• 생명과학회지
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• 제28권12호
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• pp.1432-1437
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• 2018

클라라 세포에 의해 생산되는 클라라 세포 분비 단백질(CCSP)은 폐를 염증으로부터 보호하는데 중요한 역할을 한다. 이 연구는 CCSP 유전자 발현에 관여하는 프로모터 부위에서 repressor에 결합할 수 있는 cis-element를 밝히는데 있다. DNaseI footprinting법을 사용하여 mCCSP 프로모터의 -812에서 -768 bp (45 bp) 사이에서 3 개의 보호된 motif를 찾았고, 그 중 하나인 D3 모티프(GCCTGGGAA)는 다른 3 가지 동물들과 염기서열이 100% 일치하였다. 45 bp를 사용한 EMSA 분석에서 D3 모티프(GGCCTGGGAA)는 45 bp에 높은 경쟁을 보였으나, 변이된 D3 모티프가 ($G{\underline{AA}}TG{\underline{TT}}AA$)를 사용되었을 때, 경쟁은 상당히 감소되었다. 이는 mCCSP 프로모터의 45 bp의 D3 모티프가 단백질과 DNA 상호 작용을 위한 중요한 element임을 시사한다. -756-Luc과 -812-Luc을 이용한 transient transfection 분석 결과, -756-Luc은 -812-Luc보다 CCSP의 발현이 현저하게 감소되었다. 이는 mCCSP 프로모터의 45 bp부위가 repressor의 결합 부위로서 기능을 할 수 있음을 의미한다. -812-Luc에 KLF4를 co-transfection 한 결과, KLF4는 CCSP 발현을 현저하게 저해(repression)함을 밝혔다. 그러나 -768-Luc이 사용되었을 때 KLF4에 의한 repression은 관찰되지 않았다. 이것은 KLF4가 CCSP 유전자의45 bp에 결합할 수 있고, 전사 억제자 역할을 하여 mCCSP 발현을 억제 할 수 있음을 명확히 보여 준다. 또한 이는 45 bp 중, D3 모티프가 KLF4의 결합에 강하게 관여 함을 시사한다. 이 반응에 KLF4에 대한 항체가 첨가되었을 때는 super-shifted 밴드가 관찰되었으나, SP1에 대한 항체가 사용되었을 때는 관찰되지 않았다. 이는 KLF4가 CCSP 프로모터의 45 bp 영역에 결합하여 repressor기능 할 수 있고, D3 모티프가 KLF4의 특이적 결합에 관여 할 수 있음을 시사한다.

• 한방재활의학과학회지
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• 제34권2호
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• pp.15-28
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• 2024

Objectives We used the D-galactose (D-gal) induced C2C12 myoblast senescence model to investigate whether ethanol extract of Perilla. fructescens leaves (EEPF) could delay cellular senescence and regulate related mechanisms. Methods C2C12 myogenic cells were cultured in an incubator under 37 ℃ and 5% CO₂ conditions. EEPF, dried perilla leaves were pulverized and extracted at 1:10 (v/v) at 50 ℃ for 4 hours. Cell counting kit-8 and western blot analysis was performed. Annexin V-FITC apoptosis detection kit and DAPI staining was applied. Catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde analysis kits were used. To measure the level of reactive oxygen species generation, staining and flow cytometry was used. To analyze the mitochondrial activity, membrane potential changes were measured using JC-1. 𝛽-gal activity was analyzed using SA-𝛽-gal staining solution, and DNA damage was analyzed by using 𝛾-H2AX. Quantikine ELISA kit was used to analyze inflammatory cytokine production. Results According to the results of this study, EEPF significantly alleviated the decrease in cell viability in C2C12 cells treated with D-gal and suppressed the decrease in the expression of proliferating cell nuclear antigen. EEPF also markedly blocked D-gal-induced C2C12 cell apoptosis and restored reduced activity of CAT, GSH-Px, T-AOC, SOD. In addition, EEPF suppressed the decrease in 𝛽-galactosidase activity, the induction of DNA damage and the increase in expression of senescence-associated secretory phenotype proteins such as p16, p53 and p21 in D-gal-treated C2C12 cells. Furthermore, EEPF significantly attenuated D-gal-induced production and expression of inflammatory cytokines such as interleukin (IL)-6 and IL-18. Conclusions The results of this study indicate that EEPF can be used as a potential candidate for the prevention and treatment of muscle aging.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권1호
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• pp.83-85
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• 2007

A glycine and proline-rich antibacterial protein was cloned from larvae of a beetle, Protaetia brevitarsis. The DNAs encoded a deduced propeptide of 127 amino acid residues with predicted molecular weight of 14.0 kDa and PI of 7.89. Structural analysis of this protein indicated the presence of a recognition sequence for the cleavage site within the constitutive secretory pathway(Arg-Xaa-Lys/Arg-Arg), suggesting that mature portion(72 amino acid residues) is produced by cleavage of signal peptide and propeptide from 127 amino-acid-long precursor protein. Mature portion sequence of this protein showed 72% similarity to that of Oryctes rhinoceros Rhinocerosin and 91% to that of Holotrichia diomphalia holotricin 2. The mRNA expression was reached the highest level at 4 hrs after E. coli injection and then declined gradually.

