The capacity for liver regeneration involves a variety of nutritional factors. Vitamin C has multiple metabolic and antioxidant functions. In this study, we investigated the role of vitamin C in liver regeneration following hepatectomy in senescence marker protein (SMP)-30 knockout (KO) mice. Partial hepatectomy was performed by resecting the median and left lateral lobes of mice. Vitamin C accelerated liver recovery in SMP30 KO mice treated with vitamin C (KV). The livers of the KV mice exhibited lower levels of aspartate aminotransferase and lower injury than those of the KO mice. Increased type II transforming growth factor-β receptor (TGF-βRII)-mediated regeneration signaling was accompanied by HGF and cMet in the KV but not the KO mice. Consistent with this, the expression of cell cycle regulatory proteins, including cyclin D1 and proliferating cell nuclear antigen (PCNA), increased rapidly in the KV mice. Enhanced activation of ERK and GSK-3β proteins and a significantly increased number of binuclear hepatocytes were also detected in the livers of the KV mice. Moreover, the KV mice synthesized the highest levels of albumin. These data suggest that treating SMP30 knockout mice with vitamin C resulted in earlier recovery and liver regeneration by activation of the regeneration system.
Objective: Zearalenone (ZEA) has estrogen-like effects. Our previous study has shown that ZEA (0.5 to 1.5 mg/kg) could induce abnormal uterine proliferation through transforming growth factor signaling pathway. To further study the other regulatory networks of uterine hypertrophy caused by ZEA, the potential mechanism of ZEA on porcine endometrial epithelial cells (PECs) was explored by the Illumina Hiseq 2000 sequencing system. Methods: The PECs were treated with ZEA at 0 (ZEA0), 5 (ZEA5), 20 (ZEA20), and 80 (ZEA80) µmol/L for 24 h. The collected cells were subjected to cell cycle, RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and western blot analysis. Results: The proportion of cells in the S and G2 phases decreased (p<0.05), but the proportion of cells in the G1 phase increased (p<0.05) in the ZEA80 treatment. Data analysis revealed that the expression of Wnt pathway-related genes, estrogen-related genes, and mitogen-activated protein kinase pathway-related genes increased (p<0.05), but the expression of genetic stability genes decreased (p<0.05) with increasing ZEA concentrations. The relative mRNA and protein expression of WNT1, β-catenin, glycogen synthase kinase 3β (GSK-3β) were increased (p<0.05) with ZEA increasing, while the relative mRNA and protein expression of cyclin D1 (CCND1) was decreased (p<0.05). Moreover, our immunofluorescence results indicate that β-catenin accumulated around the nucleus from the cell membrane and cytoplasm with increasing ZEA concentrations. Conclusion: In summary, ZEA can activate the Wnt/β-catenin signaling pathway by up-regulating WNT1 and β-catenin expression, to promote the proliferation and development of PECs. At the same time, the up-regulation of GSK-3β and down-regulation of CCND1, as well as the mRNA expression of other pathway related genes indicated that other potential effects of ZEA on the uterine development need further study.
Kim, Jung-Im;Kim, Seong-Yun;Seo, Min-Jeong;Lim, Hak-Seob;Lee, Young-Choon;Joo, Woo-Hong;Choi, Byung-Tae;Jeong, Yong-Kee;Choi, Yung-Hyun
Journal of Life Science
/
v.18
no.6
/
pp.884-890
/
2008
Genistein, an isoflavone in soybean products, is a potential chemopreventive agent against various types of cancer. There are several studies documenting molecular alterations leading to cell cycle arrest at G2/M phase and induction of apoptosis; however, its mechanism of action and its molecular targets on the prostaglandin $E_2$ ($PGE_2$) production and telomere length regulation in human cancer remain unclear. In this study, we investigated the effect of genistein on the levels of cyclooxygenases (COXs) and telomere regulatory components of several human cancer cell lines (T24, human bladder carcinoma cells; U937, human leukemic cells; AGS, human stomach adenocarcinoma cells and SK-MEL-2, human skin melanoma cells). Genistein treatment resulted in the inhibition of cancer cell proliferation in a concentration-dependent manner. It was found that genistein treatment markedly decreased the levels of COX-2 mRNA and protein expression without significant changes in the expression of COX-1, which was correlated with a decrease in $PGE_2$ synthesis. Genistein treatment also partly inhibited the levels of human telomerase reverse transcriptase (hTERT) as well as human telomerase RNA (hTR) and telomerase-associated protein (TEP)-1, and the activity of telomerase. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of genistein.
Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development.
