• The Korean Journal of Pharmacology
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• v.25 no.1
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• pp.53-58
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• 1989

For several years, we investigated the pharmacological action of several substances isolated from Buxus microphylla var koreana Nakai, which had been used as folk remedies of malaria and venereal disease. Cyclobuxine $D(C_{25}H_{42}ON_2)$, a steroidal alkaloid, exerted an antiinflammatory action, hypotensive and bradycardic effects in rats. In the present study, we isolated alkaloid from the acetone-insoluble fraction of the strong bases of this plants. This alkaloid $(C_{25}H_{38}ON_2)$ was identified as a steroidal alkaloid contained a cyclopropane ring by physical and chemical methods. It is a derivative of cyclobuxine D and named cyclobuxine E. We examined the effect of cyclobuxine E on the contractile response induced by acetylcholine and two distinct types of potassium-activated calcium channels in an intestinal smooth muscle of the rat. Cyclobuxine E inhibited significantly the Ach-induced contraction. The isolated longitudinal muscle from the rat duodenum was immersed calcium-depleted potassium depolarizing solution. Ten minutes after, 1.8 mM $CaCl_2$ was added to muscle bath and elicited a biphasic increase in muscle tension. Cyclobuxine E produced an appreciable inhibition of both components of the mechanical response. In addition, Cyclobuxine E introduced at a point when the tonic response had reached its maximum level, caused the muscle to exhibit a rapid loss of tension. Based on these experimental results, we proposed the possibility that the inhibitory action of cyclobuxine E on the isolated rat duodenum may be due to inhibiting the transmembrane fluxes of calcium ion in potassium-activated calcium channels.

• Development and Reproduction
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• v.4 no.1
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• pp.67-77
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• 2000

Snailfish usually lives at the bottom of the sea and showed typical retrogressive change with specialized tissue structures of skin and skeletons. In order to obtain the specific genes of snailfish, highly expressed in the body, we made subtracted cDNA library and analyzed 200 clones. Totally 200 clones were obtained and sequenced, and among them 62 clones were turned out to be homologous to the known gene, i.e., thioesterase (9), myosin (8), creatine kinase (7), skeletal alpha-actin (6), parvalbumin b (5), ribosomal protein (5), type I collagen (3), muscle troponin (3), dopamine receptor (2), histatin (2), and heat shock protein (2), cystatin (1), lectin (1), statherin (1), secretory carrier membrane protein (1), keratin type I (1), desmin (1), chloroplast (1), muscle tropomyosin (1), reticulum calcium ATPase (1), ribonucleoprotein (1). The remaining 138 clones were low homologous or non-redundant genes through Genbank search. Especially 5 clones were novel and specifically expressed in the body tissues of Snailfish by in situ hybridization. Therefore, we analysed these 5 clones to identify the C-terminal protein structures and motifs, and partly defined the roles of these proteins in comparison with the expression patterns by in situ hybridization. C9O-77, about 5000 bp, was supposed to be a matrix protein expressed strongly positive in epithelium, myxoid tissue, fibrous tissue and collagenous tissue. C9O-116, about 1500 bp, was supposed to be a transmembrane protein which was weakly expressed in the fibrous tissue, epithelium tissue, and myxoid tissue, but strong in muscle tissue. C9O-130, about 1200 bp, was supposed to be an intracytoplasmic molecule usually in the epithelial cells. C9O-161, about 2000 bp, was weakly expressed in epithelium, muscle tissue and myxoid tissue, but specially strong in epithelium. C9O-171, about 1000 bp, was supposed to be a transcription factor containing zinc finger like domain, which was intensely expressed in the epithelium, muscle tissue, fibrous tissue, and in collagenous tissue.

