• BMB Reports
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• v.28 no.6
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• pp.515-522
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• 1995

The effects of the thyroid hormone ($T_3$) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in a baby hamster kidney cell line, C100. The cells cultured in MEM were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 82.5% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS-MEM). When $T_3$ was supplemented to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM containing $T_3$ at a concentration of $10^{-6}$ M, the level of HMG-CoA reductase activity was 91.8% relative to the cells grown in FBS-MEM. These changes in HMG-CoA reductase activity seemed to be at least partly due to the changes of HMG-CoA reductase mRNA levels. The level of HMG-CoA reductase mRNA in cells incubated in THDS-MEM decreased to 76.2% relative to the cells grown in FBS-MEM, while the level of reductase mRNA in cells incubated in THDS-MEM containing $T_3$ at a concentration of $10^{-6}$ M increased to 243.4% relative to the cells grown in FBS-MEM. The increase of HMG-CoA reductase mRNA level after $T_3$ treatment may have been due to the increased stability of reductase mRNA, because the transcriptional rate of the reductase gene did not change significantly in the presence or absence of $T_3$. These results indicate that $T_3$ stabilizes HMG-CoA reductase mRNA at the posttranscriptional level and regulates HMG-CoA reductase activity in a dose-dependent manner.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.61-70
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• 1983

Synchronized cat kidney cells chronically infected with feline leukemia virus (FeLV) were used to study virus production, the synthesis of group specific antigen (gag) and envelope (env) proteins, the expression of env protein on the cell surface during the cell cycle, and the stability of viral RNA. As detecting method, we developed the radioimmunoassay (RIA) system using beta-emission of $^{131}I$ and demonstrated the validity of this system by comparison with routine RIA system using gamma-emission of $^{125}I$. The produced virus was analysed by developed RIA interval was determined by measuring reverse transcriptase activity. The results show that infected cells produce the complete virus particle containing products of gag, env and pol genes of FeLV, and maximum virus production occurs during mitosis of synchronized cells. Labeling of the cell surface of synchronized cells with $^{131}I$ shows that the amount of $gp70^{env}$ on the cell surface parallels cellular gorwth. Therefore, the cell cycle-dependent release of virus is not petition RIA of synchronized cells with $^{131}I$ labeled viral proteins synthesis during the cell cycle. The rate of synthesis of gag protein shows three peaks, corresponding to the $G_1,\;late\;S\;and\;late\;G_2$ phases of cell cycle. But the rate of synthesis of env protein dose not change, suggesting that in these cells the synthesis of these two gene products in controlled seperately. In Actionomycin D treated cells, the synthesis of viral proteins decreased sharply from 8 hours after treatment, and the late S and $G_2$ peaks of gag protein synthesis were disappeared. This shows the stability of viral RNA for about 6 hours in the absence of continuing viral RNA synthesis.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.3
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• pp.423-432
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• 2013

Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don't know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ${\beta}$-actin (ACTB), ${\beta}$-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese.

• KSBB Journal
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• v.20 no.5 s.94
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• pp.387-391
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• 2005

Totally 16,299 conservative genes, commonly found in 13 thermophilic and hyperthermophilic bacteria, were analyzed. All genes were belong to W 67 COGs (clusters of orthologous groups of proteins). COGs related to protein metabolism were 80 among 167 COGs. Conservative genes were not limited only thermophiles and hyperthermophiles, meaning thermal stability is independent of specific protein. However reverse gyrase was only found in all hyperthermophilic archaebacteria and eubacteria, meaning DNA stability is important in hyperthermophiles. Hyperthermophilic eubacteria and thermophilic archaebacteria had different position between phylogenetic tree of gene content and 165 rRNA gene. Thermophilic archaebacteria hyperthermophilic eubacteria and archaebacteria had similar values by the statistical analysis of distance values with 167 COGs in each organism.

• Molecules and Cells
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• v.38 no.2
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• pp.163-170
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• 2015

Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria. Its effects on living organisms are different from those of lipopolysaccharide (LPS) found in Gram-negative bacteria. LTA contributes to immune regulatory effects including anti-aging. In this study, we showed that LTA isolated from Lactobacillus plantarum (pLTA) inhibited melanogenesis in B16F10 mouse melanoma cells. pLTA reduced the cellular activity of tyrosinase and the expression of tyrosinase family members in a dose-dependent manner. The expression of microphthalmia- associated transcription factor (MITF), a key factor in the synthesis of melanin, was also decreased by pLTA. Further, we showed that pLTA activated melanogenesis signaling, such as extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinse (PI3K)/AKT. In addition, the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and HuR, which are important RNA-binding proteins (RBPs), was reduced. pLTA likely degrades MITF via regulation of melanogenic signaling and RNA stability of melanogenic proteins, resulting in the reduction of melanin. Thus, our data suggest that pLTA has therapeutic potential for treating hyperpigmentation disorders and can also be used as a cosmetic whitening agent.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.722-729
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• 2004