• Molecules and Cells
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• 제19권2호
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• pp.185-190
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• 2005

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin and its cells undergo cell proliferation and differentiation. There has been no study of the cellular mechanism involved in cell death in the chicken oviduct. Therefore, this study has focused on the study of apoptosis in primary oviduct cells. Because ceramide is known to activate apoptosis in tumor cells and is produced in the oviduct, we used an exogenous ceramide analog to induce cell death. The viability of ceramide-treated chicken oviduct cells decreased in a dose-dependent manner and apoptotic cells were detected by staining with annexin V. The expression of apoptosis-related genes was assessed by RT-PCR and bcl-2 mRNA was found to decrease after exposure to ceramide while Bcl-x mRNA increased 12 h post-treatment. In addition, caspase-3 was expressed strongly in the early stages of apoptosis, while caspase-1 and -9 transcripts increased at later times. We conclude that ceramide induces apoptosis in oviduct-derived primary cells via a caspase- and bcl-2-dependent pathway.

• 대한의생명과학회지
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• 제10권3호
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• pp.219-230
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• 2004

This experiment was performed to examine the in vitro and in vivo affects of the two different haplotype strains of mice, BALB/c and C3H/HeN infected with Echinostoma hortense, and the manifestation of the profiles of cytokine in the splenocytes. In the in vitro experiment, the two mice's splenocytes were divided and stimulated with antigen of crude extracts and the antigen of excretory and secretory products of an adult warm and the manifestation of cytokine mRNA was verified with RT-PCR. As a result, the two different strains of mice both strongly manifested the Th2 cytokine rather than the Thl cytokine and in the case of the Th2 cytokine, the BALB/c mice manifested more strongly than the C3H/HeN mice. In the experiment using the ELISA method, the protem cytokine manifestation had the same result as the mRNA experiment. In the in vivo experiment, the mice was infected via oral route with the metacercaria of the Echinostoma hortense and the manifestation of cytokine was verified by RT-PCR and ELISA and the results were the same as the in vitro experiment. Therefore, in the two strains of BALB/c and C3H/HeN, the C3H/HeN showed a higher susceptivity to the Echinostoma hortense.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1232-1238
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• 2004

To investigate the production and characteristics of thermostable levan sucrase from Rahnella aquatilis ATCC 33071, the levan sucrase gene from R. aquatilis was cloned and expressed in Escherichia coli without induction system. Expression of levansucrase gene in E. coli had no notable or detrimental effect on the growth of host strain, and the recombinant levan sucrase exhibited levan synthesis activity. Levansucrase was secreted to the periplasm in E. coli, and addition of $0.5\%$ glycine yielded further secretion of levansucrase to the growth medium and resulted in an increase of total levansucrase activity. Furthermore, the cellular levansucrase was evaluated for the production of levan by using toluene­permeabilized whole-cells. The levansucrase was thermostable at $37^{\circ}C$. The molecular size oflevan was $1{\times}\;10^{6}$ Da, as determined by HPLC, and the degree of polymerization of levan varied with incubation temperatures: Low incubation temperature was preferable for the production of high-molecular size levan. The present study demonstrated that the mass production of levan and levan oligosaccharides can be achieved by glycine supplementation to the growth medium or by toluene­permeabilized whole-cells.

• 한국임상수의학회지
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• 제28권1호
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• pp.113-121
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• 2011

Foot-and-mouth disease (FMD) is a severe vesicular disease of cloven-hoofed animals including domesticated ruminants and pigs. Acute clinical signs may be mild in sheep and goats but are associated with lameness in pigs and mouth lesions with vesicles in cattle. The required condition for a successful pathogen appears to be the ability to counteract both the host innate and adaptive immune response. FMD virus (FMDV) inhibits the induction of antiviral molecules and interferes with the secretory pathway in the infected cell. The surface expression of Major Histocompatibility Complex (MHC) class I molecules is reduced in infected cells. Thus, the ability of the host to recognize and eliminate virus infected cells is decreased. Furthermore, FMDV infection results in a rapid, but transient lymphopenia, reducing the number of T and B cells, and affecting T cell function. The virus appears to premature apoptosis-mediated cell death because it has a very short replication cycle and is able to rapidly produce large amounts of virus. FMDV engages the host protective response at multiple steps to ensure its effective replication and pathogenesis. This review describes the recent pathological and immunological studies to overcome the powerful abilities of FMDV to counteract defense mechanism of host.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2007년도 춘계학술발표회
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• pp.65-73
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• 2007

