Earticle

Immunotherapy using cytotoxic T lymphocyte (CTL) is researched actively to treat various types of cancers and viral infections. For an efficient therapy, obtaining enough cells is very important. Clinical trials involving the adoptive transfer of antigen-specific CTL have shown that efficacy can be achieved with CTL doses of 107-109 cells per kilogram of body mass.1) To expand sufficient cells, 10% serum supplemented RPMI1640 media is widely used in research or clinical use. However, all sera contain poorly characterized substances, including grwoth factors, antibodies, and other immunologically active substances.2) Through several culture experiments, our laboratory developed a serum-free medium(SFM) which resulted in cell expansion comparable to 10% serum supplemented media. To make a more efficient SFM, we added other supplements to the SFM, which was to be used as the basal SFM. By using fractional factorial methods, we screened the effective supplements between 4 candidates. According to the Design-Expert software(version 7.1.2), supplements B and D show a positive effect on the cell expansion. Optimization of the amounts of these 2 supplements was conducted. Through several culture experiments and analysis using the above software, composition of a powerful SFM for cytotoxic T lymphocytes was adjusted.

The main goal of this project is to study in vitro construction and in vivo application of combined skin substitutes based on the uses of cell populated gelatin and fibrin. We prepared skin equivalents with cell populated gelatin and fibrin, and then performed immunohistochemistry (IHC) analysis with bio-markers to evaluate the artificial skin in vitro and in vivo. Skin equivalent models were prepared by combining gelatin-based dermal substitutes and fibrin-based cultured epithelium and by raising them up to the air-liquid interface. Skin substitute models were evaluated by marker proteins specific to individual dermis and epidermis. The basal membrane protein, type IV collagen and cytokeratin 14 were expressed in parallel with the basal layer protein on both skin substitute models. Also two spinous layer proteins, involucrin and cytokerain 10 were stained. These results suggest that the skin equivalents combined with cell populated gelatin and fibrin differentiate well in vitro and in vivo and may be applicable as new skin substitutes.

In order to develop a nanoparticle which has specificity at tumor site, a biocompatible polysaccharide was employed. The complex formed nanoparticle easily. The cationic polymer has cytotoxicity,1) but the nanoparticle from the conjugate with vitamin slightly reduced the toxicity. The polysaccharide which can be degraded by an enzyme from tumor site (BP) covered with cationic polymer.2) The cytotoxicity of the nanoparticle measured by MTT assay depended on the enzyme concentration. The interaction between the nanoparticle and cancer cells was observed by confocal microscopy as a function of BP concentration. Although the cell cytosole was perfectly stained by the red color (RI-TC labeling nanoparticles) in the presence of the enzyme, RI-TC labeling nanoparticles only stained in the cell membrane without the enzyme. The result indicated that BP can enhance the specificity of tumor site due to inhibit the internalization of the nanoparticle in the normal tissue.

Mast cells are so widely recognized as critical effecter cells in allergic disorders and other Immunogloblin(Ig)E-associated acquired immune response.(1,2) In addtion, Mast cells (MCs) play an essential role in allergic inflammation, including asthma, atopic dermatitis and allergic rhinitis.(3,4) This study was designed to evaluate whether the Houttuynia cordata and Ulmus davidiana var. japonica water extracts(HUD) could modulate the production of pro-inflammatory cytokines and mechanism in phorbol 12-myristate 13-acetate(PMA) plus calcium inopore A23187 treated human mast cell line, HMC-1, to prevent the inflammatory response. HUD inhibited inflammatory cytokines such as TNF-α, IL-6 and IL-8 stimulated by PMA pus A23187 from HMC-1 cells. In addition, HUD did not significantly affect cell viability and had no toxicity on HMC-1 cells. Based on these results, HUD can be used for treatment of allergic inflammation response.

