• Journal of Microbiology and Biotechnology
• /
• 제31권8호
• /
• pp.1079-1087
• /
• 2021

Gentisic acid (GA), a benzoic acid derivative present in various food ingredients, has been shown to have diverse pharmaceutical activities such as anti-carcinogenic, antioxidant, and hepatoprotective effects. In this study, we used a co-culture system to investigate the mechanisms of the anti-inflammatory and anti-adipogenic effects of GA on macrophages and adipocytes, respectively, as well as its effect on obesity-related chronic inflammation. We found that GA effectively suppressed lipopolysaccharide-stimulated inflammatory responses by controlling the production of nitric oxide and pro-inflammatory cytokines and modulating inflammation-related protein pathways. GA treatment also inhibited lipid accumulation in adipocytes by modulating the expression of major adipogenic transcription factors and their upstream protein pathways. Furthermore, in the macrophage-adipocyte co-culture system, GA decreased the production of obesity-related cytokines. These results indicate that GA possesses effective anti-inflammatory and anti-adipogenic activities and may be used in developing treatments for the management of obesity-related chronic inflammatory diseases.

• Pediatric Infection and Vaccine
• /
• 제23권3호
• /
• pp.194-201
• /
• 2016

목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription polymerase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA)를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학자료 수집에 큰 도움이 될 것으로 기대한다.

• 식물조직배양학회지
• /
• 제24권1호
• /
• pp.1-35
• /
• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• 대한한방소아과학회지
• /
• 제32권3호
• /
• pp.1-15
• /
• 2018

Objectives Alismatis Rhizoma has been known to suppress inflammation and allergic reaction. However, the cellular target of Alismatis Rhizoma and its mechanism of action remain unclear. This study was designed to examine the effect of Alismatis Rhizoma extract (ALC) on the RBL-2H3 mast cells in vitro and on the OVA/alum sensitized mice ex vivo. Methods In the study, RBL-2H3 mast cells were cultured in minimal essential medium (MEM) for 24 hours, and treated separately with cyclosporin A and varying doses of ALC, and then stimulated with Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and Ionomycin ($0.5{\mu}M$). The levels of IL-13, IL-4 were measured by ELISA analysis. The mRNA levels of IL-4, IL-5, IL-6, IL-13, GM-CSF, $TNF-{\alpha}$ were analyzed with Real-time PCR. Also, manifestations of MAPKs transcription factors and $NF-{\kappa}B$ p65 translocation were analyzed by western blotting in vitro. Subsequently, for ex vivo experiment, we induced allergic inflammation on Balb/c mice by OVA/alum and administered ALC orally. And we measured serum OVA-specific IgE level and IL-4, IL-13 in the splenocyte culture supernatant by ELISA analysis. Results ALC was shown to suppress mRNA expression of IL-4, IL-5, IL-6, IL-13, GM-CSF, $TNF-{\alpha}$, and to inhibit the IL-13, IL-4 production. Also ALC reduced an activation of mast cells specific signal MAPKs transcription factors and $NF-{\kappa}B$ p65 from the western blot analysis in in vitro experiment. In ex vivo, ALC oral adminstration decreased the level of OVA-specific IgE in serum, and IL-4, IL-13 in the splenocyte culture supernatant. Conclusions ALC is shown to reduce inflammation and allergic response by suppressing Th2 cytokines through the regulation of transcription factors MAPKs and $NF-{\kappa}B$ p65 in mast cells. Administration of ALC suppressed OVA-specific IgE in ovalbumin allergy model through the inhibition of Th2 cytokine. In conclusion, ALC can be considered as an effective treatment for allergic diseases such as atopic dermatitis.

• 생명과학회지
• /
• 제24권7호
• /
• pp.713-720
• /
• 2014

본 연구에서는 Ardisia arborescens 에탄올 추출물(AAEE)의 항산화능과 항염증 생리활성을 in vitro assay system 및 cell culture model system을 이용하여 분석하였다. 먼저 AAEE의 항산화능을 DPPH radical 소거능으로 분석한 결과 양성 대조군으로 사용한 ascorbic acid와 유사한 정도의 높은 활성을 보여 매우 강한 항산화능을 보유함을 확인하였다. 또한 RAW 264.7 세포주를 이용한 $H_2O_2$ 및 LPS의 유도에 의해 생성된 ROS에 대한 소거능을 분석한 결과에서도 농도의존적인 강한 소거능을 보였다. 뿐만 아니라 대표적인 항산화효소로 천연물에 의한 항산화능 활성에 의해 발현이 유도되는 HO-1, TrxR1 및 그 전사 인자인 Nrf2의 단백질 발현이 AAEE의 처리에 의해 농도의존적으로 증가됨을 보였으며 이러한 HO-1, TrxR1 및 Nrf2의 발현 변화는 상위신호전달계인 MAPKs에 의해 조절될 가능성을 보였다. 한편 AAEE가 LPS에 의해 유도된 NO 생성에 미치는 영향을 분석한 결과 세포독성 없이 농도의존적인 NO 생성 저해능을 보였으며 이는 NO 생성 단백질인 iNOS의 발현 저해에서 기인함을 확인하였다. 이와 같은 AAEE의 NO 생성 억제 효과는 염증 상위신호전달계인 NF-${\kappa}B$ 및 AP-1의 조절을 통해 일어날 가능성을 보였다. 이러한 결과를 통해 AAEE의 높은 항산화능과 항염증 활성을 처음으로 확인하였으며 향후 기능성 소재로서 유용하게 활용될 수 있을 것으로 판단된다.

