• Food Science and Biotechnology
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• v.16 no.3
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• pp.343-348
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• 2007

Starch contents play important roles in determining the fruit quality. Stawberry accumulates starch in the early stages and then mobilized into soluble sugars during fruit ripening. To date the molecular studies on the ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, were not reported. cDNAs encoding small (FagpS) and large (FagpL1 and FaspL2) AGPase subunits were isolated from strawberry (Fragaria ${\times}$ ananassa Duch. cv. Niyobou). Both FagpS and FagpL1 cDNAs have open reading frames deriving 55-58 kDa polypeptides, where FagpL2 contains a partial fragment. Sequence analyses showed that FagpS has a glutamate-threonine-cysteine-leucine (ETCL) instead of a glutamine-threonine-cysteine-leucine (QTCL) motif found in all the dicot plants except for Citrus. In fruits, FagpS and FagpL1 were expressed in all stages with a little change in the amounts of transcripts. In the case of FagpL2, we were not able to detect any signal from all stages of fruit development and all tissues except for very a weak signal from the leaf. The results indicate that FagpL1 and FagpL2 show ubiquitous and leaf-specific expression patterns, respectively. The studies suggest that the starch contents in strawberry might be controlled by the expression of AGPase gene at both the transcriptional and post-transcriptional levels during fruit development.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1620-1627
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• 2009

The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.

• Preventive Nutrition and Food Science
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• v.22 no.1
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• pp.50-55
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• 2017

Raphanus sativus L. (RS) is a cruciferous vegetable that is widely consumed in Korea. The anticancer activity of leaves of RS (RSL) extract has been investigated; however, no studies focused on its anti-inflammatory effects. Therefore, the aim of the current study was to evaluate the anti-inflammatory effects of RSL extract. In brief, RSL powder was fractionated into n-hexane, chloroform, ethyl acetate, n-butanol, and water-soluble fractions. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were treated with each fraction for initial screening. It was found that the chloroform fraction significantly inhibited nitric oxide release in LPS-stimulated RAW264.7 cells with a half maximal inhibitory concentration value of $196{\mu}g/mL$. In addition, the mRNA and protein expression levels of inducible nitric oxide synthase, measured using reverse transcriptase-polymerase chain reaction and western blotting, respectively, were reduced in a concentration-dependent manner. Moreover, the inflammatory cyclooxygenase-2 enzyme expression decreased. Furthermore, the expression of nuclear factor-kappa B ($NF-{\kappa}B$), the key regulator of the transcriptional activation of the inflammatory cytokine genes, was reduced by the RSL chloroform fraction. Therefore, the results of our study suggest that RSL exhibits anti-inflammatory effects in LPS-stimulated macrophages via $NF-{\kappa}B$ inactivation.

• Toxicological Research
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• v.19 no.1
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• pp.67-72
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• 2003

Cholestatic liver injury results from the accumulation of toxic bile salts within the liver. The aim of the present study is to elucidate the changes in expression and cellular localization of apoptosis related proteins in the liver of bile duct-ligated (BDL) rat. Extrahepatic cholestasis was induced by double ligation of the common bile duct and cut between the ligatures. Animals were sacrificed at day 3 and at week 1, 2, 4, 6, and 8 after BDL. The number of TUNEL positive cells was increased significantly after 3 days of BDL, decreased over 2 weeks and remained constant thereafter. Fas expression was not changed and activation of caspase 8 did not occur. Fas immunoreactivity was exclusively observed in the cytoplasm of hepatocytes, indicating that Fas expressed in rat hepatocytes is a soluble form. Hepatocyte apoptosis was associated with Bax expression, which showed a peak at day 3 and decreased over time gradually. Immnunostaining of Bax was observed in hepatocytes and bile duct epithelial cells (BEC) of control and BDL rats. Bcl-2 was increased over time in BDL rats. These results suggest that apoptosis of hepatocytes in BDL rats is independent of Fas and controlled by Bax expression.

