• Journal of Embryo Transfer
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• v.33 no.3
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• pp.177-183
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• 2018

The identification of biomarkers of a living tissues is essentially required to understand specific functions of the cells. In previous study, we reported IGFBP 3 as one of the putative biomarkers, by showing specific expression at porcine spermatogonial stem cells (SSCs) of early stage of porcine testis. In this study, we analyzed the expression of seven members of IGFBP family (IGFBPs) in SSCs and histological expression pattern of pregnancy-associated plasma protein-A (PAPP-A), which plays a role on the growth promoting enzyme by cleavage of IGFBPs in testis of 5 days old pig. RT-PCR analysis showed that IGFBP 1, 2, 3, 4, and 6 were expressed at high level specifically in porcine SSCs compared with whole testis. We performed immunohisotochemical staining of testis sections with PAPP-A and protein gene product 9.5 (PGP9.5) which are the known biomarkers for SSCs. We were not able to find co-expression of PAPP-A and PGP9.5; PAPP-A was expressed only in Sertoli cells and PGP9.5 expression was confirmed in spermatogonium. Additionally, we were able to confirm the GATA4 expression in Sertoli and Leydig cells as a regulator of Sertoli cell function was not detected PGP9.5 expressing cells, indicating indirect evidence of that cytolocalization of PAPP-A expression is limited in Sertoli cells. These results suggested that the PAPP-A expressed in Sertoli cells may play role on regulation of development and differentiation of testicular cells through the IGF axis in neonatal porcine testis.

• Journal of Korean Society on Water Environment
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• v.28 no.2
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• pp.313-319
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• 2012

Various nanomaterials may flow into the aquatic ecosystem via production, use, and treatment processes. Especially, gold nanoparticles (AuNPs) were categorized as manufactured nanomaterials presented by the Organization for Economic Cooperation and Development Working Party on Manufactured Nanomaterials (OECD WPMN) in 2010. AuNPs have been used in medical area, however, they were reported to induce cytotoxicity and oxidative DNA damage, as well as down-regulation of the DNA repair gene in mice and human cell lines. In this study, the aquatic toxicity data of AuNPs and gold ions were collected, with the specific test methods analyzed with respect to the form and size of AuNPs, test species, exposure duration, and endpoints. Currently, aquatic toxicity data of AuNPs and gold ions have been presented in 14 studies including 4 fish, 6 crustacean, 2 green algae, and 2 macrophytes studies, as well as a further 8 studies including 4 fish, 4 crustacean, 1 platyhelminthes, and 1 green algae, respectively. The AuNPs were 0.8-100 nm in size, as gold nanoparticles, gold nanorod, glycodendrimer-coated gold nanoparticles, and amine-coated gold nanoparticles. The tested endpoints were the individual toxicities, such as mortality, malformation, reproduction inhibition, growth inhibition and genetic toxicity such as oxidative stress, gene expression, and reactive oxygen species formation. The accumulation of AuNPs was also confirmed in the various receptor organs. These results are expected to be useful in understanding the aquatic toxicity of AuNPs and gold ions, as well as being applicable to future toxicity studies on AuNPs.

• Journal of Radiation Protection and Research
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• v.38 no.4
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• pp.166-171
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• 2013

While high-dose ionizing radiation results in long term cellular cytotoxicity, chronic low-dose (<0.2 Gy) of X- or ${\gamma}$-ray irradiation can be beneficial to living organisms by inducing radiation hormesis, stimulating immune function, and adaptive responses. During chronic low-dose-rate radiation (LDR) exposure, whole body of mice is exposed to radiation, however, it remains unclear if LDR causes changes in gene expression of the whole brain. Therefore, we aim to investigate expressed genes (EGs) and signaling pathways specifically regulated by LDR-irradiation ($^{137}Cs$, a cumulative dose of 1.7 Gy for total 100 days) in the whole brain. Using microarray analysis of whole brain RNA extracts harvested from ICR and AKR/J mice after LDR-irradiation, we discovered that two mice strains displayed distinct gene regulation patterns upon LDR-irradiation. In ICR mice, genes involved in ion transport, transition metal ion transport, and developmental cell growth were turned on while, in AKR/J mice, genes involved in sensory perception, cognition, olfactory transduction, G-protein coupled receptor pathways, inflammatory response, proteolysis, and base excision repair were found to be affected by LDR. We validated LDR-sensitive EGs by qPCR and confirmed specific upregulation of S100a7a, Olfr624, and Gm4868 genes in AKR/J mice whole brain. Therefore, our data provide the first report of genetic changes regulated by LDR in the mouse whole brain, which may affect several aspects of brain function.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.2
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• pp.68-74
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• 2011

Objective: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of $Obox4$ in oocyte maturation by evaluating downstream signal networking. Methods: The $Obox4$ dsRNA was prepared by $in$ $vitro$ transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by $in$ $vitro$ maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix Gene-Chip$^{(R)}$ Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Results: Total 424 genes were up (n=80) and down (n=344) regulated after $Obox4$ RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by $Obox4$ RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. Conclusion: From the results of this study, it is concluded that $Obox4$ is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.

• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• Animal cells and systems
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• v.3 no.2
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• pp.207-213
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• 1999

During the differentiation of Naegleria gruberi amoebas into flagellates, the amoebas undergo sequential changes in cell shape and form new cellular organelles. To understand the nature of the signal which initiates this differentiation and the signal transduction pathway, we treated cells with four agents, PMA, retinoic acid (RA), okadaic acid, and cAMP. Retinoic acid and cAMP had specific effects on the differentiation of N. gruberi depending on the time of the drug treatment. Addition of (100$\mu$M) retinoic acid at the initiation of differentiation inhibited differentiation by blockinq the transcription of differentiation specific genes (e.g., $\beta$-tubulin). This inhibition of differentiation by retinoic acid was overcome by co-treatment with cAMP (or dbcAMP, 20 $\mu$M). Addition of retinoic acid at later stages (30 and 70 min) had no effect on the transcriptional regulation of the $\beta$-tubulin gene, however the differentiation was inhibited by different degrees. Co-treatment of cAMP at these stages did not overcome the inhibitory effect of retinoic acid. These results suggest that the role of retinoic acid as a transcriptional regulator might be conserved throughout the evolution of eukaryotes.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.4
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• pp.199-205
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• 2006

The functional expression of potassium $(K^+)$ channels has electrophysiologically been studied in bone cells from several species, however, their identity and regulation of gene expressions in bone cells are not well known. In the present study, to investigate how $K^+$ channel expressions are regulated by estrogen, we measured changes of transcript levels of various $Ca^{2+}$-activated ($K_{Ca}$) and ATP-sensitive $K^+$ channels in rat osteoblastic ROS 17/2.8 cells after treatment with estrogen. Application of 17${\beta}$-estradiol $(E_2)$ for 24 h and 48 h increased mRNA and protein expressions of inwardly rectifying $K^+$ channel (Kir) 6.2 and type 2 small conductance $K_{Ca}$ channel (SK2), respectively. Combined treatment of cells with 17${\beta}-E_2$ and ICI 182,780, a pure antiestrogen, suppressed 17${\beta}-E_2$-induced alterations of SK2 and Kir6.2 mRNA levels. In addition, treatment of cells with U0126, a specific inhibitor of extracellular receptor kinases (ERK)1/2, and SP600125, a specific inhibitor of c-jun N-terminal kinase (JNK) blocked the enhancing effects of 17${\beta}-E_2$ on SK2 and Kir6.2 protein expressions. On the other hand, blocking of p38 mitogen-activated protein kinase had no effect. Taken together, these results indicate that 17${\beta}-E_2$ modulates SK2 and Kir6.2 expressions through the estrogen receptor, involving ERK1/2 and JNK activations.

• BMB Reports
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• v.43 no.6
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• pp.438-444
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• 2010

Dnd (dead end) gene encodes an RNA binding protein and is specifically expressed in primordial germ cells (PGCs) as a vertebrate-specific component of the germ plasma throughout embryogenesis. By utilizing a technique of specific nucleic acids associated with proteins (SNAAP), 13 potential target mRNAs of zebrafish Dnd (ZDnd) protein were identified from 8-cell embryo, and 8 target mRNAs have been confirmed using an RT-PCR analysis. Of the target mRNAs, the present study is focused on the regulation of geminin, which is an inhibitor of DNA replication. Using electrophoretic mobility shift assay (EMSA), we demonstrated that ZDND protein bound the 67-nucleotide region from 864 to 931 in the 3'UTR of geminin mRNA, a sequence containing 60.29% of uridine. Results from a dual-luciferase assay in HEK293 cells showed that ZDND increases the translation of geminin. Taken together, the identification of target mRNA for ZDnd will be helpful to further explore the biological function of Dnd in zebrafish germ-line development as well as in cancer cells.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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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.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.199-206
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• 1994

We present here a new PCR-based cloning technique that allows the different PCR products during mouse embryogenesis. Recently, mRNA differential display described by Liang & Pardee (Science 257, 1992) and re-confirmed by Zimermann & Schultz (PNAS 91,1994). This method will detect the appropriate changes in the temporal patterns of expression or in the transition from maternal control to zygotic control as well as the functional difference of embryo with polyspermy or monospermy, the difference of expression between successfully hatched blastocyst and blastocyst failed to hatching, response to agents, and cell cycle regulation. By this methods, we have cloned an eDNA, which showed mouse 2 cell specific expression. Genomic DNA digested with EcoRI showed approximately 15 kb and then showed higher expression in fetal liver rather than adult liver. Furthermore, this gene is likely to have 2 mRNA by alternative splicing.