• 미생물학회지
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• 제38권3호
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• pp.213-220
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• 2002

일본뇌염바이러스(Japanese encephalitis virus, JEV)의 구조단백질 capsid (C), precursor membrane (prM/M), 및 envelop (E) 단백질의 독립적인 발현을 위한 inducible expression system을 구축하였다. 발현세포주로는 BHK-21을 사용하였으며 발현의 induction에는 tetracycline analog인 doxycycline이 사용되었다. Transfectant BHK-21/IV(vector대조구), BHK21/IC(C), BHK-21/IP (prM/M),및 BHK-21/IE는 G418과 hygromycin 존재하에 클로닝되었으며 doxycycline induction에 따른 각 유전자의 mRNA 전사를 확인하였다. 세포의 성장곡선, chromatin condensation, internucleosomal DNA fragmentation, 및 flow cytometry에 의한 DNA content profile 분석을 통해 induction에 의한 각 구조단백질의 발현이 숙주세포에 미치는 영향을 조사하였다. 세 transfectants 모두 세포성장이 감소하고 chromatin이 응축되었다. 그러나 DNA fragmentation 및 DNA content profile 분석에서는BHK-21/IC만이 induction에 따라 상응하여 반응하였다. 이상의 결과는 JEV 감염에 의한 apoptotic 세포사멸 유도기전에서 capsid 단백질이 직접적이고 독립적인 영향요인이 될 수 있음을 제시한다.

• 생명과학회지
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• 제17권3호통권83호
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• pp.381-385
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• 2007

FACT(facilitates chromatin transcription)은 크로마틴을 주형으로 하는 전사에 필요한 크로마틴 특이적 전사 진행 인자이다. FACT는 Saccharomyces Cerevisiae Spt16/Cdc68의인간 유사체와 high mobility froup-1-like protein structure-specific recognition protein-1(SSRP-1)의 이종단백질 복합체이다 본 논문에서는 FACT의 단위체인 hSpt16의 발현이 휴지기의 T98G 세포에서 급격히 감소됨을 면역형광 분석법과 Western blot 분석법을 이용하여 관찰하였다. 이와 반대로 증식기의 T98C 세포에서는 hSpt16이 높은 수준으로 발현되고 있는 것이 관찰되었다. FACT의 또 다른 단위체 SSRP-1의 발현은 휴지기나 증식기의 세포에서 변화가 없음이 관찰되었다. 이상의 결과로부터 hSpt16이 발현이 세포의증식과 연관이 되어 있음을 알 수 있었고 세포 증식 표지 인자로 사용될 수 있을 것으로 사료된다.

• Molecules and Cells
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• 제43권12호
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• pp.1011-1022
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• 2020

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.

• Clinical and Experimental Reproductive Medicine
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• 제48권1호
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• pp.27-33
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• 2021

Objective: The chief outcome of testicular torsion in clinical and experimental contexts is testicular ischemia. Curcumin, a compound with anti-inflammatory and antioxidant properties, has fascinated researchers and clinicians for its promise in the treatment of fertility diseases. Methods: Thirty-five fully grown male mice were randomly classified into five groups: control, sham, testicular torsion, treatment group 1 (testicular torsion+short-term curcumin), and treatment group 2 (testicular torsion+long-term curcumin). Thirty-five days later, spermatozoa from the right cauda epididymis were analyzed with regard to count and motility. Toluidine blue (TB), aniline blue (AB), and chromomycin A3 (CMA3) staining assays were used to evaluate the sperm chromatin integrity. In addition, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) test was used to assess apoptosis. Results: Treatment group 1 exhibited a remarkably elevated sperm count compared to the testicular torsion group. Additionally, notably lower sperm motility was found in the testicular torsion group compared to the control, treatment 1, and treatment 2 groups. Staining (CMA3, AB, and TB) and the TUNEL test indicated significantly greater testicular torsion in the torsion group compared to the control group (p<0.05). The data also revealed notably lower results of all sperm chromatin assays and lower apoptosis in both treatment groups relative to the testicular torsion group (p<0.05). Significantly elevated (p<0.05) AB and TB results were noted in treatment group 1 compared to treatment group 2. Conclusion: Curcumin can compensate for the harmful effects of testicular ischemia and improve sperm chromatin quality in mice.

• Clinical and Experimental Reproductive Medicine
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• 제47권4호
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• pp.277-283
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• 2020

Objective: The sperm DNA fragmentation index (DFI) guides the clinician's choice of an appropriate assisted reproductive technology (ART) procedure. The DFI can be determined using commercially available methodologies, including sperm chromatin dispersion (SCD) kits and sperm chromatin structure assay (SCSA). Currently, when DFI is evaluated using SCD kits, the result is analyzed in reference to the SCSA-derived threshold for the choice of an ART procedure. In this study, we compared DFI values obtained using SCSA with those obtained using SCD and determined whether the difference affects the choice of ART procedure. Methods: We compared SCSA to two SCD kits, CANfrag (n=36) and Halosperm (n=31), to assess the DFI values obtained, the correlations between tests, the technical repeatability, and the impact of DFI on the choice of ART. Results: We obtained higher median DFI values using SCD kits than when using SCSA, and this difference was significant for the CANfrag kit (p<0.001). The SCD kits had significantly higher coefficients of variation than SCSA (p<0.001). In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) would be chosen for a significantly higher proportion of patients if a decision were made based on DFI derived from SCD rather than DFI determined using SCSA (p=0.003). Conclusion: Our results indicate that SCD kit-specific thresholds should be established in order to avoid the unnecessary use of IVF/ICSI based on sperm DNA damage for the management of infertility. Appropriate measures should be taken to mitigate the increased variability inherent to the methods used in these tests.