• Tuberculosis and Respiratory Diseases
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• v.59 no.1
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• pp.30-38
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• 2005

Background : Arsenic trioxide ($As_2O_3$) has been used to treat acute promyelocytic leukemia, and it induces apoptosis in a variety of solid tumor cell lines including non-small cell lung cancer cells. However, nonsteroidal antiinflammatory drugs (NSAID) can enhance tumor response to chemotherapeutic drugs or radiation. It was previously demonstrated that a combination treatment with $As_2O_3$ and sulindac induces the apoptosis of NCI-H157 human lung carcinoma cells by activating the caspase cascade. This study aimed to determine if a combination treatment augmented its apoptotic potential through other pathways except for the activation of the caspase cascade. Material and Methods : The NCI-H157 cells were treated with $As_2O_3$, sulindac and antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC). The cell viability was measured by a MTT assay, and the level of intracellular hydrogen peroxide ($H_2O_2$) generation was monitored fluorimetrically using a scopoletin-horse radish peroxidase (HRP) assay. Western blotting and mitochondrial membrane potential transition analysis were performed in order to define the mechanical basis of apoptosis. Results : The viability of the cells was decreased by a combination treatment of $As_2O_3$ and sulindac, and the cells were protected using antioxidants in a dose-dependent manner. The increased $H_2O_2$ generation by the combination treatment was inhibited by antioxidants. The combination treatment induced changes in the mitochondrial transmembrane potential as well as the expression of the Bcl-2 family proteins, and increased cytochrome c release into the cytosol. However, the antioxidants inhibited the effects of the combination treatment. Conclusion : Combination treatment with $As_2O_3$ and sulindac induces apoptosis in NCI-H157 human lung carcinoma cells via ROS generation with a mitochondrial dysfunction.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.246-250
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• 2009

Retroviruses enter host cells by membrane fusion between the viral Env proteins on the virus membrane and a virus receptor on the cellular membrane. The envelope protein of the ecotropic Moloney murine leukemia virus is synthesized as a gp85 precursor and is proteolytically cleaved into an extracellular surface unit (SU) and the transmembrane protein (TM). The cytoplasmic tail (16 amino acid; R peptide) of the TM protein is further cleaved by the viral protease during virion maturation. Unlike the wild type Env protrin bearing the R peptide, R peptide-truncated Envelope induces syncytia in susceptible cells. To understand the mechanism of R peptidetruncated Env in syncytium formation, R peptide-truncated Env expressing full-length molecular clone containing EGFP in PRR (proline rich region) of Env was constructed. This molecular clone induced syncytia in transfected NIH3T3 cells, fluorescence was detected in the cytoplasm and at the plasma membrane, while the nuclei did not stain and appeared black by fluorescence microscopy. Interestingly, virions with truncated envelope produced from transfected NIH3T3 cells induced syncytia in NIH3T3 cells, but fluorescence was not detected in the same infected cells. It is believed that cell-free viruses direct the fusion of neighboring cells without infection. Our data suggests that use of EGFP-tagged envelope for monitoring syncytium is a sensitive and convenient method. We also found that virion incorporated the R peptide-truncated Env is able to induce the formation of syncytia by fusion from without.

• Journal of Life Science
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• v.21 no.7
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• pp.1032-1038
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• 2011

Recently identified Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) consists of an ion channel-like transmembrane domain (VSD) and a phosphatase-like domain. Ci-VSP senses the change of membrane potential by its VSD and works as a phosphoinositide phosphatase by its phosphatase domain. In this study, we present the construction of His-tagged phosphatase-like domain of Ci-VSP, its recombinant expression and purification, and its enzymatic activity behavior in order to examine the biochemical behavior of phosphatase domain of Ci-VSP without interference. We found that Ci-VSP(248-576)-His can be eluted with an elution buffer containing 25 mM NaCl and 100 mM imidazole during His-tag purification. In addition, we found the proper measurement condition for kinetics study of Ci-VSP(248-576)-His against p-nitrophenyl phosphate (pNPP). We measured the kinetic constant of Ci-VSP(248-576)-His at $37^{\circ}C$, pH 5.0 or 5.5, under 30 min of reaction time, and less than $2.0\;{\mu}g$ of protein amount. With these conditions, we acquired that Ci-VSP(248-576)-His has $K_m$ of $354{\pm}0.143\;{\mu}M$, $V_{max}$ of $0.0607{\pm}0.0137\;{\mu}mol$/min/mg and $k_{cat}$ of $0.359{\pm}0.009751\;min^{-1}$ for pNPP dephosphorylation. Therefore, we produced a pure form of Ci-VSP(248-576)-His, and this showed a higher activity against pNPP. This purified protein will provide the road to a structural investigation on an interesting protein, Ci-VSP.

