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Mesenchymal stem cells (MSCs) are new cell-therapeutic solution due to their capacity that differentiate into cells of connective tissues such as bone, cartilage, adipocyte, tendon and neural cells. MSCs are capable of self-renewing and have the multipotential nature. MSCs characterized were: (1) MSCs have to be plastic-adherent if maintained in standard culture condition (2) MSCs have to capability to differentiation various types other tissue. We are looking for maintain stemness of MSCs can reduce the aging conditions in the expansion media, the experimental groups: (1) general meia (2) general media with antioxidants (3) commercial media. Stemness analysis was passage 5 and passage 10 in the osteogenesis, chondrogenesis, adipogenesis, neurogenesis and differentiation potential have been confirmed. As a results, MSCs in antioxidant-added group had similar activities to commercial media, but those in general media did not. Therefore, it is strongly supposed that antioxidants play a crucial role in maintainin.H.

Among various elements that influence axonal outgrowth in neural cells in vitro, cell-adhesion is one of the most important factors. To improve cell adhesion and neural differentiation, several experiments have explored the effects of the adhesive surface characters. Polydopamine has the potential to be used as a cell- or tissue-adhesive. Surface modification by polydopamine-coating is extremely useful because it does not require the time consuming synthesis of complex linkers and the process is solvent-free and biocompatible. We hypothesized that polydopamine coated surface would enhance neural cell viability and neural differentiation through improving cell adhesion. To test the hypothesis, cell viability and neural differentiation of pheochromocytoma 12 (PC12) cells cultured on polydopamine-coated glass coverslips were examined. Compared to other conventionally produced cell-adhesion material such as gelatin, Polydopamine showed enhanced cell adhesion, which is important for cell viability, as well as better cell differentiation. Polydopamine could be a suitable cell-adhesion material for cell culture or tissue engineering..

TypeⅠ diabetes is caused by progressive autoimmune destruction of insulin-producing cells (IPCs). Various kinds of stem cells can be used for the preparation IPCs. However, application of stem cells for cell therapy is usually dependent upon donor ages. Accordingly, the growth and differentiation potential of periosteum-derived progenitor cells (PDPCs) obtained from aged fifty, sixty, and seventy into IPCs was investigated in this study. PDPCs have been known to be another kind of mesenchymal stem cells (MSCs) and are expected to be used as a cell source to make IPCs. Our results demonstrated that the cell number, viability and morphology of PCPCs were not age-dependent. Immunofluorescence analysis of b-cell marker (insulin) demonstrated no significant differences in the differentiation by donor age. Insulin was detectable at mRNA levels after induction. Moreover, induced PDPCs could release insulin in response to glucose in vitro. In conclusion, it was found that the differentiation capability of PDPCs into IPCs was not affected by donor ages.

Bone marrow mononuclear cells (BMMNCs) can be used to treat patients with myocardial infarction, since BMMNCs can differentiate in vitro toward cardiomyogenic lineages when treated with transforming growth factor-β1 (TGF-β1). In this study, we hypothesize that BMMNCs implanted in cardiomyogenically undifferentiated state to myocardial infarction site would differentiate cardiomyogenically in situ when exogenous TGF-β1 is delivered to the cell implantation site. Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) suspended in fibrin gel were used as a TGF-β1 delivery system. BMMNCs were labeled with a green fluorescent dye (PKH-67) and implanted into the rat myocardium using fibrin gel containing HCPNs and TGF-β1. Four weeks after implantation, the expression of cardiomyogenic marker proteins by the implanted BMMNCs was dramatically greater in the TGF-β1 delivery group than in the control group. This method can significantly improve the stem cell therapy technology for myocardial regeneration, since it can remove in vitro cell culture step for cardiomyogenic differentiation prior to cell implantation.

The immobilization of biomolecules on biomaterials is a widely researched to control cell and tissue responses. We experimented to find the most effective calcium chelating agents which can enhance adhesion of human BM-MSCs on nano-hydroxyapatite (nHAp) coated titanium substrates by having covalent immobilization of BMP-2. The nHAp solution was spin-coated onto titanium disc surface and all samples were totally immersed in 1 mg/ml calcium chelating agent for 4 hours. The chitosan seemed to hold many BMP-2 because of plentiful amine groups by performing the BMP-2 assay. We confirmed that toxicity of alendronate by measuring the BrdU assay of T-lymphocyte cultured with modified titanium and TNF-α assay. BM-MSCs on chitosan calcium chelated nHAp surface and those on chitosan calcium chelated BMP-2 immobilized nHAp surface seemed to be healthy because of showing spindle-like fibroblastic morphology. We suggest that the chitosan can be used as an effective calcium chelating agent for implant.