The development of magnetic nanoparticles for specific targeting of cell therapeutics has been the main issue in biomedical research. A common problem in therapeutic application of stem cells is that the cells rapidly disappear in circulatory system after the injection of the cells. In this study, we conducted an experimental study in microfluidic system with magnetic nanoparticles, isolated from the Magnetospillum sp. AMB-1. Magnetic nanoparticles were delivered to the stem cells (Endothelial Progenitor Cell, EPC) ranged from 0 to 20 µg per 104 cells and their behaviors were controlled by a NdBFe magnet. A blood vessel was modeled by a single microchannel and the flow rate was uniformly controlled by a syringe pump. We investigated the cell behaviors according to the flow rate and the amount of magnetic particles taken up into the cells. The magnetic nanoparticles-introduced cells at a concentration of 50 µg per 104 cells under 0.4 T were fixed at a flow rate of 1.6 µl min-1. This microfluidic system would be a useful tool for better understanding of the behavior of magnetic nanoparticle-introduced cells in the human circulatory system for clinical applications.

In living organisms, reactive oxygen species arise from endogenous oxidative metabolism or exogenous stresses, such as UV light, ionizing radiation, and some types of redox agents, causing oxidative damage to proteins, nucleic acids, and unsaturated fatty acids in lipids. Reactive oxygen species and other free radical emissions by cells and tissue are often taken as an indicator and a cause of aging in vitro and in vivo. In the case of MSC however, ROS was also shown to be involved in signaling, down-regulating proliferation and stimulating apoptosis or differentiation processes. Mesenchymal stem cells (MSC) are of great therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. A hot new topic in medical treatment is the use of MSC in therapy. So we confirmed the changes of hMSC apoptosis caused by ROS using various tools for apoptosis assay. And we performed proteome profiling to find apoptosis associated regulators.

Adipogenic differentiation needs many factors to regulate protein or RNA expression. Among them, factor to regulate extracelluar matrix were confirmed, which is expected to affect adipogenic differentiation. The protein profiles from the adipocytes differentiated from hMSC were determined by 2-D gel electrophoresis (2-DE), and the differentially expressed proteins were identified by MALDI-TOF and ESI-Q-TOF MS/MS analysis. Among these, the three up-regulated proteins (Calgizzarin, Kallikrein-9, and Ubiquitin) were newly identified from differentiated hMSC, which might prove to be useful biomarkers for the differentiation of hMSC into adipocytes. Over expression of Kallikrein-9 was reconfirmed in adipogenic cells by RT-PCR, Western blotting. This protein was expected to play important roles for adipogenic-differentiation which will be able to help understanding of differentiation related in its extracellular matrix (ECM) not in signal transduction

A major cause of diabetes is impaired insulin action. Specially, the liver plays important roles in the absorption, metabolism and excretion of glucose. In early studies, it was found that diabetes mellitus is associated with an increase on the risk of acute liver failure by oxidative stress. In states of oxidative stress, vitamins may have an antioxidant role. In this study, we tested that effect of vitamin A on the risk of acute liver failure induced by diabetes mellitus. The changes in the liver tissue may reflect pathologic alterations during Vitamin A (retinol) treatment. To produce hyperglycemia environment, the rats were injected with STZ. After STZ injection, rats were fed either a retinol contained diet or normal rodent chow. We measured glucose concentrations and body weight to show the diabetes and measured GPT and TG levels to identify liver injury. In addition homogenized liver samples were consecutively undergone IEF and SDS-PAGE. The obtained gels were stained with "silver staining" to visualize the specific proteins. After image analysis, several proteins were found to be significantly changed in comparison to normal and retinol treated groups.