• Radiation Oncology Journal
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• v.12 no.3
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• pp.271-284
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• 1994

Purpose : Phospholipase C(PLC) isozymes play significant roles in transmembrane signal transduction. PLC-${\gamma}1$ acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU. Materials and Methods : To determine the effect of the administration mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group I had no treatment. Group II received radiation(8 Gy) only. Group III received radiation(8 Gy) and 5-FU(150mg/kg) continuous intravenous (iv) infusion for 12 hours. Group IV received radiation(8 Gy) and 5-FU(750mg/kg) iv bolus injection. Group V received only 5-FU(150mg/kg) continuous iv infusion for 12 hours, Group VI received only 5-FU (150mg/kg) iv bolus injection. Through immunoblotting and immunohistochemistry, we examined the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU adminstration. We also investigated the histological findings using hematoxylin and eosin stain. Results : In the immunohistochemistry study, PLC-${\gamma}1$ expression was the highest in group III followed by groups II and VI in that order and was weakly positive in groups V and VI. PLC-${\gamma}1$ was hardly detected in the control group. The expression of ras oncoprotein was the same as the PLC-${\gamma}1$ expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-${\gamma}1$ expression was the highest in group III followed by group IV and II in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-${\gamma}1$ on the damaged surface epithelium rather than to its expression in the regeneration region as observed in the immunohistochemistry study for group IV. The expression of PLC-${\delta}1$ was positive only in group V and VI, which received both radiation and 5-FU, and the expression of PLC-${\beta}1$ was negligible for all groups. Conclusion : These results suggest that PLC-${\gamma}1$ mediated signal transduetion and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous iv infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition, the expression of PLC-${\delta}1$ in combined group of radiation and 5-FU implies that PLC-${\delta}1$ may be involved in signal transduction mediated by concerted action between radiation and 5-FU.

• Journal of Life Science
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• v.21 no.12
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• pp.1772-1777
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• 2011

Cystic fibrosis transmembrane conductance regulator (CFTR) gene and sodium-independent $Cl^-/HCO_3^-$ anion exchanger (AE) genes are expressed in a wide variety of mammalian tissues including cholangiocytes. They play an important role in the regulation of intracellular pH (pHi) as well as in transepithelial acid/base transport necessary for biliary bicarbonate secretion. The aim of this study was to examine the expression level of CFTR gene and AE genes (AE1, AE2 and AE3) in the cholangiocytes and the hepatocytes, and also measure AE2 gene expression level after bile duct ligation (BDL). As we previously described, isolated hepatocytes and cholangiocytes from the liver of normal and BDL rats were prepared and gene expression levels were measured by using RT-PCR. We found that AE1, AE2, and AE3 genes were expressed in both hepatocytes and cholangiocytes, but CFTR was only in cholangiocytes. AE2 gene expression level was higher in the BDL hepatocytes than normal hepatocytes, which was significantly different between two groups. AE2 gene expression level was lower in the BDL cholangiocytes than normal cholangiocytes. However, AE2 gene expression level in both hepatocytes and cholangiocytes were not changed with a longer duration of BDL. These results suggest that CFTR and AE2 may play an important role in the pathogenetic mechanism of biliary cholestatic liver disease.