Leptin is an adipocyte-secreted hormone and its plasma levels correlate with total body fat mass, however, it also plays a regulatory role in immunity, inflammation, and hematopoiesis. Chemokine is known as a chemoattractant cytokine in inflammatory reaction, but its role in leptin reaction has not been well studied. In this study, the direct effect of leptin on the expression of chemokine mRNAs and lipopolysaccharide (LPS)-induced chemokine KC mRNA in mouse peritoneal macrophages was investigated. Leptin did not induce the expression of lymphotactin, RANTES, eotaxin, MIP-1$\beta$, MIP-1$\alpha$, MIP-2, MCP-1, IP-10, TCA-3, and KC mRNA in mouse peritoneal macrophages, and had no direct effect on the expression of these LPS-induced chemokine mRNAs except KC mRNA. The synergistic effect of leptin on the expression of LPS-induced KC mRNA occurred late in the time course of response to LPS. The increased expressions of Ob-Rb mRNA and leptin receptor protein were detected during the LPS treatment. Leptin produced a substantial increase in the stability of the LPS-induced KC mRNA, and the synergistic effect of leptin on LPS-induced KC mRNA expression was further augmented by cycloheximide (CHX). Pyrrolidine dithiocarbamate (PDTC) did not block the synergistic effect of leptin on LPS-induced KC mRNA expression in mouse peritoneal macrophages. These data suggest that although leptin has no direct effect on the expression of lymphotactin, RANTES, eotaxin, MIP-1$\beta$, MIP-1$\alpha$, MIP-2, MCP-1, IP-10, TCA-3, and KC mRNA in mouse peritoneal macrophages, the synergistic effect of leptin on the expression of LPS-induced KC mRNA has the possibility that LPS might induce the expression of the Ob-Rb receptor or an unknown gene(s) that sensitizes macrophages to the synergistic function of leptin. Therefore, further studies are necessary to examine leptin as a regulatory factor of chemokine production.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.8
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• pp.417-428
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• 2020

Housekeeping genes are expressed in cells of all organisms and perform basic cellular functions such as energy generation, substance synthesis, cell death, and cell defense. Accordingly, the expression levels of housekeeping genes are relatively constant, and thus they are used as reference genes in gene expression studies, such as protein expression and mRNA expression analysis of target genes. However, the levels of expression of these genes may be different among various tissues or cells and may change under certain circumstances. Therefore, it is important to select the best reference gene for specific gene expression research by exploring the stability of housekeeping gene expression. This review summarizes housekeeping genes found in humans, chickens, pigs, and rats in the literature and estimates expression stability using geNorm, NormFinder, and BestKeeper software. The most suitable reference housekeeping gene can selected based on expression stability according to the experimental conditions of the gene expression study and can thus be applied to data normalization.

• The Journal of Internal Korean Medicine
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• v.25 no.1
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• pp.28-45
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• 2004

