• Molecules and Cells
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• v.26 no.2
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• pp.140-145
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• 2008

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides ($OmpASP_{tr}$) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP ($OmpASP_{tr}$) and a factor Xa cleavage site (Xa) [$OmpASP_{tr}{\mid}(xxx){\mid}Xa$], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ HepI fusion proteins revealed that the transmembrane-like domains derived from the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.

• Biomolecules & Therapeutics
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• v.28 no.2
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• pp.163-171
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• 2020

Silibinin exhibits antidiabetic potential by preserving the mass and function of pancreatic β-cells through up-regulation of estrogen receptor-α (ERα) expression. However, the underlying protective mechanism of silibinin in pancreatic β-cells is still unclear. In the current study, we sought to determine whether ERα acts as the target of silibinin for the modulation of antioxidative response in pancreatic β-cells under high glucose and high fat conditions. Our in vivo study revealed that a 4-week oral administration of silibinin (100 mg/kg/day) decreased fasting blood glucose with a concurrent increase in levels of serum insulin in high-fat diet/streptozotocin-induced type 2 diabetic rats. Moreover, expression of ERα, NF-E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in pancreatic β-cells in pancreatic islets was increased by silibinin treatment. Accordingly, silibinin (10 μM) elevated viability, insulin biosynthesis, and insulin secretion of high glucose/palmitate-treated INS-1 cells accompanied by increased expression of ERα, Nrf2, and HO-1 as well as decreased reactive oxygen species production in vitro. Treatment using an ERα antagonist (MPP) in INS-1 cells or silencing ERα expression in INS-1 and NIT-1 cells with siRNA abolished the protective effects of silibinin. Our study suggests that silibinin activates the Nrf2-antioxidative pathways in pancreatic β-cells through regulation of ERα expression.

• BMB Reports
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• v.38 no.3
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• pp.328-333
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• 2005

A human homologue of Sar1, named Sara2, was shown to be preferentially expressed during erythropoiesis in a culture stimulated by EPO. Previous studies, in yeast, have shown that secretion-associated and Ras-related protein (Sar1p) plays an essential role in protein transport from the endoplasmic reticulum to the Golgi apparatus. Here, we report the molecular analysis of Sara2 in erythroid cell culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very similar to Sar1 proteins from other organisms, was obtained. Furthermore, we also report a functional study of Sara2 with Real-time quantitative PCR analysis, demonstrating that expression of Sara2 mRNA increases during the initial stages of erythroid differentiation with EPO and that a two-fold increase in expression occurs following the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was observed to have a constant expression and the addition of HU also up-regulated the expression in these cells. Our results suggest that Sara2 is an important gene in processes involving proliferation and differentiation and could be valuable for understanding the vesicular transport system during erythropoiesis.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.2
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• pp.95-111
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• 2022

Chronic obstructive pulmonary disease (COPD) is an important healthcare problem worldwide. Often, glucocorticoid (GC) resistance develops during COPD treatment. As a classic hypoglycemic drug, metformin (MET) can be used as a treatment strategy for COPD due to its anti-inflammatory and antioxidant effects, but its specific mechanism of action is not known. We aimed to clarify the role of MET on COPD and cigarette smoke extract (CSE)-induced GC resistance. Through establishment of a COPD model in rats, we found that MET could improve lung function, reduce pathological injury, as well as reduce the level of inflammation and oxidative stress in COPD, and upregulate expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), multidrug resistance protein 1 (MRP1), and histone deacetylase 2 (HDAC2). By establishing a model of GC resistance in human bronchial epithelial cells stimulated by CSE, we found that MET reduced secretion of interleukin-8, and could upregulate expression of Nrf2, HO-1, MRP1, and HDAC2. MET could also increase the inhibition of MRP1 efflux by MK571 significantly, and increase expression of HDAC2 mRNA and protein. In conclusion, MET may upregulate MRP1 expression by activating the Nrf2/HO-1 signaling pathway, and then regulate expression of HDAC2 protein to reduce GC resistance.

• Molecules and Cells
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• v.27 no.5
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• pp.577-582
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• 2009

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon ${\gamma}$, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

• The Journal of the Korean Society for Microbiology
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• v.35 no.1
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• pp.41-48
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• 2000

Bacteroides fragilis and Escherichia coli, normal colonic inhabitants, are the most frequently isolated bacteria in infected tissues, particularly in intraabdominal abscesses. This study was designed to determine whether enteric bacteria may alter the B. fragilis-induced expression of pro inflammatory cytokines in mouse peritoneal tissue (MPT). After C57BL/6 mice were inoculated with abscess-forming mixture containing B. fragilis in the presence or absence of E. coli, RNA was extracted from MPT. Expression of interleukin (IL)-$1{\alpha}$ and tumor necrosis factor $(TNF){\alpha}$ mRNA was assessed using RT-PCR and standard RNA. Each cytokine protein was also measured by ELISA. The co-inoculation of E. coli into mouse peritoneal cavity advanced the onset of abscess development by B. fragilis infection. When mouse was co-infected with E. coli and B. fragilis intraperitoneally, there was a synergistic increase in the expression of IL-$1{\alpha}$ and $TNF{\alpha}$ mRNA in MPT and this was paralleled by increased cytokine protein secretion. Mixed inoculation of heat-killed E. coli and B. fragilis did not cause a synergistic increase in those cytokine mRNA expression. These results suggest that enteric bacteria may significantly affect proinflammatory cytokine signal produced by host peritoneal cavity in response to B. fragilis infection.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.425-435
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• 2024