Transplantation of beta cells is considered to be the best treatment for type 1 diabetes. However, due to the shortage of donor pancreas, the generation of insulin-producing cells (IPCs) from various stem cells has been studied as an alternative source of beta cells. In previous study, we accomplished the differentiation of periosteum-derived progenitor cells (PDPCs) known as a kind of mesenchymal stem cells (MSCs) into IPCs. During the differentiation, decrease of cell viability has been a problem to be overcome. In order to be used in transplantation in vivo, this problem should be solved to improve the viability and feasibility. For this reason, we screened various antioxidants to find an efficacy in enhancing the viability by adding into the culture medium. MTT assay was used for evaluating toxicity of various antioxidants. Viable cells were counted by using trypan blue dye exclusion method. It was found that the addition of antioxidants during the differentiation improve the viability of the cells in culture.

Magnetic field (MF) therapy is an emerging method of treatment for numerous neurodegenerative diseases. However, the exact mechanism of how and to what degree a magnetic field affects neurological function remains controversial. In Alzheimer’ disease, hyperphosphorylation of tau protein is known as one of the main causes of neurofibilary tangles leading to neuronal death. In this study, effects of a range of static magnetic fields (SMF) and electromagnetic fields (EMF) on phosphorylation of tau protein have been investigated. PC12 cells treated with nerve growth factor (NGF) have been exposed to SMF of 3 mT, 15 mT, and 170 mT and an EMF of 1 mT at 50 Hz for 4 days. Tau protein phosphorylation level of each sample was examined, and the results indicated that the phosphorylation level increased with low MF exposures (1, 3 mT) and decreased with high MF exposures (15, 170 mT) when compared with the control. To examine more specifically where this effect takes place, expression levels of some of the upstream genes related to tau phosphorylation have been observed. This study suggests that MF may open up a new venue for the treatment of Alzheimer’ disease.

The essential operating parameters in virus vaccine production are multiplicity of infection (MOI), harvest time, and infection time. Stimulating agents also can be applied in order to improve vaccine productivity further. We investigated the optimum operating conditions in porcine transmissible gastroenteritis virus (TGEV) vaccine production and the applicability of sodium butyrate (NaBu) as a stimulating agents for the improvement of vaccine productivity. The optimum MOI, infection time, and harvest time for high production of TGEV by swine testicle (ST) cells were found to be 0.0001 pfu/cell, 3 day after cell inoculation, and 24 hpi, respectively. NaBu is known as a histone deacetylase inhibitor that has been widely used for the high expression of recombinant protein using mammalian cells and for the enhancement of virus propagation. So we tried to examine the potential of NaBu as a stimulating agent and to determine the optimum concentration by comparing TGEV titers with different range of NaBu concentration. TGEV titer with 5 mM NaBu was 1.5 times higher than control. Therefore, we concluded that NaBu can be a promising agent for stimulating various vaccine production including TGEV and the optimum NaBu concentration for TGEV production was determined to be 5 mM.

Traditionally, mushrooms are widely used as nutritional supplements and encouraged as to be beneficial for health. Inonotus Obliquus is known to not only anti-autoimmune diseases effect but also anti-oxdative stress effect. Damage induced by oxidative stress is associated with intracellular ROS. Reactive oxygen species (ROS) are known to prime mediators of cell damage by oxidative stress. Excess production of intracellular ROS brings about on cellular tissues oxidation. Oxidation of cellular tissues leads to diseases such as aging, arteriosclerosis, inflammation, rheumarthritis and diabetes. From among these diseases, diabetes was occurred by loss of pancreatic function (destruction of pancreatic beta-cells). In this study, we investigated cytoprotect and anti-oxidative effect on mouse insulin-producing pancreatic beta-cells (MIN6N) by hot water extracts from Inonotus Obliquus.

