• Asian-Australasian Journal of Animal Sciences
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• v.29 no.12
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• pp.1702-1709
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• 2016

The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.

• Korean Journal of Clinical Laboratory Science
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• v.40 no.1
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• pp.31-40
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• 2008

Despite of the importance of the primordial follicle (PMF) recruitment, factors and mechanisms for process are poorly understood. To evaluate expression and role of the follicular transition from PMF to PMF/primary follicles (PMIF) in the present study, we evaluated expression of lats1, lats2, cyclin A1, and cyclin A2 mRNA and protein, and elucidated and role of lats1-cyclin A in the follicular transition from PMF to PRIF. To analysis of differential expression in PMF and PMIF, each stage follicles were collected by day1 and day5 of immuno-compromised rats (ICR) and analyzed by real-time PCR for the genes. For localization of mRNAs and proteins of the genes, in situ hybridization and immunohistochemistry were performed. We confirmed that the lats1, lats2, cyclin A1, and cyclin A2 mRNA were more expressed in PMF than PMIF. Localization of the four genes expression were observed in nuclei of oocytes from the arrested primordial, and in the surrounding granulosa cells of the growing follicles. The mRNA expressions were gradually decreased with follicular development. From immunohistochemistry studies, Cyclin A1 protein expression were observed in oocyte cytoplasmas of early stage follicles, while observed in granulose cells and oocyte nucleoli during growing follicles. This study suggested that the presence of lats gene family might perform negatively regulation of cell proliferation by modulation of the CDC2/Cyclin A complex activity. lats-cyclin A genes in oocytes of the early stage follicles might play a role in the meiotic cell cycle arrest of the primary oocytes at the primordial follicle stage as well as the follicular growth.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.201-208
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• 2015

The role of RNA-binding proteins (RBPs) to regulate expression of genes seems to be very important. RBPs play important roles in RNA related bioprocess such as transcription, pre-mRNA splicing, polyadenylation, transport, localization, translation, turn over and maintenance of structure. Despite of many researches on RNA binding proteins, detailed mechanisms of these proteins have not been fully understood. It seems that many parts of RBPs remains unknown and should be characterized for the better understanding of gene expression. Recently, genetic, biochemical, and bioinformatic analysis of genomes revealed a vast array of RBPs and many parts are interesting to understand bioprocessing including gene expression.

• Korean Journal of Ecology and Environment
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• v.52 no.4
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• pp.394-402
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• 2019

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

• Journal of Cancer Prevention
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• v.21 no.3
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• pp.135-143
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• 2016

Background: Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods: Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results: Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase $(AMPK){\alpha}$ and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and $AMPK{\alpha}$ abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions: Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or $AMPK{\alpha}/Nrf2$ pathway in HaCaT cells.

• International Journal of Oral Biology
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• v.43 no.4
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• pp.177-183
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• 2018

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary $2^{nd}$ molar germs of rats. We used the maxillary $2^{nd}$ molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary $2^{nd}$ molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Journal of Ginseng Research
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• v.45 no.6
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• pp.734-743
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• 2021

Background: The underlying mechanisms of the potential tumor-suppressive effects of ginsenoside Rh2 are complex. N6-methyladenosine (m6A) RNA methylation is usually dysregulated in cancer. This study explored the regulatory effect of ginsenoside Rh2 on m6A RNA methylation in cancer. Methods: m6A RNA quantification and gene-specific m6A RIP-qPCR assays were applied to assess total and gene-specific m6A RNA levels. Co-immunoprecipitation, fractionation western blotting, and immunofluorescence staining were performed to detect protein interactions and distribution. QRT-PCR, dual-luciferase, and ChIP-qPCR assays were conducted to check the transcriptional regulation. Results: Ginsenoside Rh2 reduces m6A RNA methylation and KIF26B expression in a dose-dependent manner in some cancers. KIF26B interacts with ZC3H13 and CBLL1 in the cytoplasm of cancer cells and enhances their nuclear distribution. KIF26B inhibition reduces m6A RNA methylation level in cancer cells. SRF bound to the KIF26B promoter and activated its transcription. SRF mRNA m6A abundance significantly decreased upon KIF26B silencing. SRF knockdown suppressed cancer cell proliferation and growth both in vitro and in vivo, the effect of which was partly rescued by KIF26B overexpression. Conclusion: ginsenoside Rh2 reduces m6A RNA methylation via downregulating KIF26B expression in some cancer cells. KIF26B elevates m6A RNA methylation via enhancing ZC3H13/CBLL1 nuclear localization. KIF26B-SRF forms a positive feedback loop facilitating tumor growth.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.781-787
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• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.