• 농업과학연구
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• 제49권1호
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• pp.103-112
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• 2022

Fipronil is a popular insecticide used in both agricultural and domestic fields. Factors that affect sperm and eggs have a direct influence on reproductive outcomes. This study was undertaken to assess the effect of varying concentrations (10 - 200 μM) of fipronil and incubation times (30 min and 2 hrs) on boar spermatozoa. Spermatozoa were evaluated for motility, motion kinematics, viability, chromatin stability, and for the generation of intracellular reactive oxygen species (ROS) and the results were compared to those from corresponding controls. The findings revealed a significant, dose-dependent reduction in sperm motility in all fipronil treatment groups at 30 min of incubation (p < 0.05). A similar dose-dependent reduction in sperm motility was observed subsequent to fipronil exposure for 2 hrs of incubation (p < 0.05). Groups treated with fipronil showed a gradual reduction in motion kinematics (p < 0.05). Moreover, a significantly higher percentage of dead sperm was observed at 200 μM fipronil, as compared to the highest live percentage obtained in controls (p < 0.05). Evaluating the sperm chromatin integrity revealed a significantly higher percentage of damaged chromatin in spermatozoa incubated with 200 μM of fipronil. Moreover, ROS production was significantly higher in fipronil-exposed sperm (p < 0.05). In conclusion, boar spermatozoa incubated with fipronil showed decreased levels of sperm motility and viability, weaker chromatin integrity, and increased levels of intracellular ROS generation, all of which indicate that exposure to fipronil potentially impairs the fertilization competence of boar spermatozoa.

• 식물조직배양학회지
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• 제24권1호
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• pp.1-35
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• 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.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.181-189
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• 2018

Candida albicans is a major pathogenic fungus in humans, and meets at first the innate immune cells, such as macrophages, in its host. One important strategy of the host cell to kill C. albicans is to produce reactive oxygen species (ROS) by the macrophages. In response to ROS produced by the macrophages, C. albicans operates its defense mechanisms against them by expressing its oxidative stress response genes. Although there have been many research studies explaining the specific transcription factors and the expression of the oxidative stress genes in C. albicans, the regulation of the oxidative stress genes by chromatin structure is little known. Epigenetic regulation by the chromatin structure is very important for the regulation of eukaryotic gene expression, including the chromatin structure dynamics by histone modifications. Among various histone modifications, histone acetylation is reported for its direct relationship to the regulation of gene expression. Recent studies reported that histone acetyltransferases regulate genes to respond to the oxidative stress in C. albicans. In this review, we introduce all histone acetyltransferases that C. albicans contains and some papers that explain how histone acetyltransferases participate in the oxidative stress response in C. albicans.

• Molecules and Cells
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• 제37권12호
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• pp.851-856
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• 2014

ATAD2, a remarkably conserved, yet poorly characterized factor is found upregulated and associated with poor prognosis in a variety of independent cancers in human. Studies conducted on the yeast Saccharomyces cerevisiae ATAD2 homologue, Yta7, are now indicating that the members of this family may primarily be regulators of chromatin dynamics and that their action on gene expression could only be one facet of their general activity. In this review, we present an overview of the literature on Yta7 and discuss the possibility of translating these findings into other organisms to further define the involvement of ATAD2 and other members of its family in regulating chromatin structure and function both in normal and pathological situations.

• Animal cells and systems
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• 제13권4호
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• pp.405-409
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• 2009

When DNA double-strand breaks (DSBs) were induced in mammalian cells, many DNA damage response proteins are accumulated at damage sites to form nuclear foci called IR-induced foci. Although the formation of foci has been shown to promote repair efficiency, the structural organization of chromatin in foci remains obscure. BAF53 is an actin-related protein which is required for maintenance of chromosome territory. In this study, we show that the formation of IR-induced foci by $\gamma$-H2AX and 53BP1 were reduced when BAF53 is depleted, while DSB- activated ATM pathway and the phosphorylation of H2AX remains intact after DNA damage in BAF53 knockdown cells. We also found that DSB repair efficiency was largely compromised in BAF53 knockdown cells. These results indicate that BAF53 is critical for formation of foci by $\gamma$-H2AX decorated chromatin at damage sites and the structural organization of chromatin in foci is an important factor to achieve the maximum efficiency of DNA repair.