Maintenance of mitochondria integrity by overexpression of Bcl-2, a typical anti-apoptotic protein, is a critical point in recombinant Chinese hamster ovary (rCHO) cell culture treated with sodium butyrate (NaBu) known as a typical specific productivity-enhancing factor. Bcl-2 distributes and functions in multiple intracellular organelles such as nucleus, mitochondria, and ER. To evaluate the effect of organelle -specific overexpression of Bcl-2 on NaBu-induced apoptosis, Bcl-2 expression was restricted at mitochondria or ER by employing mitochondrial insertion sequence of ActA or ER-specific sequence of cytochrome b5 to its sequence. The rCHO cell lines over -expressing mitochondrial Bcl-2 (MT-Bcl-2) or ER-targeted Bcl-2 (ER-Bcl-2) were established. Overexpression of MT-Bcl-2 or ER-Bcl-2 could increase cell viability and decrease LDH release under NaBu-treated condition. Also, it could suppress NaBu-induced apoptosis as evidenced from DNA fragmentation assay. Mitochondria membrane potential assay revealed that ER-Bcl-2 overexpression can maintain the mitochondria membrane integrity without helping MT-Bcl-2 overexpression, indicating the role of ER should be considered in alleviating NaBu-induced apoptosis by genetic modulation strategy. Taken together, it was found that restricted Bcl-2 overexpression at ER could inhibit the NaBu-induced apoptosis via maintaining mitochondria integrity in rCHO cells.

Cyclo (His-Pro) (CHP) is a naturally occurring cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). The major goal of the current study was to determine whether the CHP plays important role in Type I diabetes. The experiment was designed to investigate the effect of CHP on glucose metabolism in vivo (Type I diabetes model). CHP (approx. 11 mg/g of dry weight) was efficiently obtained from soybean meal by proteolytic hydrolysis using the mixture of Flavourzyme and Alcalase. When Type I rats (Sprague-Dawley) induced with streptozotocin (50 mg/kg BW) were administrated by CHP (4 mg/kg BW), blood glucose level in the CHP-fed rats was remarkably decreased by approximately 56% as compared to the diabetes group. In the oral glucose tolerance test, blood glucose levels were restored to its baseline after 120 min by CHP treatment, although blood glucose levels increased significantly after 30 min. Insulin level in plasma was also restored by 29%, compared to the STZ-treated rat and triglyceride levels were normalized in CHP-treated rats, whereas no effects of CHP treatment on cholesterol level were observed. Moreover, a histopathological examination showed that CHP administration could restore the impaired β-cells of pancreas to the normal level. Accordingly, the CHP exhibited an excellent hypoglycemic effect by lowering average plasma glucose levels, increasing insulin secretion, and restoring viability of pancreatic β-cells in diabetic rats. Considering these results, we suggest that the CHP might be a candidate material to control Type I diabetes mellitus.

Classical swine fever virus causes significant economical losses in pig industry in many countries. So the development of porcine cholera marker vaccine is urgently required. The baculovirus-insect cell system has been established as a powerful technology for the production of high value recombinant proteins. Baculovirus is safe virus because it has a limited host range, and it produces heterologous recombinant protein under polyhedrin promoter. Insect cells are an attractive host for recombinant protein expression via infection with a genetically modified baculovirus as this system allows for high levels of production of proteins that are functionally similar to the native protein. So insect cell-baculovirus vector system was used in this study for producing porcine cholera maker vaccine. The effects of agitation speed and infection time, pH on marker vaccine production were investigated in each case of individual cell line. As a result, the highest productivity of porcine cholera marker vaccine achieved was 61.26 mg/L at the condition of 1 day infection time with sf21 cells. And highest productivity of porcine cholera marker vaccine achieved was 169.29 μ g/mL at the condition of 100 rpm with sf9 cell. The productivity of 194.74 mg/L was obtained by the sf9 cell in case of pH shift from 6.1 to pH 6.5.

Primary human hepatocytes are clinically used for transplantation or in bioartificial liver devices for the treatment of patients with liver failure. Bioartificial liver support systems (BALSS), which employ freshly isolated primary hepatocytes, presently severe logistic difficulties in the continuous supply of primary hepatocytes. Cryopreserved primary hepatocytes that are thawed as required would answer this problem.1)-3) Investigation was carried out with different concentrations of dimethyl sulfoxide (DMSO) with hormonally-defined medium (HDM). Various solutions of histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS) and sucrose, were used as cryopreservation solutions. In this study, initial dispersed hepatocytes. After thawing viability, plating efficiency, urea synthesis and albumin secretion were measured to assess the effects of freezing methods. The result shows that cryopreserved thawed hepatocytes with HTK solutions enhance hepatocytes cell viability and cell attachement,hence good for hepatocyte cryopreservation. HTK solution in presence of 200mM sucrose showed significantly increase in urea secreation, on the other hand substitution of BME amino acids and BME vitamins does not show any significant.