The objective of this study is to understand how regulatory mechanisms respond to sugar status for more efficient carbon utilization and source-sink regulation in plants. So, we need to identify and characterize many components of sugar-response pathways for a better understanding of sugar responses. For this end, genes responding change of sugar status were screened using Arabidpsis cDNA arrays, and confirmed thirty-six genes to be regulated by sucrose supply in detached leaves by RNA blot analysis. Eleven of them encoding proteins for amino acid metabolism and carbohydrate metabolism were repressed by sugars. The remaining genes induced by sugar supply were for protein synthesis including ribosomal proteins and elongation factors. Among them, I focused on three hydrolase genes encoding putative $\beta$-galactosidase, $\beta$-xylosidase, and $\beta$-glucosidase that were transcriptionally induced in sugar starvation. Homology search indicated that these enzymes were involved in hydrolysis of cell wall polysaccharides. In addition to my results, recent transcriptome analysis suggested multiple genes for cell wall degradation were induced by sugar starvation. Thus, I hypothesized that enzyme for cell wall degradation were synthesized and secreted to hydrolyze cell wall polysaccharides producing carbon source under sugar-starved conditions. In fact, the enzymatic activities of these three enzymes increased in culture medium of Arabidopsis suspension cells under sugar starvation. The $\beta$-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved condition with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. Further, contents of cell wall polysaccharides especially pectin and hemicellulose were markedly decreased associating with sugar starvation in detached leaves. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These results supported my idea that cell wall has one of function to supply carbon source in addition to determination of cell shape and physical support of plant bodies.

• The Korean Journal of Physiology and Pharmacology
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• 제22권5호
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• pp.555-566
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• 2018

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are used in tissue repair and regeneration; however, the mechanisms involved are not well understood. We investigated the hair growth-promoting effects of hUCB-MSCs treatment to determine whether hUCB-MSCs enhance the promotion of hair growth. Furthermore, we attempted to identify the factors responsible for hair growth. The effects of hUCB-MSCs on hair growth were investigated in vivo, and hUCB-MSCs advanced anagen onset and hair follicle neogeneration. We found that hUCB-MSCs co-culture increased the viability and up-regulated hair induction-related proteins of human dermal papilla cells (hDPCs) in vitro. A growth factor antibody array revealed that secretory factors from hUCB-MSCs are related to hair growth. Insulin-like growth factor binding protein-1 (IGFBP-1) and vascular endothelial growth factor (VEGF) were increased in co-culture medium. Finally, we found that IGFBP-1, through the co-localization of an IGF-1 and IGFBP-1, had positive effects on cell viability; VEGF secretion; expression of alkaline phosphatase (ALP), CD133, and ${\beta}-catenin$; and formation of hDPCs 3D spheroids. Taken together, these data suggest that hUCB-MSCs promote hair growth via a paracrine mechanism.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.7-16
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• 2002

점액질이 풍부한 꼼치 조직에서 NIH 3T3 세포주를 이용하여 subtracted cDNA 라이브러리를 얻어 200례의 클론을 제작하였다. 이 클른 중에서 비반복성 유전자를 선택하고, RNA in situ hybridization을 실행하여 꼼치 조직에서 특이하게 발현되는 곰신 클론(C90-171)을 선택하였다. 이 클론은 사람의 타액선 조직에서도 특이하게 발현되는 유전자로서 이를 확인하기 위하여 C90-171(곰신) 항체를 제작하였다. 꼼치의 cDNA 라이브러리에서 곰신의 항체를 통하여 스크리닝한 결과 PRP(proline-rich protein)와 가장 많이 교차반응하며, 면역조직화학적 염색으로 PRP와 유사한 양성반응으로 나타나 PRP와 유사한 기능을 하는 단백질로 사료된다. 또한 타액 내에서의 꼼치 단백질의 분해에 대한 실험결과 거의 분해가 일어나지 않는 것으로 보아, 곰신은 꼼치의 몸통을 보호하는 유전물질일 뿐만 아니라, PRP와 유사하게 조직을 보호하는 안정된 새로운 기능성 단백질로 사료된다.