• The Korean Journal of Pharmacology
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• v.31 no.1 s.57
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• pp.85-93
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• 1995

In order to examine the effect of testosterone of the cell growth, using a primary rabbit kidney proximal tubule cell culture system, we observed the effect of 3 growth factors and testosterone supplementation on the growth of primary rabbit kidney proximal tubule cells in the serum-free medium. 1 nM of testosterone showed a potentiation of the effect on the growth of the proximal tubule cell in serum-free medium, but higher concentration (>10 nM) of testosterone indeed inhibited the growth. In the absence of hydrocortisone as a growth supplement in serum-free medium, testosterone caused to potentiate the growth of the cell. In the presence of hydrocortisone, testosterone also potentiated the grwoth of the proximal tubule cells. According to the Northern analysis, testosterone increased significantly the level of ${\beta}-actin$ mRNA in proximal tubular cells of rabbit kidney. Consequently we may suggest that growth stimulatory effect of testosterone on the primary rabbit kidney proximal tubule cell in serum-free and hormonally defined media ascribed to increase the synthesis of ${\beta}-actin$, which is an important protein consisting of cellular microfilament.

• Journal of Life Science
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• v.22 no.12
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• pp.1651-1657
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• 2012

Plectin and microtubule actin cross-linking factor 1 (MACF1) are architectural proteins that contribute to the function of skeletal muscle as generators of mechanical force. However, the influence of insulin- like growth factor-I (IGF-I), a master regulator of skeletal muscle cells, on plectin and MACF1 in skeletal muscle cells has not been demonstrated. The effect of IGF-I on plectin and MACF1 gene expression was investigated by treating differentiated C2C12 murine skeletal muscle cells with 20 ng/ml of IGF-I at different time points. The IGF-I treatment increased plectin protein expression in a dose-dependent manner. The mRNA level of plectin was measured by real-time quantitative PCR to determine if plectin induction was regulated pretranslationally. IGF-I treatment resulted in a very rapid induction of plectin mRNA transcript in C2C12 myotubes. Plectin mRNA increased by 140 and 180% after 24 and 48 hours of IGF-I treatment, respectively, and returned to the control level after 72 hours of IGF-I treatment. MACF1 mRNA increased 86 and 90% after 24 and 48 hours of IGF-I treat-ment, respectively, and returned to the control level after 72 hours of IGF-I treatment. These results suggested that the plectin gene is regulated pretranslationally by IGF-I in skeletal muscle cells. In conclusion, IGF-I induces a rapid transcriptional modification of the plectin and MACF1 genes in C2C12 skeletal muscle cells and has modulating effects on a cytolinker protein as well as on contractile proteins.

• Korean Journal of Ecology and Environment
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• v.37 no.1 s.106
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• pp.122-129
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• 2004

In an effort to develop the biomarker for monitoring the contamination of xenoestrogen in the freshwater environment of Korea, reverse transcription-polymerasechain reaction (RT-PCR) analysis of vitellogenin (VTG) gene expression was optimized in Hearisarsus Iaseo, Based on the homology of the VTG cDNA sequences between the common carp and zebra fish, a set of PCR primers for VTG mRNA amplification for H; labo was designed. VTG mRNA level in livers from female and male fishes was analyzed by RT-PCR following single injection of 17 beta estradiol($E_2$ 10 mg $kg^{-1}$ B.W.). As an internal control, beta actin mRNA was amplified. One us of total liver RNA was subjected to RT-PCR. In female the amount of PCR productof VfC gradually increased in the range from 16 to 34 cycles of amplification. On the contrary, in control male, PCR product first detected at 32 cycles of amplification and linearly increased up to 40 cycles of amplification. In $E_2$ injected male liver, the VTC mRNA level was similar to that in the female. Taken together, this result suggests that liver of male H. labo expresses minute amount of VTG mRNA which are2-l6 equivalent of female and that induction of VTG mRNA occurs in male liver after estrogen treatment. In conclusion, the optimized protocol for RT-PCR analysis of VTG mRNA expression in liver of male H. labo will provide the environmental monitoring method for the xenoestrogen contamination in the rivers in Korea.