Cyclo (His-Pro) (CHP) is a naturally occurring cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). CHP was efficiently obtained from soybean meal by hydrolysis using flavourzyme and alcalase. In this study, anti-diabetic effects of CHP were investigated in a Type II diabetic animal model (C57BL/6J ob/ob). When the ob/ob mice were administrated with CHP (2 mg/kg BW, 24 days), blood glucose levels decreased to approximately 57% that of the control mice. In the oral glucose tolerance test, blood glucose levels were restored to its baseline after 120 min by CHP treatment, although blood glucose levels increased significantly after 30 min. Total plasma cholesterol and triglyceride concentrations in ob/ob mice treated with CHP were significantly decreased (2-fold), compared with those in control ob/ob mice. In contrast, insulin levels in plasma were significantly increased (2-fold) in CHP-treated ob/ob mice. Moreover, a histopathological examination showed that CHP administration could restore the impaired β -cells of pancreas to the normal level. In addition, pancreatic β-cells (in CHP-treated and stopped groups) were strikingly restored on 72nd and 130th day, respectively. From these results, it is demonstrated that CHP could be effective for regulating blood glucose to normal level by increasing plasma insulin level in ob/ob mice. Conclusively, we suggest that this compound could be a candidate material as a dietary supplement to control hyperglycemia in patients suffering from type II diabetes.

Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic plant cell lines (Oryza sativa L.) using RAmy3D promoter. Successful production of proteins in bioreactor requires sufficient oxygen transfer into the culture medium. However, shear damage by agitation and aeration in induction phase causes cell lysis and consequently lowers productivity. Culture fluid after the beginning of cell lysis contains diverse endogenous enzymes, such as proteases and nucleases, which are capable of degrading the secreted proteins in the culture medium. In this study, to identify the types of secreted proteases, various protease inhibitors such as AEBSF, bestatin, E-64, 1,10- phenanthroline, pepstatin A and protease inhibitor cocktail were tested under the same conditions. Effects of protease inhibitors at diverse corresponding concentrations were confirmed by comparing the levels of target protein, degree of protein degradation and protease activity.

Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. To increase productivity of rCHO cells, gene amplification system, adding stepwise increment of methotrexate, is generally utilized. The clone did not show im proved productivity proportional to increased MTX concentration, which can be called as rate-limiting step. We speculated that promoter methylation may be one reason, which limits full production capacity. Generally, in mammalian cells, cytomegalovirus (CMV) promoter is used for transcription of gene of interest. 36 CpG sites were identified within CMV promoter, which are candidates for de novo methylation. Here, we hypothesize that methylationat CMV promoter might lead to reduced gene transcription. It is well known that methylation of DNA inhibits gene expression. The results indicated that specific productivity decreased at 320nM amplified clone compared to 80 nM amplified clone. Significant methylation difference at CMV promoter was identified between parental cloneand MTX amplified clones. For future studies, we will focus on correlating promoter methylation with transcription level. To support the hypothesis, we will analyze gene copy number through real-time PCR. Taken together, we provide first hints that amplified gene copy number could be silenced by promoter methylation, which can be proved by reduced mRNA transcription.

Due to the need for a faster and cheaper approach for therapeutic protein production, Transient gene expression (TGE) system has been highlighted. TGE can also serve as a rapid mammalian protein expression system for the evaluation of therapeutic protein candidates and subsequent high-throughput screening, without the lengthy time periods and resources required for stable cell line development. While CHO cells have been the most widely used mammalian host in the field of biotechnology industry, TGE in CHO cells has been hampered by low titers. In this report, episomal plasmid replication in CHO cells was accomplished by the use of polyomavirus large T-antigen (PyLT)/ori system. CHO DG44 cell line was stably tranasfected with the PyLT gene and Polyomavirus origin was inserted to expression vector for autonomous replication. Also, Epstein-Barr Virus nuclear antigen-1 and OriP were encoded in expression vector for plasmid retention. With PyLT/ori system, CHO cells were engineered to inducibly overexpress anti apoptotic protein Bcl-xL using the doxycycline Tet-off system. Culture longevity was enhanced by overexpression of Bcl-xL in fed-batch culture and under NaBu added condition causing enhancement of a recombinant protein production. The results demonstrate the use of Bcl-xL was combined with the PyLT/ori system can enhance the production of recombinant protein by transient expression in CHO cells.