Proceedings of the Korean Society of Crop Science Conference
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2017.06a
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pp.36-36
/
2017
Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.
MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3'-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3'-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.
Kim, Chung-Hee;Kim, Jong-Soo;Van Cuong, Dang;Kim, Na-Ri;Kim, Eui-Yong;Han, Jin
Journal of Life Science
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v.13
no.3
/
pp.241-247
/
2003
While the DNA-protein kinase (DNA-PK) complex, comprised of DNA-PKcs and Ku80, is primary involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes. Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type (Wt) or DNA-PKcs-null (DNA-$PKcs^{-/-}$) mice with various stress inducing agents revealed that adriamycin was markedly more cytotoxic for $Ku80^{-/-}MEFs$ and led to their long-term accumulation in the $G_2$/M phase. This differential response was not due to differences in DNA repair, since adrimycin-triggered DNA damage was repaired with comparable efficiency in both Wt and $Ku80^{-/-}MEFs$, but was associated with differences in the expression of important cell cycle regulatory genes. Our results support the notion that Ku80-mediated cytoprotection and $G_2$/M-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression.
Objective: Inbreeding depression of reproduction is a major concern in the conservation of native chicken genetic resources. Here, based on the successful development of strongly inbred (Sinb) and weakly inbred (Winb) Langshan chickens, we aimed to evaluate inbreeding effects on reproductive traits and identify candidate genes involved in inbreeding depression of reproduction in Langshan chickens. Methods: A two-sample t-test was performed to estimate the differences in phenotypic values of reproductive traits between Sinb and Winb chicken groups. Three healthy chickens with reproductive trait values around the group mean values were selected from each of the groups. Differences in ovarian and hypothalamus transcriptomes between the two groups of chickens were analyzed by RNA sequencing (RNA-Seq). Results: The Sinb chicken group showed an obvious inbreeding depression in reproduction, especially for traits of age at the first egg and egg number at 300 days (p<0.01). Furthermore, 68 and 618 differentially expressed genes (DEGs) were obtained in the hypothalamus and ovary between the two chicken groups, respectively. In the hypothalamus, DEGs were mainly enriched in the pathways related to vitamin metabolism, signal transduction and development of the reproductive system, such as the riboflavin metabolism, Wnt signaling pathway, extracellular matrix-receptor interaction and focal adhesion pathways, including stimulated by retinoic acid 6, serpin family F member 1, secreted frizzled related protein 2, Wnt family member 6, and frizzled class receptor 4 genes. In the ovary, DEGs were significantly enriched in pathways associated with basic metabolism, including amino acid metabolism, oxidative phosphorylation, and glycosaminoglycan degradation. A series of key DEGs involved in folate biosynthesis (gamma-glutamyl hydrolase, guanosine triphosphate cyclohydrolase 1), oocyte meiosis and ovarian function (cytoplasmic polyadenylation element binding protein 1, structural maintenance of chromosomes 1B, and speedy/RINGO cell cycle regulator family member A), spermatogenesis and male fertility (prostaglandin D2 synthase 21 kDa), Mov10 RISC complex RNA helicase like 1, and deuterosome assembly protein 1) were identified, and these may play important roles in inbreeding depression in reproduction. Conclusion: The results improve our understanding of the regulatory mechanisms underlying inbreeding depression in chicken reproduction and provide a theoretical basis for the conservation of species resources.
Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-I to LC3-II and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.
Ubiquitination is a post-translational modification that is involved in the quality control of proteins and responsible for modulating a variety of cellular physiological processes. Protein ubiquitination and deubiquitination are reversible processes that regulate the stability of target substrates. The ubiquitin proteasome system (UPS) helps regulate tumor-promoting processes, such as DNA repair, cell cycle, apoptosis, metastasis, and angiogenesis. The UPS comprises a combination of ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-ligase enzymes (E3), which complete the degradation of target proteins. Ubiquitin-conjugating enzymes (UBE2s) play an inter-mediate role in the UPS process by moving activated ubiquitin to target proteins through E3 ligases. UBE2s consist of 40 members and are classified according to conserved catalytic ubiquitin-conjugating (UBC) domain-flanking extensions in humans. Since UBE2s have specificity to substrates like E3 ligase, the significance of UBE2 has been accentuated in tumorigenesis. The dysregulation of multiple E2 enzymes and their critical roles in modulating oncogenic signaling pathways have been reported in several types of cancer. The elevation of UBE2 expression is correlated with a worse prognosis in cancer patients. In this review, we summarize the basic functions and regulatory mechanisms of UBE2s and suggest the possibility of their use as therapeutic targets for cancer.
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