• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.322-328
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• 2013

In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

• Journal of Cancer Prevention
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• v.21 no.2
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• pp.95-103
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• 2016

Background: Excess energy supply induces chronic low-grade inflammation in association with oxidative stress in various tissues including intestinal epithelium. The objective of this study was to investigate the effect of high-fat diet (HFD) on intestinal cell membrane integrity and intestinal tumorigenesis in $Apc^{Min/+}$ mice. Methods: Mice were fed with either normal diet (ND) or HFD for 12 weeks. The number of intestinal tumors were counted and biomarkers of endotoxemia, oxidative stress, and inflammation were determined. Changes in intestinal integrity was measured by fluorescein isothiocyanate (FITC)-dextran penetration and membrane gap junction protein expression. Results: HFD group had significantly higher number of tumors compared to ND group (P < 0.05). Blood total antioxidant capacity was lower in HFD group, while colonic 8-hydroxy-2'-deoxyguanosine level, a marker of oxidative damage, was higher in HFD group compared to that of ND group (P < 0.05). The penetration of FITC-dextran was substantially increased in HFD group (P < 0.05) while the expressions of membrane gap junction proteins including zonula occludens-1, claudin-1, and occludin were lower in HFD group (P < 0.05) compared to those in ND group. Serum concentration of lipopolysaccharide (LPS) receptor (CD14) and colonic toll-like receptor 4 (a LPS receptor) mRNA expression were significantly higher in HFD group than in ND group (P < 0.05), suggesting that significant endotoxemia may occur in HFD group due to the increased membrane permeability. Serum interleukin-6 concentration and myeloperoxidase activity were also higher in HFD group compared to those of ND group (P < 0.05). Conclusions: HFD increases oxidative stress disrupting intestinal gap junction proteins, thereby accelerating membrane permeability endotoxemia, inflammation, and intestinal tumorigenesis.

• Animal Bioscience
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• v.37 no.8
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• pp.1377-1386
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• 2024

Objective: Embryonic interferon-tau (IFNT) and progesterone affect expression of interferon-stimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Methods: Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16, and 25 of pregnancy, and real-time quantitative polymerase chain reaction assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR, and PIBF. Results: Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and signal transducer and activator of transcription 1 (STAT1) decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Conclusion: Early pregnancy affects expression of ISGs, PGR, and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

• Journal of Acupuncture Research
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• v.31 no.2
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• pp.87-98
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• 2014

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in $HT_{29}$ human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

• Journal of Life Science
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• v.20 no.12
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• pp.1784-1791
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• 2010

In this study, we investigated the effects of ethyl alcohol extracts of Hizikia fusiforme (EHF) on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in human gastric adenocarcinoma AGS cells. Inhibitory effects of EHF on cell proliferation, motility, and invasiveness were found to be associated with increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance. Activities of matrix metalloprotease (MMP)-2 and -9 in AGS cells were dose-dependently inhibited by treatment with EHF, and this was also correlated with a decrease in expression of their mRNA and proteins; however, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 mRNA levels were increased. Additionally, immunoblotting results indicated that EHF repressed the levels of claudin proteins (claudin-1, -3, and -4), major components of TJs that play key roles in control and selectivity of paracellular transport. Furthermore, EHF decreased expression of insulin such as growth factor-1 receptor proteins, while concurrently increasing that of thrombospondin-1 and E-cadherin. In conclusion, these results suggest that EHF treatment may inhibit tumor cell motility and invasion, and therefore act as a dietary source to decrease the risk of cancer metastasis.

• Molecules and Cells
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• v.38 no.10
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• pp.829-835
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• 2015

It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) ($SCF^{TIR1/AFB}$) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional $SCF^{TIR1/AFB}$ auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.134-139
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• 2021

Under physiological conditions, calcium (Ca²⁺) regulates essential functions of polarized secretory cells by the stimulation of specific Ca²⁺ signaling mechanisms, such as increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the store-operated Ca²⁺ entry (SOCE) and the receptor-operated Ca²⁺ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP₃) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca²⁺ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca²⁺ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2^-/-) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2^-/- mice. Furthermore, the response of Ca²⁺ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2^-/- mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca²⁺]_i via SOCE and ROCE in mouse pancreatic acinar cells.