• 한국수정란이식학회지
• /
• 제32권4호
• /
• pp.343-351
• /
• 2017

Porcine spermatogonial stem cells (SSCs) prefer three-dimensional (3D) culture systems to 2D ones for the maintenance of self-renewal. Of the many 3D culture systems, agar-based hydrogels are candidates for supporting porcine SSC self-renewal, and there are various types of agar powder that can be used. In this study, we sought to identify an agar-based 3D hydrogel system that exhibited strong efficacy in the maintenance of porcine SSC self-renewal. First, 3D hydrogels with different mechanics were prepared with various concentrations of Bacto agar, lysogeny broth (LB) agar, and agarose powder, and the 3D hydrogel with the strongest alkaline phosphatase (AP) activity and greatest increase in colony size was identified for the different types of agar powder. Second, among the porcine SSCs cultured in the different 3D hydrogels, we analyzed the colony formation, morphology, and size; AP activity; and transcription and translation of porcine SSC-related genes, and these were compared to determine the optimal 3D hydrogel system for the maintenance of porcine SSC self-renewal. We found that 0.6% (w/v) Bacto agar-, 1% (w/v) LB agar-, and 0.2% (w/v) agarose-based 3D hydrogels showed the strongest maintenance of AP activity and the most pronounced increase in colony size in the culture of porcine SSCs. Moreover, among these hydrogels, the strongest transcription and translation of porcine SSC-related genes and largest colony size were detected in porcine SSCs cultured in the 0.2% (w/v) agarose-based 3D hydrogel, whereas there were no significant differences in colony formation and morphology. These results demonstrate that the 0.2% (w/v) agarose-based 3D hydrogel can be effectively used for the maintenance of porcine SSC self-renewal.

• Journal of Animal Science and Technology
• /
• 제65권3호
• /
• pp.664-678
• /
• 2023

To improve culture efficiency of Hanwoo myosatellite cells, these cells were cultured at different temperatures. Hanwoo myosatellite cells were compared with C2C12 cells to observe proliferation and differentiation at culture temperatures of 37℃ and 39℃ and determine the possibility of using them as cultured meat. Immunofluorescence staining using Pax7 and Hoechst, both cells cultured at 37℃ proliferated better than cultured at 39℃ (p < 0.05). When differentiated cells were stained with myosin and Hoechst, there was no significant difference in myotube thickness and Fusion index (p > 0.05). In Western blotting analysis, Hanwoo myosatellite cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). C2C12 cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). In reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis, Hanwoo myosatellite cells cultured at 39℃ had significantly (p < 0.05) higher expression levels of MyHC, MYF6, and MB than those cultured at 37℃. C2C12 cells cultured at 39℃ showed significantly (p < 0.05) higher expression levels of MYOG and MB than those cultured at 37℃. To increase culture efficiency of Hanwoo myosatellite cells, proliferating at 37℃ and differentiating at 39℃ are appropriate. Since results of temperature differences of Hanwoo myosatellite cells were similar to those of C2C12 cells, they could be used as a reference for producing cultured meat using Hanwoo satellite cells.

• Journal of Microbiology and Biotechnology
• /
• 제18권3호
• /
• pp.434-442
• /
• 2008

We observed that an inclusion body (IB) of recombinant ${\beta}$-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the ${\beta}$-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific ${\beta}$-galactosidase production, although ${\beta}$-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of ${\beta}$-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of ${\beta}$-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

• 대한한방내과학회지
• /
• 제26권4호
• /
• pp.853-863
• /
• 2005

Object : This study was performed to investigate the anti-fibrogenic effect of Artemisiae Capillaris Herba(ACH) on cultured rat hepatic stellate cells. Methods : Hepatic Stellate Cells were obtained from a 350gm Sprague-Dawley rat by tissue perfusion system, and the cells for the study were selected after 3 passages of culture on non-coated plastic culture dishes which enable the cells to activate, thus producing collagens in the cell media. Cells were treated with various concentrations of Artemisia Capillaris Herba(ACH) extract powder for 24 or 48 hours. After the treatment, Soluble collagen, procollagen levels and the mRNA of the procollagen type I C were measured by using assay kit and RT-PCR method. Results : Procollagen production by the hepatic stellate cells decreased after the treatment in a time-dependent dose-dependent manner. The mRNA expression decreased consistently with the volume of the secreted procollagen which indicates the herb hat inhibitory effect on fibrogenesis of the liver by regulating one of the fibrosis associated genes in transcription. Conclusion : These results suggest that ACH is beneficial in the treatment of cirrhotic patients as well as for the patients with chronic hepatitis.

• 한국미생물·생명공학회지
• /
• 제19권4호
• /
• pp.348-355
• /
• 1991

Bacillus subtilis의 Beta-1,4-glucanase 유전자와 Saccharomyces cerevisiae의 alcohol dehydrogenase isoenzyme I 유전자 (ADHI)의 promoter와 mouse $\alpha$-amylase의 분비신호를 연결하여 분비형 플라스미드를 구성하였으며 이를 산업용 알콜생산 효모인 Saccharomyces cerevisiae 54에 도입하여 형질전환시켰다. 한편 $\beta$-glucanase 유전자의 발현정도를 증대시키기 위해 CYC1 유전자의 전사종결신호를 부가한 후 역시 Saccharomyces cerevisiae 54에 도입하였다. 형질전체들은 carboxymethyl cellulose가 함유된 평판배지에서 $\beta$-glucanase를 분비하고 있음을 확인할 수 있었다. 전사종결 신호가 부가된 경우엔 전체역가가 2배 정도 증가하였다. 형질전환체들이 세포밖으로 분비한 효소역가는 전체의 60 정도였다.