Objectives : The main purpose of this study is to evaluate the effect of Injinchunggan-tang on $TNF-{\alpha}$ signal transmission system. Materials and Methods : We analyzed the following with quantitative RT-PCR method; the effect of Injinchunggan-tang on secretion of $TNF-\alpha$ mRNA/protein and stability, the effect on gene revelation that consists of signal transmission system (TRAIL, NIK, A20, TRADD, RAIDD, RIP TNFR-I, TNFR-II, TRAF1, TRAF2, FADD), the one on activation of p38, Erk1/2 MAPK and the rate of nuclear $NF-{\kappa}B/cytosolic\;NF-{\kappa}B$ in HepG2 cell. We also analyzed the inhibitory effect of Injinchunggan-tang on the apoptosis of HepG2 cell that $TNF-{\alpha}$ induces and the $NF-{\kappa}B$ restraint effected by transfection of $I{\kappa}B{\Delta}N$ through tryphan blue exclusion assay. Results : Injinchunggan-tang prohibits revelation of $TNF-{\alpha}$ mRNA in HepG2 cell and the creation of protein. However, it has no effect on the stability of $TNF-{\alpha}$ mRNA. While it did not have any effect on the generation of TRAIL, NIK, A20, TRADD, RAIDD and RIP genes, Injinchunggan-tang reduces the revelation of TNFR-I, TNFR-II, TRAF1, TRAF2 and FADD genes. It has been confirmed that Injinchunggan-tang restraints the revelation of $TNF-{\alpha}$ mRNA that is promoted by ethanol, acetaldehyde, lipopolysaccharide, in proportion to the treatment density and time. It activated $NF-{\kappa}B$ of HepG2 cell and promoted activation of $NF-{\kappa}B$ that is occurred by $TNF-{\alpha}$. It has been observed that the restraint effect against the $TNF-{\alpha}$ inducing apoptosis is lost when it is intercepted the function of $NF-{\kappa}B$ in HepG2 cell. Conclusion: It has been confirmed that Injinchunggan-tang has restraining effect against the revelation of $TNF-{\alpha}$ and mRNA that is constituent element of TNF-a signal transmission system. It also has been revealed that it restraints the activation of p38, Erk1/2 by $TNF-{\alpha}$. Through this prohibiting effect, it is inferred that it restraints signal transmission among various cells that are related to inflammation reaction. Meanwhile, Injinchunggan-tang protects liver cell from apoptosis that is caused by $TNF-{\alpha}$, by maintaining the activating function for $NF-{\kappa}B$.

• Molecules and Cells
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• v.43 no.4
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• pp.373-383
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• 2020

Our previous study revealed a novel role of Fas-associated death domain-containing protein (FADD) in islet development and insulin secretion. Insulin-degrading enzyme (IDE) is a zinc metalloprotease that selectively degrades biologically important substrates associated with type 2 diabetes (T2DM). The current study was designed to investigate the effect of FADD phosphorylation on IDE. We found that the mRNA and protein levels of IDE were significantly downregulated in FADD-D mouse livers compared with control mice. Quantitative real-time polymerase chain reaction analysis showed that FADD regulates the expression of IDE at the transcriptional level without affecting the stability of the mRNA in HepG2 cells. Following treatment with cycloheximide, the IDE protein degradation rate was found to be increased in both FADD-D primary hepatocytes and FADD-knockdown HepG2 cells. Additionally, IDE expression levels were reduced in insulin-stimulated primary hepatocytes from FADD-D mice compared to those from control mice. Moreover, FADD phosphorylation promotes nuclear translocation of FoxO1, thus inhibiting the transcriptional activity of the IDE promoter. Together, these findings imply a novel role of FADD in the reduction of protein stability and expression levels of IDE.

• Clinical and Experimental Vaccine Research
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• v.12 no.2
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• pp.156-171
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• 2023

Purpose: The development of vaccines that confer protection against multiple avian influenza A (AIA) virus strains is necessary to prevent the emergence of highly infectious strains that may result in more severe outbreaks. Thus, this study applied reverse vaccinology approach in strategically constructing messenger RNA (mRNA) vaccine construct against avian influenza A (mVAIA) to induce cross-protection while targeting diverse AIA virulence factors. Materials and Methods: Immunoinformatics tools and databases were utilized to identify conserved experimentally validated AIA epitopes. CD8⁺ epitopes were docked with dominant chicken major histocompatibility complexes (MHCs) to evaluate complex formation. Conserved epitopes were adjoined in the optimized mVAIA sequence for efficient expression in Gallus gallus. Signal sequence for targeted secretory expression was included. Physicochemical properties, antigenicity, toxicity, and potential cross-reactivity were assessed. The tertiary structure of its protein sequence was modeled and validated in silico to investigate the accessibility of adjoined B-cell epitope. Potential immune responses were also simulated in C-ImmSim. Results: Eighteen experimentally validated epitopes were found conserved (Shannon index <2.0) in the study. These include one B-cell (SLLTEVETPIRNEWGCR) and 17 CD8⁺ epitopes, adjoined in a single mRNA construct. The CD8⁺ epitopes docked favorably with MHC peptidebinding groove, which were further supported by the acceptable ∆G_bind (-28.45 to -40.59 kJ/mol) and Kd (<1.00) values. The incorporated Sec/SPI (secretory/signal peptidase I) cleavage site was also recognized with a high probability (0.964814). Adjoined B-cell epitope was found within the disordered and accessible regions of the vaccine. Immune simulation results projected cytokine production, lymphocyte activation, and memory cell generation after the 1st dose of mVAIA. Conclusion: Results suggest that mVAIA possesses stability, safety, and immunogenicity. In vitro and in vivo confirmation in subsequent studies are anticipated.