• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.579-585
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• 2007

Formation of inclusion bodies is usually observed when foreign proteins are overexpressed in E. coli. The formation of inclusion bodies might be prevented by lowering the rate of protein synthesis, and appropriate regulation of the protein expression rate may lead to the soluble expression. In this study, human growth hormone (rhGH) was expressed in a soluble form by slowing down the protein synthesis rate, which was controlled in the transcriptional and translational levels. The transcriptional level was controlled by the regulation of the amount of RNA polymerase specific to the promoter in front of the rhGH gene. For lowering the rate of translation, the T7 transcription terminator-deleted vector was used to synthesize the longer mRNA of the target gene because the longer mRNA is expected to reduce the availability of tree ribosomes. In both methods, the percentage of soluble expression increased when the expression rate slowed down, and more than 93% of rhGH expressed was a soluble form in the T7 transcription terminator-deleted expression system.

• YAKHAK HOEJI
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• v.55 no.3
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• pp.203-212
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• 2011

Adipocytes have been known to secrete a number of important proteins called adipokines with roles in energy metabolism, reproduction, cardiovascular function and immunity. In this study we have attempted to identify intensively secretory proteins from 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into mature adipocytes and then the cells were left in serum-free medium. The supernatant was filtrated and dialyzed. Lyophilized secretome was fractionated by two different methods, 1-D SDS PAGE and RP-FPLC. The tryptic peptides from the gel slices and the FPLC fractions were analyzed by nanoLC/ESI-MS/MS. We identified a total of 303 identical proteins from two methods, 251 proteins from 1-D gel and 184 proteins from RP-FPLC. 86 of them were listed as a secretory protein Finally, we identified many known or unknown secreted proteins existed in the low level including adiponectin, angiotensinogen, bone morphogenetic protein-1 (BMP-1), macrophage migration inhibitory factor (MIF), insulin like growth factor-II (IGF-II), interleukin-6 (IL-6), follistatin-related protein-1, minecan, and resistin. The existence of some of secreted proteins has been confirmed in RNA level. This proteomic experiment is useful for the intensive screening of secretory proteins in many kinds of other cells.

• Molecules and Cells
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• v.26 no.4
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• pp.323-328
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• 2008

Eukaryotic cells continuously integrate intrinsic and extrinsic signals to adapt to the environment. When exposed to stressful conditions, cells activate compartment-specific adaptive responses. If these are insufficient, apoptosis ensues as an organismal defense line. The mechanisms that sense stress and set the transition from adaptive to maladaptive responses, activating apoptotic programs, are the subject of intense studies, also for their potential impact in cancer and degenerative disorders. In the former case, one would aim at lowering the threshold, in the latter instead to increase it. Protein synthesis, consuming energy for anabolic processes as well as for byproducts disposal, can be a significant source of stress, particularly when difficult-to-fold proteins are produced. Recent work from our and other laboratories on the differentiation of antibody secreting cells, revealed a regulatory circuit that integrates protein synthesis, secretion and degradation (proteostasis), into cell lifespan determination. The apoptotic elimination - after an industrious, yet short lifetime - of terminal immune effectors is crucial to maintain immune homeostasis. Linking proteostasis to cell death, this paradigm might prove useful for biotechnological purposes, and the design of novel anti-cancer therapies.

• Journal of Cancer Prevention
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• v.21 no.3
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• pp.135-143
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• 2016

Background: Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods: Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results: Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase $(AMPK){\alpha}$ and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and $AMPK{\alpha}$ abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions: Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or $AMPK{\alpha}/Nrf2$ pathway in HaCaT cells.

• International Journal of Molecular Medicine
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• v.43 no.6
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• pp.2516-2522
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• 2019

Particulate matter (PM), a widespread airborne contaminant, is a complex mixture of solid and liquid particles suspended in the air. Recent studies have demonstrated that PM induces oxidative stress and inflammatory reactions, and may cause certain skin diseases. Afzelin is a flavonoid isolated from Thesium chinense Turcz, which has anti-inflammatory, anticancer and antibacterial properties. Therefore, the present study aimed to investigate if afzelin affected inflammatory responses in human keratinocytes exposed to PM. HaCaT cells were treated with PM (25 ㎍/cm2) in the presence or absence of afzelin (200 µM). Here, standard reference material 1649b was used as PM. Cell viability was assessed using the water-soluble tetrazolium salt-1 assay. The generation of reactive oxygen species (ROS) was measured using the dichloro-dihydro-​fluorescein diacetate assay. Gene and protein expression were investigated using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Levels of secreted inflammatory cytokines were measured using ELISA. The results suggested that afzelin inhibited PM-induced proinflammatory cytokine mRNA expression and protein secretion in HaCaT cells. In addition, afzelin suppressed PM-induced intracellular ROS generation, and p38 mitogen-activated protein kinase and transcription factor activator protein-1 component c-Fos and c-Jun activation. The results indicated that afzelin exerts anti-inflammatory and antioxidant effects in PM-exposed HaCaT. Afzelin may have potential for preventing PM-induced inflammatory skin diseases.