In animal cell culture aurintricarboxylic acid (ATA) effects on antiaggregation, anti-apoptosis, and it acts insulin-like growth factor under serum free condition. ATA is used in biological experiments as a protein synthesis inhibitor. In the meantime, we found another function of ATA that it can decrease antibody production titer in serum free condition. Addition and deletion of ATA experiments showed that it is important component in antibody production inhibition. We processed with two different medium, PF CHO MPS (Hyclone, commercial medium) and Protein free medium (In-house medium) and it showed similar results that antibody production titer decreased in ATA contained condition from 40~50 mg/L to below 10 mg/L.In short, ATA can be an antibody production inhibitor in some specific serum free condition.

To alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of unfolded protein response, was attempted. The functioning of the stably GADD153-silenced clones under four ER stress inducing conditions were investigated. Under thapsigargin and brefeldin A treatment, the GADD153-silenced clones showed lesser incidence of apoptosis (about 38%) and lesser non-viable cells (about 58%) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153 induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the mechanism revealed that elevated GADD153 expression results in the exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, the data presented here suggest that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidant injury and by manipulating the ER client protein load in rCHO cells.

Animal cells are sensitive to both DO and shear stress. First of all, we investigated O2 transfer rate and shear stress of bubble column bioreactors. In the reactors, kLa increased with aeration rate and decreased with the size of bubbles. As far as the bubble size is concerned, small bubbles showed higher O2 transfer than large bubbles. Small bubbles, however, caused shear stress problem that damaged CHO cells. Similar phenomenon was observed in shaking flasks. In well plates or T-flasks, O2 transfer rate was higher in twist-shaking than in tilted-shaking. However, cell growth and viability of twisted flasks were lower than that of tilted ones. At the same time, it was observed that cells were aggregated more in twisting than in tilting. In order to investigate the shear stress marker of CHO cells, 2-D analysis was performed. HSP90-β and G protein could be included in several proteins that might be used as anchorage or shear index proteins. Relationship between oxygen transfer efficiency and shear stress will be presented in this paper.

Phage display has now become well established technology and enabled construction and selection of large size of antibody library in vitro. Millions of candidate clones are commonly obtained following rounds of phage-displayed library panning, and labor-intensive expression of those selected clones is required for secondary screening to identify functionally positive clones with therapeutic potential. Here, we designed integrated vector system for phage display and mammalian expression for rapid expression and characterization of selected antibody clones from library. We took advantage of mammalian/bacterial hybrid signal peptide for mammalian expression which accommodates compatible restriction enzyme sites with bacterial signal peptide, which enables the direct transfer of even unknown antibody sequences from phagemid to mammalian expression vector. Its utility was demonstrated for antibody library selection against several targets. First, the vector system was applied for the construction and selection of antibody library recognizing cancer-related aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1). The selected antibody clones could track down subtle change of glycan structure on the TIMP-1. Then the system was used for the selection of antibodies recognizing membrane proteins on cell surface. Both TM4SF5 and CD9 belong to tetraspanin superfamily and are reported as candidate cancer therapeutic targets. TM4SF5 was prepared as recombinantly expressed Fc-fusion form and used for library panning and selection. On the contrary, CD9 was expressed on cell surface as native form, and the recombinant cell line was directly used for cell panning and selection. For both targets, we could successfully isolate and identify specific binders. Their binding characteristics were confirmed by using ELISA and FACS. Further, we investigated their therapeutic potentials using cell-based assays.

With effective cancer treatment or immunotherapy by target specificity and high affinity, therapeutic antibodies have been hot issues. Unlike other recombinant proteins ; erythropoietin (EPO), interferons and growth factors, dosage range of therapeutic antibodies is needed above 5 mg/kg. In addition, productivity of recombinant Chinese hamster ovary (rCHO) cells compared with microorganism is too low, thus experiments to improve productivity is essential. rCHO cells expressing chimeric antibody against CD20 antigen were amplified and selected with methotrexate (MTX) in adherent cultures ,and rCHO cells later adapted in serum-free, protein-free and chemically defined media. First of all, media selection experiment is carried out. After medium with high productivity is chosen, the effects of low temperature conditions with glucose feeding were studied in an attempt to increase antibody production from suspension adapted rCHO cells. When the culture temperature was shifted from 37 ℃ to 30 ℃ at a culture time of 72 h with glucose concentration driving up to 3.5 g/L, final rAb concentration was increased about 7.5 times higher than production at 37 ℃. Establishing a culture condition of high concentration of rAb production, mass production is attempted by Pneumatic bioreactor system (PBS) performed 2 L scale. These scale up culture processes were successfully performed. Based on the results, foundation of development for culture process of rCHO cells producing recombinant antibody was established.

