells. Various images of GFP transfected HEK-293 cell were grabbed by collecting the diffracted light using a CCD over a dynamic range of frequency of 129-171 MHz with an interval of 3 MHz. Subsequently, for in vivo image analysis of GFP transfected cells in mouse, a whole-body-imaging system was constructed. The blue light of 488 nm wavelength was obtained from a Xenon arc lamp using an appropriate filter and transmitted through an optical cable to a ring illuminator. To check the efficacy of the newly developed whole-body-imaging system, a comparative imaging analysis was performed on a normal mouse in presence and absence of Xenon arc irradiation. The developed hyperspectrum imaging analysis with AOTF showed the highest intensity of green fluorescent protein at 153 MHz of frequency and 494 nm of wavelength. However, the fluorescence intensity remained same as that of the background below 138 MHz (475 nm) and above 162 MHz (532 nm). The mouse images captured using the constructed whole-body-imaging system appeared monochromatic in absence of Xenon arc irradiation and blue when irradiated with Xenon arc lamp. Nevertheless, in either case mouse images appeared clearly.