• Food Science of Animal Resources
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• v.37 no.5
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• pp.617-625
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• 2017

The effects of replacing pork meat with yellow mealworms on the physicochemical properties and sensory characteristics of frankfurters were investigated in this study. The control (50% pork ham), T1 (45% pork ham + 5% yellow mealworm), T2 (40% pork ham + 10% yellow mealworm), T3 (35% pork ham + 15% yellow mealworm), T4 (30% pork ham + 20% yellow mealworm), T5 (25% pork ham + 25% yellow mealworm), and T6 (20% pork ham + 30% yellow mealworm) were prepared, replacing lean pork meat with yellow mealworm. The moisture content, lightness, sarcoplasmic protein solubility, hardness, gumminess, chewiness, and apparent viscosity of frankfurters with yellow mealworm were lower than those of the control (p<0.05), whereas the content of protein and ash, pH, and yellowness of frankfurters with yellow mealworm were higher than those of the control (p<0.05). The fat content of frankfurters in T1 (p<0.05) was the highest, and the fat content of treatments decreased with increasing yellow mealworm concentrations (p<0.05). Frankfurters with increasing yellow mealworm concentrations had lower color, flavor, off-flavor, and juiciness scores. The overall acceptability was not significantly different in the control, T1, and T2 (p>0.05). Thus, the results of this study showed that replacing lean pork meat with up to 10% yellow mealworm successfully maintained the quality of frankfurters at a level similar to that of the regular control frankfurters.

• Journal of Nutrition and Health
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• v.28 no.7
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• pp.585-594
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• 1995

Attempts were made to determine the effects of 5-Korean wild vegetabel consumptions on blood glucose leveles and orgen-energy metabolisms in streptozotocin-induced diabetic rats. The 5-Korean wild vegetables were : Cassia tora L.(C.t), Lycium chinese Mill(L.c), Trichosanthes kirilowii Max(T.k), Polygonatum odoratum var. Pluriflorum Ohwi(P.o) and Arctium lappa L(A.l). Sixty male Spargue-Dawley rats(160-220g) were induced diabetes mellitus by streptozotocin injection into the tail vein and were devided into 6 groups : a diabetic control and 5-experimental groups. All groups of the rats were fed on a AIN-76 diet, and the 5-experimental groups were fed with each wild vegetable (10%) for four weeks. An increased tendency in body weight of all the groups was observed and the tendency was more significant in L.c, T.k. and P.o. groups. The organ weight of liver and kidney were higher in L.c. and A.l. groups and lower in T.k. group which has shown the improvement from diabetes. Plasma glucose levels were markedly decreased from the 1st week in C.t, T.k. and P.o. groups and the tendency has lasted throughout the four weeks experimental period. The consumption of P.o. has decreased plasma cholesterol level while any significant difference was not seen in plasma protein levels from all the experimental groups. The level of plasma triglyceride was decreased in P.o. group and the levels of plasma free fatty acids were also significantly lower in P.o. and T.k. groups. The liver protein levels were significantly higher in P.o. and T.k. groups and these two groups also showed the negative or relatively small amount of urinary glucose excretion. The experimental group of T.k. has revealed the decreased level of muscles protein and the increased level of muscle glycogen. The 5-Korean wild vegetables contained dietary fiber and 9-analyzed minerals comparable to the ordinary use vegetables.

• International Journal of Oral Biology
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• v.34 no.1
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• pp.43-48
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• 2009

Thrombin-induced platelet microbicidal protein (tPMP) is a small cationic peptide that exerts potent in vitro microbicidal activity against a broad spectrum of human pathogens, including Staphylococcus aureus and Streptococcus rattus BHT. Earlier evidence has suggested that tPMP targets and disrupts the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacteria or whether subsequent, presumably intracellular, events are also involved in this process. In this study, we investigated the microbicidal activity of rabbit tPMP toward S. rattus BHT cells in the presence or absence of a pretreatment with antibiotics that differ in their mechanisms of action. The streptocidal effects of tPMP on control cells (no antibiotic pretreatment) were rapid and concentration-dependent. Pretreatment of S. rattus BHT cells with either penicillin or amoxicillin (inhibitors of bacterial cell wall synthesis) significantly enhanced the anti-S. rattus BHT effects of tPMP compared with the effects against the respective control cells over most tPMP concentration ranges tested. On the other hand, pretreatment of S. rattus BHT cells with tetracycline or doxycycline (30S ribosomal subunit inhibitors) significantly decreased the streptocidal effects of tPMP over a wide peptide concentration range. Furthermore, pretreatment with rifampin (an inhibitor of DNA-dependent RNA polymerase) essentially blocked the killing of S. rattus BHT by tPMP at most concentrations compared with the respective control cells. These results suggest that tPMP exerts anti-S. rattus BHT activity through mechanisms involving both the cell membrane and intracellular targets.

• Food Science of Animal Resources
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• v.38 no.6
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• pp.1203-1215
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• 2018

The antioxidant effects of Allium hookeri root (AHR) were investigated by evaluating lipid and protein oxidation in meatballs during refrigerated storage at $4{\pm}1^{\circ}C$. AHR was mixed at concentrations of 0.5% (w/w, T2) and 1% (w/w, T3) with minced longissimus dorsi muscle. Meatballs containing AHR (T2 and T3) were compared to those containing 0.05% (w/w) ascorbic acid (T1) as a reference and without antioxidant as a control. The 2-thiobarbituric acid reactive substances (TBARS) value, disulfide bond formation, carbonyl contents, and volatile basic nitrogen (VBN) value of T2 were lower than those of the control during storage (p<0.05). The pH values of T2 and T3 were higher than that of the control (p<0.05). Texture profile analysis of T2 revealed a lower value compared to the control (p<0.05). Therefore, the VBN value, TBARS value, disulfide bond formation, and carbonyl content in meatball containing AHR were lower than those of the control meatball. These results indicate that AHR improves the quality of meat products and functions as an antioxidant.

• BMB Reports
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• v.44 no.4
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• pp.279-284
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• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.