Recently, many recombinant therapeutic proteins are produced using transgenic plant cell cultures due to their advantages as a host for the production of therapeutic proteins, such as low production cost, safety from animal pathogens and capacity for post-translational modifications (PTMs). Especially N-linked glycosylation is one of the most important PTMs associated with protein stability, efficacy and half-life in serum. Transgenic rice cell cultures (Oryza sativa L.) with human b1,4-galactosyltransferase (hGalT) gene and inducible RAmy3D promoter were used to express recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin with hGalT (hCTLA4Ig-Gal), a soluble fusion protein formed as a dimer by disulfide bond. RAmy3D promoter was strongly induced by sugar starvation and the hCTLA4Ig-Gal was secreted into the medium. Disulfide bond is one of the most important factors for maintaining product quality. It is related with binding property, biological activity and potency. To maintain the quality of hCTLA4Ig-Gal, effects of cupric sulfate (CuSO4) on the prevention of reduction were investigated. The copper ions are known as a direct enzyme inhibitor as well as an oxidizing agent. CuSO4 was added at 10, 20 and 40 mM after purification, respectively. From the results of SDS-PAGE under non-reducing condition, it was clear that the reduction of hCTLA4Ig-Gal could be prevented by adding optimized concentration of CuSO4 after the purification of target protein.

Dimethyl sulfoxide (DMSO) can increase the specific productivity (q) of foreign proteins in mammalian cells, while it can also induce cell death, particularly apoptosis. Bcl-xL is a typical anti-apoptotic protein that inhibits the apoptosis in recombinant Chinese hamster ovary (rCHO) cell culture. To evaluate the potential role of Bcl-xL overexpression on DMSO-mediated erythropoietin (EPO) production, we used EPO-producing rCHO cells with regulated Bcl-xL overexpression (EPO-off-Bcl-xL) by doxycycline. Although DMSO addition enhanced specific EPO productivity (qEPO), it also induced cell death in EPO-off-Bcl-xL cells. Bcl-xL overexpression reduced the DMSO-induced cell death followed by release of various enzymes from plasma membrane-damaged cells as evidenced from LDH assay, resulting in decreased EPO degradation. However, it did not significantly improve the maximum EPO production. In addition, Bcl-xL overexpression suppressed DMSO-induced apoptosis, characterized by DNA fragmentation and Annexin V staining. Taken together, Bcl-xL overexpression could inhibit DMSO-induced apoptosis, thereby delaying the degradation of EPO.

Biological effects of nanomaterials with a focus on toxicity should receive great attention since commercial products as well as medical tools increasingly utilize them. Especially, a fundamental understanding of nanotoxicology is highly desirable both from the material’ stand point as well as from the biological system’ point of view. Nanocrystalline titanium dioxide (nano-TiO2) is an important material used in commerce today : for example, cosmetics of sun-cream to block UV radiation. However, despite its wide array of common applications, its pathogenetic role was always suggested under certain conditions. In this work, the toxicity of TiO2 nanoparicles on human skin- and lung cell was investigated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay- and confocal microscopy for live &dead of cells. The human skin- and lung-cell were purchase from from Korean Cell Line Bank. We observed that TiO2 nanoparticles impedes the cell growth using MTT, and dead cells increase depending on the concentration of TiO2 nanoparticles using confocal microscopy. Based on the results mentioned above, we discuss the toxicity and the safety of TiO2 nanoparicles for the human cells.