• The Journal of Korean Medicine
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• v.24 no.1
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• pp.74-83
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• 2003

Background : Production of cytokines by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma. In many recent studies, molecular biological methods have been used to investigate the role of cytokines in pathogenesis and new therapeutic targets of asthma. Objective : We aimed to identify the dose-dependent inhibitory effects of Socheongryong-tang and Socheongryong-tang plus Sasam (Adenophorae Radix) on the mRNA expressions of Interleukin (IL)-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) involved in the asthma model. Materials and Methods : In this study, BEAS-2B cell lines, human epithelial cells, were used. These cells were stimulated by tumor necrosis factor $(TNF)-{\alpha},{\;}IL-1{\beta}$ and histamine for artificial inflammatory expression. ${\beta}-actin$ messenger RNA (mRNA) was used for the internal standard. After each 24 hours of the Socheongryong-tang (小靑龍湯) and Socheongryong-tang plus Sasam (小靑 龍湯加沙蔘) treatment, total cellular RNAs were collected by applying RNAzol directly to the living cells. Then the transcriptional activities of IL-6, IL-8 and GM-CSF were measured by RT-PCR with electrophoresis. Results : In the Socheongryong-tang (小靑龍湯) study, the mRNA expressions of IL-6, IL-8 and GM-CSF were significantly inhibited compared to that of the control group (p<0.05). In the Socheongryong-tang plus Sasam (小靑龍湯加沙蔘) study, the mRNA expressions of IL-6, IL-8 and GM-CSF were significantly inhibited compared to that of the control group (p<0.05). Conclusions : This study shows that Socheongryong-tang (小靑龍湯) and Socheongryong-tang plus Sasam (小靑龍湯加沙蔘) have dose-dependent inhibitory effects on the mRNA expressions of IL-6, IL-8 and GM-CSF in human epithelial cells, so these herbal medicines may inhibit the inflammatory process of asthma. Advanced studies are required to investigate the mechanisms of inhibition by herbal medicine in the asthma model.

• The Journal of Korean Medicine
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• v.23 no.1
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• pp.11-23
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• 2002

Objectives: We aimed to identify the dose-dependent inhibitory effects of Maekmundongcheongpye-eum and Liriopis Tuber on the mRNA expression of IL-6, IL-16, GM-CSF involved in the asthma model. Methods: In the study BEAS-2B cell lines, human epithelial cells were used. These cells were stimulated with tumor necrosis factor $(TNF)-{\alpha}$ for artificial inflammatory expression. ${\beta}-actin$ messenger RNA (mRNA) was used by internal standard. After 24 hours of Maekmundongcheongpye-eum, Liriopis Tuber-treatment, total cellular RNAs were collected, treating RNAzol directly on the alive cells. Then the transcriptional activities of IL-6, 16, GM-CSF were measured by RT-PCR with electrophoresis. Results: In the Maekmundongcheongpye-eum study, the mRNA expression of IL-6 showed 48% transcriptional inhibitory effect compared to the control group in the $100{\;}{\mu}l/ml$ category (P<0.001). In the IL-16, there was 53% and 57% transcriptional inhibitory effect compared to the control group in the $20{\;}{\mu}l/ml$ and $100{\;}{\mu}l/ml$ categories (P<0.001). In the GM-CSF, there was no inhibitory effect. In the Liriopis Tuber study, the mRNA expression of IL-6 showed 43% transcriptional inhibitory effect compared to the control group in the $100{\;}{\mu}l/ml$ category (p<0.005). In the IL-16, 34% and 26% of transcriptional inhibitory effect was shown compared to the control group in the $20{\;}{\mu}l/ml$ and $100{\;}{\mu}l/ml$ categories, respectively (P<0.05). In the GM-CSF, there was no inhibitory effect. Conclusions: This study shows that Maekmundongcheongpye-eum and Liriopis Tuber have dose-dependent inhibitory effects on the mRNA expression of IL-6 and IL-16 in BEAS-2B cell lines, human epithelial cells. Advanced studies are required to investigate the mechanisms of inhibition by herbal medicine in the asthma model.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.12
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• pp.1830-1836
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• 2003