• Korean Journal of Soil Science and Fertilizer
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• v.36 no.3
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• pp.134-144
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• 2003

A rhizobacterium Pseudomonas cholororaphis O6 produced several secondary metabolites, such as phenazines, protease, and HCN that may be involved in inhibition of the growth of phytopathogenic fungi. In field study, P. chlororaphis O6 treatment on wheat seed suppressed root rot disease caused by Fusarium culmorum. The major organic acids of cucumber root exudates were fumaric acid, malic acid, benzoic acid, and succinic acid. Glucose and fructose were major monosaccharides in cucumber root exudates. The total amount of organic acids was ten times higher than that of the sugars. P. chlororaphis O6 grew well on cucumber root exudates. The dctA gene of P. chlororaphis O6 consisted of a 1,335 bp open reading frame with a deduced amino acid sequence of 444 residues, corresponding to a molecular size of about 47 kD and pI 8.2. The deduced dctA sequence has ten putative transmembrane domains, as expected of a membrane-embedded protein. Our results indicated that organic acids in cucumber root exudates may play an important role in providing nutrient source for root colonization of biological control bacteria, and the dctA gene of P. chlororaphis O6 may be an important bacterial trait that is involved in utilization of root exudates.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.3
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• pp.360-369
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• 2019

Cadherins are essential transmembrane proteins that promote cell-cell adhesion and maintain the corpus luteum structure in the ovary. This study examined the influence of prostaglandin F2 alpha ($PGF2{\alpha}$) on E-cadherin, N-cadherin, and adhesion in luteal theca cells (LTCs). The luteal cells were isolated from the mid-phase corpus luteum, and the LTCs were cultured separately from the luteal heterogeneous cells according to the morphology of the mesenchymal cells and to determine if steroidogenic and endothelial cells of LTCs, 3beta-hydroxysteroid dehydrogenase ($3{\beta}$-HSD), and vascular endothelial growth factor receptor 2 (VEGFR2) mRNA were used. The LTCs were then incubated in the culture medium supplemented with 0.01, 0.1, and 1.0 mM $PGF2{\alpha}$ for 24 h, and the E-cadherin and N-cadherin proteins in the LTCs were detected by confocal laser scanning microscopy. The results revealed $3{\beta}$-HSD mRNA expression in the LTC but no VEGF2R mRNA expression. The E-cadherin and N-cadherin proteins of the LTCs were damaged in the 0.01, 0.1, and 1.0 mM $PGF2{\alpha}$ treatment groups, and the expression of the N-cadherin protein was reduced significantly in 0.01 mM $PGF2{\alpha}$ compared to the 0 mM $PGF2{\alpha}$ treatment groups (P<0.05). In addition, the number of attached LTCs were significantly lower in the 0.01 mM $PGF2{\alpha}$ treatment group than in the 0 mM $PGF2{\alpha}$ treatment group (P<0.05). In conclusion, $PGF2{\alpha}$ affected the disruption of cadherin proteins and cell adhesion in LTCs. These results may help better understand the cadherin and adhesion mechanism during corpus luteum regression in the ovary.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.67 no.1
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• pp.41-52
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• 2022

As a cultivar of Korean wheat, 'Keumgang' wheat variety has a fast growth period and can be grown stably. Hexaploid wheat (Triticum aestivum) has moderately high salt tolerance compared to tetraploid wheat (Triticum turgidum L.). However, the molecular mechanisms related to salt tolerance of hexaploid wheat have not been elucidated yet. In this study, the candidate genes related to salt tolerance were identified by investigating the genes that are differently expressed in Keumgang variety and examining salt tolerant mutation '2020-s1340.'. A total of 85,771,537 reads were obtained after quality filtering using NextSeq 500 Illumina sequencing technology. A total of 23,634,438 reads were aligned with the NCBI Campala Lr22a pseudomolecule v5 reference genome (Triticum aestivum). A total of 282 differentially expressed genes (DEGs) were identified in the two Triticum aestivum materials. These DEGs have functions, including salt tolerance related traits such as 'wall-associated receptor kinase-like 8', 'cytochrome P450', '6-phosphofructokinase 2'. In addition, the identified DEGs were classified into three categories, including biological process, molecular function, cellular component using gene ontology analysis. These DEGs were enriched significantly for terms such as the 'copper ion transport', 'oxidation-reduction process', 'alternative oxidase activity'. These results, which were obtained using RNA-seq analysis, will improve our understanding of salt tolerance of wheat. Moreover, this study will be a useful resource for breeding wheat varieties with improved salt tolerance using molecular breeding technology.