• International Journal of Industrial Entomology and Biomaterials
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• v.32 no.2
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• pp.90-97
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• 2016

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 4, 5, and 6. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the ORF4, ORF5, and ORF6 with SUMO (small ubiquitin-related modifier). The resulting fusion protein SUMO-ORF4, -ORF5, and -ORF6 were highly expressed in Bm5 cells. The level of protein expression using the Bombyx mori larvae was higher than that using Bm5 cells. In addition, fusion to SUMOstar, which is not processed by native SUMO proteases, significantly enhanced protein expression levels compared to SUMO fusion. This study demonstrated that SUMO or SUMOstar, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.556-561
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• 2010

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

• Journal of Marine Life Science
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• v.2 no.1
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• pp.7-11
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• 2017

Sulfated polysaccharides are known to be immune-stimulators in mammals and can be used as food additives to enhance immunity. In this study, the immune-stimulating activity of water-soluble anionic macromolecules F₂ fractionation isolated from Codium fragile using ion-exchange chromatography was tested in olive flounder, Paralichythys olivaceus, in vitro and in vivo. The gene expression of interleukin (IL)-1β was adopted to check the immune-affection. As a result, in vitro study revealed that the expression of IL-1β was significantly upregulated in head kidney cells by 1 and 5 ㎍/ml of polysaccharides 4 h and by 5 ㎍/ml of polysaccharides at 24 h. In vivo, IL-1β gene expression in head kidney was significantly upregulated by 20 and 100 ㎍ of the polysaccharides at day 1 post-i.p. injection, while downregulated at day 3 but not significant. Meanwhile, in peritoneal cells, it was upregulated by 20 ㎍ of the polysaccharides at day 1 but the upregulation was sustained until day 3 though it was not significant. These results indicate that the sulfated polysaccharides from C. fragile are an immune-stimulator and might be potential feed additives for olive flounder.

• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.12-25
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• 2003

L-camitine ($\beta$ -hydroxy-${\gamma}$ -trimethyl-ammoniumbutyric acid) is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-camitine, we investigated the effects of in vitro MMP inhibition and activity and expression of UVA-induced MMP 1 in human skin fibroblasts. Fluorometric assays of the proteolytic activities of MMP-l were performed using fluorescent collagen substrates. ELISA (enzyme linked immuno sorbent assay), gelatin-substrate zymography, and RT-PCR ELISA techniques were used for the effects of L-camitine on MMP expression and activity, MMP mRNA expression in UVA irradiated fibroblast. L-camitine inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 2.45mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP expression was reduced 40% by treated with L-carnitine, and MMP-l mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibition the MMP activity, regulation of MMP expression in protein and mRNA level. All these results suggest that L-carnitine may be useful as new anti-aging cofactor for protection against UVA induced MMP expression and activity.

• Journal of Breast Cancer
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• v.21 no.4
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• pp.399-405
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• 2018

Purpose: Vesicle-associated membrane protein 8 (VAMP8) is a soluble N-ethylmaleimide-sensitive factor receptor protein that participates in autophagy by directly regulating autophagosome membrane fusion and has been reported to be involved in tumor progression. Nevertheless, the expression and prognostic value of VAMP8 in breast cancer (BC) remain unknown. This study aimed to evaluate the clinical significance and biological function of VAMP8 in BC. Methods: A total of 112 BC samples and 30 normal mammary gland samples were collected. The expression of VAMP8 was assessed in both BC tissues and normal mammary gland tissues via a two-step immunohistochemical detection method. Results: The expression of VAMP8 in BC tissues was significantly higher than that in normal breast tissues. Furthermore, increased VAMP8 expression was significantly correlated with tumor size (p=0.007), lymph node metastasis (p=0.024) and recurrence (p=0.001). Patients with high VAMP8 expression had significantly lower cumulative recurrence-free survival and overall survival (p<0.001 for both) than patients with low VAMP8 expression. In multivariate logistic regression and Cox regression analyses, lymph node metastasis and VAMP8 expression were independent prognostic factors for BC. Conclusion: VAMP8 is significantly upregulated in human BC tissues and can thus be a practical and potentially effective surrogate marker for survival in BC patients.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.