Studies using natural resistance-associated macrophage protein (NRAMP) identification indicated that cattle could be selected for immunity. Several studies performed on intracellular organisms such as Mycobacterium, Salmonella, Brucella and Leishmania in human and mouse revealed that resistance against these bacteria was dependent on high activity of NRAMP1 in macrophages. However, hardly any researches have been done on Staphylococcus aureus in bovine mastitis, which is an intracellular organism and the main cause of bovine mastitis. The objectives of this study were to establish reverse transcriptase polymerase chain reaction (RT-PCR) methods, through which NRAMP1 mRNA expression could be compared and analyzed between mastitis-resistant and -susceptible cows. NRAMP1 gene and its expression were investigated using 20 cows (Holstein Friesian) in Korea. Cows were evenly split into two groups, with and without histories of clinical mastitis. Equivalent numbers of cows were randomly selected from each group. Monocytes were isolated from the bovine peripheral blood of each selected cows and activated with lipopolysaccharide (LPS). mRNA was separated from the monocytes and cDNA of NRAMP1 was synthesized and amplified using RT-PCR with amplification of $\beta$-actin as a control. The difference in NRAMP1 expressions of mastitis-resistant (n=10) and -susceptible (n=10) Holstein cows was analyzed. Results demonstrate that resistant cows produced more NRAMP1 mRNA than the susceptible ones, and ratios of NRAMP1:$\beta$-actin expression were higher in resistant cows with or without LPS activation. Therefore, this study could be applied to select bovine mastitis resistant cows before infection based on the expression of NRAMP1.

• Journal of fish pathology
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• v.22 no.2
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• pp.147-153
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• 2009

To study the biological activity of interleukin-$1\beta$(IL-$1\beta$), a proinflammatory cytokine, in nile tilapia, Oreochromis niliticus, the recombinant tilapia IL-$1\beta$ was produced in E. coli cells based on pQE vector. Ni-NTA (nitriloacetic acid) metal affinity chromatography was used to purify recombinant protein. The eluted fractions exhibited a single band of protein with a molecular weight of about 25kDa, which is in close agreement with 25.4 kDa predicted by the cDNA sequence. The biological activity of the purified recombinant tilapia IL-$1\beta$ was tested through its effects on IL-$1\beta$ gene expression, which are known as IL-$1\beta$ inducible genes in mammals and fishes. IL-$1\beta$ gene expression induced by poly I:C, a synthetic double stranded RNA, was also assessed in tilapia head kidney cells. IL-$1\beta$ gene expression was analysed using QPCR (quantitative polymerase chain reaction). The ratio of the indicated gene expression was expressed as the relative mRNA level to $\beta$-actin mRNA level, which is constitutively expressed in macrophages. Consequently, head kidney cells incubated for three hours with rIL-$1\beta$(10, 2, 1 $\mu{g}$/ml) showed a dose dependent increase in IL-$1\beta$ mRNA levels and 1 $\mu{g}$/ml of poly I:C was also able to induce IL-$1\beta$ gene expression in head kidney in tilapia.

• Korea Journal of Cosmetic Science
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• v.1 no.1
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• pp.31-43
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• 2019

Melanosomes are specific melanin-containing intracellular organelles of epidermal melanocytes. In epidermal melanocytes, there are three kinds of key player proteins. Rab27a, melanophilin or Slac2-a and Myosin 5a form a tripartite complex connects the melanosome. Mature melanosomes make movements through the tripartite protein complex along actin filaments.In this study, we found that the haplamine (6-Methoxyflindersine) induced melanosome aggregation around the nucleus in epidermal melanocyte. In an attempt to elucidate the inhibitory effect of haplamine on melanosome transport, effect of haplamineon the expression level of Rab27a, melanophilin and Myosin 5a was measured. The results indicated that haplamine up to 5��M effectively suppressed mRNA and protein expression level of melanophilin.To determine the upstream regulator of melanophilin regulated by haplamine, we checked the level of MITF, c-JUN and USF1. Those are possible transcription factor of melanophilin. Among them,treatment of USF1 siRNA decreased mRNA and protein expression level of USF1 as well as melanophilin. Also, treatment of haplamine decreased mRNA and protein expression level of melanophilin as well as USF1 in a dose-dependent manner. Consequently, we found the inhibitory effect of haplamine on melanosome transport in melan-a melanocyte. Treatment of haplamine reduced melanophilin expression level which is a key protein of melanosome transport. We identified that USF1 could be a major transcription factor of melanophilin regulated by haplamine.

• Journal of Animal Science and Technology
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• v.57 no.5
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• pp.18.1-18.8
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• 2015

Background: Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, ${\beta}$-actin and ${\beta}2$-microglobulin) in equine marrow- and adipose-derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Materials and methods: Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. Results: The expression levels of GAPDH were significantly different between AT- and BM-derived MSCs (p < 0.05). Differences in expression level of ${\beta}$-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, ${\beta}$-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. Conclusion: This study demonstrated that GAPDH and especially ${\beta}$-actin and B2M express in different levels in equine AT- and BM-derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

• Fisheries and Aquatic Sciences
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• v.15 no.4
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• pp.317-324
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• 2012

We characterized the cytoskeletal beta-actin (${\beta}$-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.