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최중국,정소희,김성훈,이학교,김승렬,최중국 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.4
Antimicrobial peptides are widely found in the living organisms and are known to play a critical role in the innate immunity. Numerous antimicrobial peptides from diverse species appear to be effective against pathogenic microorganisms of bacteria, fungi, protozoa and viruses. Since antibiotic resistance is a global health issue in the fight against pathogenic microorganisms, there has been an urgent need for the development of new antibiotic substances. In the present study, we sought to find new peptide antibiotics from random peptide library by yeast two hybrid screening using Beclin1 bait. Two candidate peptides from the screening were expressed in a yeast secretory system of Pichia pastoris and tested for any antimicrobial activity against Staphylococcus aureus, MRSA, MRSA2242, MRSA2250, Lactobacillus casei, Lactobacillus acidophilus. Disc clear zone assay and spectrophotometric analysis revealed that the two peptides carry a decent activity against the pathogenic bacteria, in contrast to minimal effect on the commensal Lactobacillus strains. Taken together, this study presents novel peptides with antibacterial activity against the pathogenic forms of Staphylococcus aureus and suggests a possibility that these peptides, upon further characterization, may be developed as clinically useful antibiotics.
Analysis of porcine macrophage immune response to antigenic molecules and short chain fatty acids
최중국,이학교,송기덕 충북대학교 동물의학연구소 2015 Journal of Biomedical and Translational Research Vol.16 No.2
Macrophages play an important role in both the innate and adaptive immune responses. These include phagocytosis, killing of microorganisms, antigen presentation, and induction of immune cytokines and antimicrobial genes. Macrophage activity is reported to be controlled by diverse exogenous antigenic or endogenous metabolic molecules, and the underlying mechanisms are well documented in human and mouse macrophage cells. Bacterial lipopolysaccharide (LPS) is known to be one of the most potent stimuli activating macrophages through the toll like receptor 4 (TLR4) signaling pathway. There are other antigenic molecules, such as muramyl dipeptide (MDP) and outer membrane protein A (OmpA), that are also known to activate immune cells. On the other hand, short chain fatty acids (SCFAs) such as acetate and butyrate are produced by gut microbiota and control host energy metabolism and signal transduction through GPR receptors. However, there are few studies demonstrating the effects of these molecules in macrophages from domestic animals, including domestic pigs. In this study, we attempted to characterize gene expression regulation in porcine macrophages (PoM2, Pig Monocytes clone 2) following treatment with LPS, MDP, OmpA, and two short chain fatty acids using porcine genome microarray and RT-PCR techniques. A number of novel porcine genes, including anti-microbial peptides and others, appeared to be regulated at the transcriptional level. Our study reports novel biomarkers such as SLC37A2, TMEN184C, and LEAP2 that are involved in the porcine immune response to bacterial antigen LPS and two short chain fatty acids.
Modulation of Gene Expression in Raw 264.7 Macrophage Cells by Saccharomyces exiguus and LPS
최중국 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.3
Macrophages can recognize antigens and microorganisms, then initiate an appropriate defense. However, there has been a lack of comprehensive information regarding the genes which is modulated by commensal yeasts including Saccharomyces cerevisiae or Saccharomyces exiguus. Moreover, it is not clear to what extent the beneficial yeasts modulate the immune response against microbes and/or microbial toxins. We studied interactions between host cells and yeast/bacterial toxin (LPS) by analyzing the transcriptional response of macrophages stimulated by the Saccharomyces exiguus and/or Lipopolysaccharides with DNA microarray, which contains about 25,000 genes. Thirty three genes were identified to be modulated by more than 2 folds between groups of macrophage cells. Pathway analysis provided insight into the mutual interactions. Of particular interest was the responses elicited by the fungus in the murine macrophage cells, including modulation of immunity/defense, cellular signal transduction, cell proliferation/differentiation and transport. This indicates that the yeast induces immune response pathways as well as those associated with cell proliferation and transport. Among the 33 genes identified from the DNA microarray screening, 8 genes were further checked by RT-PCR analysis using gene specific primers. Compared to those of negative control, the sequential treatment with the yeast strain then LPS apparently induced the expression of Tnfaip3, IL7R and CD86, while it inhibited the expression of Cxcl10 and CD83. In conclusion, this study identified the genes that are up-regulated by Saccharomyces exiguus and it remains to be further studied whether these genes are modulated at the protein level, and also for their roles in controlling immune responses.
Ubiquilin 1 Interacts with Orai1 to Regulate Calcium Mobilization
이정은,최중국,전인숙,한나은,송혜진,김응국,최재운,송기덕,이학교 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.1
Store-operated calcium entry (SOCE) channels com-posed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticu-lum (ER)-mediated calcium (Ca2+) depletion, Stim proteins oligomerize with Orai to initiate Ca2+ influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglu-tamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, pre-vented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca2+ influx. As we expected, intracellular Ca2+ mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca2+ mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.
BMP-4에 의한 C2C12근아세포가 골세포로 분화하는 과정에 미치는 이소플라본의 상승효과
최호산,김용민,이성진,최중국,김동수,최의성,손현철,박경진,이학교 대한정형외과학회 2007 대한정형외과학회지 Vol.42 No.4
목 적: 이소플라본은 콩의 한가지 성분으로서 골형성에 도움이 된다. 이 연구는 골형성성장인자-4가 존재할 때 이소플라본이 근아세포 C2C12세포를 골세포로 분화하는 작용 및 그 원리를 알아보려 하였다.대상 및 방법: 이소플라본과 골형성인자-4를 따로 혹은 함께 C2C12세포에 작용하였다. 72시간 후 염색방법으로 알칼리 포스파타아제의 활성을 측정하였다. 알칼리 포스파타아제의 활성은 스캔한 이미지를 비교하는 방법과 분광광도법으로 분석하였다. 이소플래본 중의 한 가지 성분인 다이드제인으로 샘플을 처리한 후 세포 표면의 리모델링을 책임진 세포외기질 관련유전자의 발현을 마이크로어레이방법으로 분석하였다.결 과: 골형성성장인자-4 혹은 이소플래본을 작용한 후 C2C12세포들의 알칼리 포스파타아제의 활성은 골형성성장인자-4의 농도에 따라 증가하였으나 이소플라본이 단독으로 작용했을 때는 변화가 없었다. 이소플라본과 골형성성장인자-4가 동시에 작용했을 때 알칼리 포스파타아제의 활성은 증가되었다. 마이크로어레이의 결과 세포외기질 관련유전자들 중 Mmp13과 Mmp3 등 발현이 증가하였으나 Col4a2, Col5a1과 Mmp9 등은 감소되었다.