• Childhood Kidney Diseases
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• v.19 no.2
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• pp.65-70
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• 2015

It is well known that proteins present in the primary urine are reabsorbed in the renal proximal tubules, and that this reabsorption is mediated via the megalin-cubilin complex and the neonatal $Fc{\gamma}$ receptor. However, the reabsorption is also thought to be influenced by an electrostatic interaction between protein molecules and the microvilli of the renal proximal tubules. By analyzing the charge diversity of urinary IgG, we showed that this reabsorption process occurs in a cationic charge-preferential manner. The charge-selective molecular sieving function of the glomerular capillary walls has long been a target of research since Brenner et al. demonstrated the existence of this function by a differential clearance study by using the anionic dextran sulfate polymer. However, conclusive evidence was not obtained when the study was performed using differential clearance of serum proteins. We noted that immunoglobulin (Ig) A and IgG have similar molecular sizes but distinct molecular isoelectric points. Therefore, we studied the differential clearance of these serum proteins (clearance IgA/clearance IgG) in podocyte diseases and glomerulonephritis. In addition, we studied this differential clearance in patients with Dent disease rather than in normal subjects because the glomerular sieving function is considered to be normal in subjects with Dent disease. Our results clearly showed that the charge-selective barrier is operational in Dent disease, impaired in podocyte disease, and lacking in glomerulonephritis.

• BMB Reports
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• v.44 no.6
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• pp.369-374
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• 2011

MHC-I molecules play a critical role in immune surveillance against viruses by presenting peptides to cytotoxic T lymphocytes. Although the mechanisms by which MHC-I molecules assemble and acquire peptides in the ER are well characterized, how MHC-I molecules traffic to the cell surface remains poorly understood. To identify novel proteins that regulate the intracellular transport of MHC-I molecules, MHC-I-interacting proteins were isolated by affinity purification, and their identity was determined by mass spectrometry. Among the identified MHC-I-associated proteins was Tmp21, the human ortholog of yeast Emp24p, which mediates the ER-Golgi trafficking of a subset of proteins. Here, we show that Tmp21 binds to human classical and non-classical MHC-I molecules. The Tmp21-MHC-I complex lacks ${\beta}_2$-microglobulin, and the number of the complexes is increased when free MHC-I heavy chains are more abundant. Taken together, these results suggest that Tmp21 is a novel protein that preferentially binds to ${\beta}_2$-microglobulin-free MHC-I heavy chains.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.3
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• pp.685-690
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• 1999

The insulin like growth factors(IGFs) are bound to several binding proteins(IGFBPs) that appear to regulate IGF transfort, receptor binding, and its action. The concentrations of these peptides are regulated by quantity and nutritional quality of dietary proteins. The aim of this study was to compare the effects of two diets, which differed in their protein source, Carassius carassius(CC), Carassius carassius hot water extract(CCHE), for 4 weeks. Body weight was significantly increased in the CC group(74.14$\pm$12.00 to 266.31$\pm$36.62g; p<0.01). Likewise, IGF I concentration of CC group(101.76$\pm$15.90 ng/ml) was significantly higher than that of CCHE group(38.50$\pm$ 11.20ng/ml; p<0.05). By western immunoblot analysis, especially IGFBP 1, 2 levels are increased, whereas IGFBP 3 level was de creased in CCHE group. After extraction of browning material from each samples, the extractive was filtered and absorbance at 420nm was measured. The absorbance of CCHE group was significantly higher than that of CC group. These results suggest that IGF I can be employed as an index of protein metabolism, particulary as a simple index in the assessing the status of protein nutrition.