Schisandra chinensis extract (SCE) protects against hypocholesterolemia by inhibiting proprotein convertase subtilisin/kexin 9 (PCSK9) protein stabilization. We hypothesized that the hypocholesterolemic activity of SCE can be attributable to upregulation of the PCSK9 inhibition-associated low-density lipoprotein receptor (LDLR). Male mice were fed a low-fat diet or a Western diet (WD) containing SCE at 1% for 12 weeks. WD increased final body weight and blood LDL cholesterol levels as well as alanine transaminase and aspartate aminotransferase expression. However, SCE supplementation significantly attenuated the increase in blood markers caused by WD. SCE also attenuated WD-mediated increases in hepatic LDLR protein expression in the obese mice. In addition, SCE increased LDLR protein expression and attenuated cellular PCSK9 levels in HepG2 cells supplemented with delipidated serum (DLPS). Non-toxic concentrations of schisandrin A (SA), one of the active components of SCE, significantly increased LDLR expression and tended to decrease PCSK9 protein levels in DLPS-treated HepG2 cells. High levels of SA-mediated PCSK9 attenuation was not attributable to reduced PCSK9 gene expression, but was associated with free PCSK9 protein degradation in this cell model. Our findings show that PCSK9 secretion can be significantly reduced by SA treatment, contributing to reductions in free cholesterol levels.

• Journal of Life Science
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• v.14 no.3
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• pp.391-395
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• 2004

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

• The Journal of Internal Korean Medicine
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• v.27 no.4
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• pp.864-878
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• 2006

Purpose : Panax ginseng(PG) is considered to have salutary effects and stimulant actions on physical capacity. However, the effects of PG on the inflammatory cytokine secretion and hypoxia condition are still not understood. This study wasto elucidate the effect of PG on inflammatory cytokine secretion such as interleukin (IL)-1, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor $(TNF)-{\alpha}$. Also, the effects on the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) were measured. Methods : The water extract of PG was administrated to HMC-1 cells before phorbol myristate acetate (PMA)+A23187 treatment. $IL-1{\beta}$, IL-6, $TNF-{\alpha}$, GM-CSF, and VEGF secretion were measured by a modified enzyme-linked immunosorbent assay (ELISA). HIF-1 activation was measured by transcription factor enzyme-linked immunoassay (TF-EIA) Results : PG significantly decreased secretion of $IL-1{\beta}$, IL-6, $TNF-{\alpha}$, and GM-CSF in PMA+A23187-induced HMC-1 cells. VEGF secretion was not changed but HIF-1 activation was decreased by the treatment of PG. Conclusions : PG inhibited the secretion of inflammatory cytokines, which impliesPG might contribute to treatment of mast cell-mediated inflammatory disease. Also, PG inhibited PMA+A23187-induced $HIF -1{\alpha}activation}$ and DNA-binding activity for HIF-1. Therefore, these data demonstrate that PG modulates inflammatory cytokines through inhibition of $HIF-1{\alpha}activation}$ activation.

• Nutrition Research and Practice
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• v.10 no.1
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• pp.11-18
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• 2016

BACKGROUND/OBJECTIVES: Type 2 diabetes (T2D) is more frequently diagnosed and is characterized by hyperglycemia and insulin resistance. $\small{D}$-xylose, a sucrase inhibitor, may be useful as a functional sugar complement to inhibit increases in blood glucose levels. The objective of this study was to investigate the anti-diabetic effects of $\small{D}$-xylose both in vitro and stretpozotocin (STZ)-nicotinamide (NA)-induced models in vivo. MATERIALS/METHODS: Wistar rats were divided into the following groups: (i) normal control; (ii) diabetic control; (iii) diabetic rats supplemented with a diet where 5% of the total sucrose content in the diet was replaced with $\small{D}$-xylose; and (iv) diabetic rats supplemented with a diet where 10% of the total sucrose content in the diet was replaced with $\small{D}$-xylose. These groups were maintained for two weeks. The effects of $\small{D}$-xylose on blood glucose levels were examined using oral glucose tolerance test, insulin secretion assays, histology of liver and pancreas tissues, and analysis of phosphoenolpyruvate carboxylase (PEPCK) expression in liver tissues of a STZ-NA-induced experimental rat model. Levels of glucose uptake and insulin secretion by differentiated C2C12 muscle cells and INS-1 pancreatic ${\beta}$-cells were analyzed. RESULTS: In vivo, $\small{D}$-xylose supplementation significantly reduced fasting serum glucose levels (P < 0.05), it slightly reduced the area under the glucose curve, and increased insulin levels compared to the diabetic controls. $\small{D}$-xylose supplementation enhanced the regeneration of pancreas tissue and improved the arrangement of hepatocytes compared to the diabetic controls. Lower levels of PEPCK were detected in the liver tissues of $\small{D}$-xylose-supplemented rats (P < 0.05). In vitro, both 2-NBDG uptake by C2C12 cells and insulin secretion by INS-1 cells were increased with $\small{D}$-xylose supplementation in a dose-dependent manner compared to treatment with glucose alone. CONCLUSIONS: In this study, $\small{D}$-xylose exerted anti-diabetic effects in vivo by regulating blood glucose levels via regeneration of damaged pancreas and liver tissues and regulation of PEPCK, a key rate-limiting enzyme in the process of gluconeogenesis. In vitro, $\small{D}$-xylose induced the uptake of glucose by muscle cells and the secretion of insulin cells by ${\beta}$-cells. These mechanistic insights will facilitate the development of highly effective strategy for T2D.