Exess ethanol intake is usually toxic in health. Chronic ethanol consumption causes oxidative damage in liver. Acer tegmentosum MAX. good for a liver has been known since ancient times in Korea and China. Ethanol-induced oxidative stress is related to the metabolism of ethanol. The major part of the ethanol is metabolized by alcohol dehydrogenase in liver. Alcohol dehydrogenase to form acetaldehyde results in the formation of free radicals. Cytochrome P450 2E1 (CYP2E1) participates to the ethanol decomposition. Excess ethanol intake affects more microsomal enzyme CYP2E1 activity than ADH resulting in causing oxidant stress. CYP2E1 has been to be a major contributor to ethanol-induced oxidant stress. Thereby, decrease of SOD, CAT, GSH. In this study, we investigated in vitro and in vivo the inhibition of ethanol-induced liver injury by Acer tegmentosum MAX. extract. In vitro studies with ROS scavenging, decreased of CYP2E1, and increased of SOD, CAT, GSH showed that verification antioxidant activity. In vivo studies, SD Rats were treated by ethanol administration. SD Rats were injected with or without ethanol (35% ethanol, 10ml/kg per day) and Acer tegmentosum MAX. extract (120mg/kg per day) continuously for 5 week. In consequence, Acer tegmentosum MAX. extract was recognized to inhibit the liver maker emzymes (AST, ALT, GGT) and increased of SOD, CAT, GSH.

In this study, to identify proteins involved in tumor progression and to deeply understand mechanism underlying melanoma metastasis, comparative proteome analysis on two melanoma cell strains with low and high metastatic potentials, WM793 and 1205LU, respectively, was performed using a combined three-dimensional difference in gel electrophoresis (3-D DIGE) technology [1,2]. The majority of protein level changes are closely associated with many aspects of the pathophysiology of cancers, such as cell death and growth, or tumorigenesis. In addition, cellular pathways related in oncogenes (JNK, c-myc, and N-myc) and tumor suppressor genes (p53 and TGF-β) were found to be prevalent metastatic melanoma progression by the Ingenuity Pathway Analysis. These networks showed substantial overlap and connectivity with the main biological theme of cell death and cancer. Thus the use of more comprehensive proteins profiling to analyze genetically very similar melanoma cell lines can contribute to the identification of novel metastatic biomarkers and provide new insights into the protein pathways involved in melanoma metastasis. [This work was supported by the Basic Science Research Program (2010-0008826) and Converging Research Center Program (2009-0093652) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology].

Phellinus linteus is a medicinal mushroom including many bioactive compounds, containing polysaccharides that have been reported to induce antitumor activity and to inhibit tumor metastasis and recurrence. Activated macropahges such as the phagocytic uptake and release of toxic molecules containing reactive oxygen species(ROS) and nitric oxide(NO) were investigated. To investigate whether polysaccharides from Phellinus linteus were able to stimulate the functional activation of macrophage, different celluar activities of macrophage, such as NO production, ROS generation and phagocytosis, and morphological changes were assessed. The cell lines used were macrophage-like cell, RAW 264.7 cells. In this study, we evaluated immunomodulatory potency of macrophage in immune response using polysaccharides from Phellinus linteus.

A human hybrid cell line named F2N78 was developed by somatic fusion of human embryonic kidney (HEK293) cells and Namalwa cells. Human cells have a human-like glycosylation profiles, so that they should be efficient for the production of human therapeutic proteins considering efficacy and stability. As a result of cell fusion, F2N78 cell line has the characteristics of both HEK 293 and Namalwa cells, such as high transfection efficiency, ease of suspension culture, and continuous expression of EBNA1 gene by EBV genome. In order to be used in commercial processes, development of optimal media for the growth and transfection of this novel host cell line is necessary. For that reason, development of an optimized medium for F2N78 cell line was performed in this study. Various media such as EXCELL 293, CD OPTI CHO, MPC002 were compared for the growth and transfection of F2N78 as well as HEK293. It was found that MPC002 medium provided the best results for growth of F2N78 cells and the modification and optimization of MPC002 medium will be carried out further.

A general method for the facile development of new biosensors for detection of liver biomarker protein targets have constructed and characterized. The peptides as recognition element selected using M13 phage display. A quartz crystal microbalance (QCM) is used as a diagnosis tool during biosensor development, and electrochemical techniques (cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)) are also used as the detection methods in the biosensor. A novel peptide capable of binding to alanine transaminase (ALT) was discovered by biopanning an M13 phage-displayed library. The selection procedure readily converged to a peptide (ALT5-8), with an amino acid sequence WHWRNPDFWYLK. The binding affinity of the peptide-containing phage particles was assessed by ELISA and the specific recognition of ALT was observed with a picomolar binding constant (Kapp= 8.14 pM). Peptides with the same sequence were chemically synthesized with a C-terminal cysteine and immobilized on a gold surface. These results demonstrate a simple platform for developing sensitive peptide-based biosensors for almost any desired protein targets.