• Journal of Embryo Transfer
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• v.32 no.1
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• pp.17-24
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• 2017

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 ${\beta}$-galactoside ${\alpha}$-2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a ($10{\mu}M$) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

• Nutrition Research and Practice
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• v.15 no.3
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• pp.294-308
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• 2021

BACKGROUD/OBJECTIVES: Allomyrina dichotoma larva (ADL), one of the many edible insects recognized as future food resources, has a range of pharmacological activities. In a previous study, an ADL extract (ADLE) reduced the hepatic insulin resistance of high-fat diet (HFD)-induced diabetic mice. On the other hand, the associated molecular mechanisms underlying pancreatic beta-cell dysfunction remain unclear. This study examined the effects of ADLE on palmitate-induced lipotoxicity in a beta cell line of a rat origin, INS-1 cells. MATERIALS/METHODS: ADLE was administered to high-fat diet treated mice. The expression of apoptosis-related molecules was measured by Western blotting, and reactive oxidative stress generation and nitric oxide production were measured by DCH-DA fluorescence and a Griess assay, respectively. RESULTS: The administration of ADLE to HFD-induced diabetic mice reduced the hyperplasia, 4-hydroxynonenal levels, and the number of apoptotic cells while improving the insulin levels compared to the HFD group. Treatment of INS-1 cells with palmitate reduced insulin secretion, which was attenuated by the ADLE treatment. Furthermore, the ADLE treatment prevented palmitate-induced cell death in INS-1 cells and isolated islets by reducing the apoptotic signaling molecules, including cleaved caspase-3 and PARP, and the Bax/Bcl2 ratio. ADLE also reduced the levels of reactive oxygen species generation, lipid accumulation, and nitrite production in palmitate-treated INS-1 cells while increasing the ATP levels. This effect corresponded to the decreased expression of inducible nitric oxide synthase (iNOS) mRNA and protein. CONCLUSIONS: ADLE helps prevent lipotoxic beta-cell death in INS-1 cells and HFD-diabetic mice, suggesting that ADLE can be used to prevent or treat beta-cell damage in glucose intolerance during the development of diabetes.

• Animal Bioscience
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• v.37 no.5
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• pp.929-943
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• 2024

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

• Journal of Life Science
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• v.24 no.10
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• pp.1039-1045
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• 2014

This study analyzed whether human chorionic gonadotropin (hCG) induces ER stress via the IRE/XBP1 pathway in mouse Leydig tumor (mLTC-1) cells. In a previous study, we demonstrated that the unfolding protein response (UPR) plays an important role in the expression of steroidogenic enzymes by modulating the ATF6 pathway, as well as ER stress-mediated apoptosis in hCG-stimulated Leydig cells. Although UPR signaling has been reported to regulate the IRE1/XBP1 pathway, it is not known whether hCG-induced ER stress in Leydig cells can activate the pathway. To investigate the activation of the IRE1/XBP1 pathway in mLTC-1 cells after hCG treatment, we performed a Western blot analysis to detect the phospho-IRE1 protein and an RT-PCR analysis to validate splicing of XBP1 mRNA. We used ER stress-activated indicator (ERAI) constructs for monitoring the activity of IRE1 and then analyzed by fluorescence microscopy and flow cytometry. The expression levels of the phospho-IRE1 protein markedly increased in response to the hCG treatment. In the mLTC-1 cells transfected with an F-XBP1-venus/F-$XBP1{\Delta}DBD$-venus construct, the hCG treatment led to the appearance of green fluorescent cells and detectable fluorescence in the nucleus and cytosol, respectively. In addition, splicing of XBP1 mRNA significantly increased after the hCG treatment. Taken together, these results indicate that hCG-induced ER stress leads to activation of the IRE1/XBP pathway in Leydig cells.

• Applied Microscopy
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• v.30 no.4
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• pp.347-355
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• 2000

Zinc is one of the most abundant oligoelements in the living cell. It appears tightly bound to some metalloproteins and nucleic acids, loosely bound to some metallothioneins or even as free ion. Small amounts of zinc ions (in the nanomolar range) regulate a plentitude of enzymatic proteins, receptors and transcription factors, thus rolls need accurate homeostasis of zinc ions. Zinc is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity at many central excitatory synapses. Growing evidences suggest that zinc may also be a key mediator and modulator of the neuronal death associated with transient global ischemia and sustained seizures, as well as perhaps other neurological disease stoles. Some neurons have developed mechanisms to accumulate zinc in specific membrane compartment ('vesicular zinc') which can be evidenced using histochemical techniques. Substances giving a bright colour or emitting fluorescence when in contact with divalent metal ions are currently used to detect them inside cells; their use leads to the so called 'direct' methods. The fixation and precipitation of metal ions as insoluble salt precipitates, their maintenance along the histological process and, finally, their demonstration after autometallographic development are essential steps for other methods, the so called 'indirect methods'. This study is a short report on the autometallograhical approaches for zinc detection in the central nervous system (CNS) by means of a modified selenium method.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.6 no.1
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• Journal of Life Science
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• v.27 no.12
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• pp.1445-1451
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• 2017

Mitochondria play a central role in energy generation by using electron transport coupled with oxidative phosphorylation. They also participate in other important cellular functions including metabolism, apoptosis, signaling, and reactive oxygen species production. Therefore, mitochondrial dysfunction is known to contribute to a variety of human diseases. Furthermore, there are various inherited diseases of energy metabolism due to mitochondrial DNA (mtDNA) mutations. Unfortunately, therapeutic options for these inherited mtDNA diseases are extremely limited. In this regard, mitochondrial replacement techniques are taking on increased importance in developing a clinical approach to inherited mtDNA diseases. In this study, green fluorescence protein (GFP)-tagged mitochondria were isolated by differential centrifugation from a mammalian cell line. Using microinjection technique, the isolated GFP-tagged mitochondria were then transferred to bovine oocytes that were triggered for early development. During the early developmental period from bovine oocytes to blastocysts, the transferred mitochondria were observed using fluorescent microscopy. The microinjected mitochondria were dispersed rapidly into the cytoplasm of oocytes and were passed down to subsequent cells of 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Together, these results demonstrate a successful in vitro transfer of isolated mitochondria to oocytes and provide a model for mitochondrial replacement implicated in inherited mtDNA diseases and animal cloning.