The hair follicle is small, but complex and a dynamic organ. In humans, it plays an important role from the esthetic aspect of social life. When alopecia caused by inheritance, male hormones or stress progresses, the dermal papilla undergoes gradual degradation, resulting in the degeneration of hair follicles. In the past, alopecia was treated typically by various methods of implanting artificial hair into hair follicle root bulbs of the scalp, but such artificial hair implant methods had led to some serious problems, and such methods are now banned. Currently, there are two methods employed to treat alopecia: drug or natural substance therapy, and human hair transplantation. The drug or natural substance therapy may retard the progress of alopecia or prevent future hair loss, but it may accelerate hair loss when the medication is stopped after a prolonged use. On the other hand, the transplantation of human hair involves taking plugs of natural hair from occipital hair growing areas and transplanting them to bald areas. Although the transplanted hair settles at the transplant area as a complete hair follicle and becomes a permanent hair that undergoes a normal growth cycle, the number of hair to be transplanted is severely limited, and in case of transplanting about 2,000 hair strands per one operation, it is generally not possible to perform more than three such operations. Thus, the methods currently used for treating alopecia have a number of limitations, and to overcome such problems, many researchers have attempted to revive hair follicles by in vitro culturing hair follicle cells and implanting them in the treatment area. In our study, by utilizing culture-expanded mesenchymal stem cells (MSCs) which don't have aggregative activity, cell-aggregated spheroidal DP tissues were produced by a special culture condition in vitro, and hair follicle inductive capacity pertinent to the aggregative activity was evaluated. The MSCs cultured as monolayer migrated and formed clumps, the reconstructed DP tissues suspended in the culture medium, their size and shape were similar to intact DP, and the spheroid type composed of similar ECM components could be generated. We were confirmed by light microscope that the reconstructed DP tissues generated by such procedure had the size and shape similar to actual DP. In addition, by immunohistochemical staining, the expression of laminin and type collagen was also Ⅳ observed, and thus it was confirmed that their ECM components were also similar. In addition, the expression of versican that appears only in cells that could induce hair follicles was confirmed, and by the in vitro mixed culture with hair follicles removed the hair bulb, it was confirmed that the reconstructed DP-like tissue could form new hair bulb structure by inducing the growth of outer root sheath cells.

It has been suggested that inflammation in the brain involves in the pathogenesis and progression of Parkinson's disease.1) However, the mechanisms of inflammatory neurotoxic effect on mesencephalic dopaminergic neurons in Parkinson's disease are not fully elucidated. In this study, we found that dipyridamole, a selective cGMP phosphodiesterase inhibitor,2) attenuated the rotenone-induced microglial inflammation in primary rat ventral mesencephalic neuron-glia cultures. Dipyridamole inhibited the rotenone-induced loss of tyrosine hydroxylase-positive neurons and production of microglia-derived proinflammatory factors including tumor necrosis factor-α (TNF-α), nitric oxide (NO) and interleukin-1β (IL-1β). These results suggest that microglial inflammation in Parkinson's disease could be mediated, at least in part, by cGMP pathway

Hottuynia cordata is known as a therapeutic drug that has been used in traditional oriental medicine for the treatment of allergy.(1) And Ulmus davidiana var. japonica is a deciduous broad-leaved tree widely distributed in oriental countries, and its stem and root barks have been used as a traditional medicine for the treatment of edema, mastitis, cancer, inflammation, and rheumatoid arthritis for a long time.(2) This study was on the inhibitory effects of Houttuynia cordata and Ulmus davidiana var. japonica water extracts (HUD) on mast cell mediated allergic reactions. Compound 48/80 was used to activate the mast cells, which is known as a potent inducer of a deregulation and for the release of histamine and other chemical mediators that are responsible for anaphylactic symptoms. HUD inhibited a compound 48/80 induced systemic anaphylactic shock and a passive cutaneous anaphylactic reaction. In addition, HUD inhibited the itching behavior by a histamine release from mouse skin mast cells stimulated by compound 48/80. These results suggest a possible therapeutic application of this agent to mast cell mediated inflammatory allergic diseases.

At present, cell-based drug screening assays have been attracted attention for various molecular interactions including induction of apoptosis. Apoptosis is the process of programmed cell death through a tightly controlled program that plays important roles in many normal processes, ranging from fetal development to adult tissue homeostasis.1) Screening of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression.2) In this study, the affinity of specific binding between annexin V and liposome that consists of several molar ratio of phosphatidylserine (PS) and phosphatidylcholine (PC) in presence of Ca2+ was measured by surface plasmon resonance (SPR) analysis. Annexin V-FITC is used for quantification to confirm efficacy of anticancer agent. Induction of apoptosis is induced by stausrosporine (SSP) as one of anticancer agents. A variety of concentration of SSP induced to be apoptotic cells with macrophage cell line (RAW 264.7 cell) and breast cancer cell line (MCF-7 cell). The apoptotic, necrotic and live cells was monitored using fluorescent probes such as annexin V-FITC and propidium iodide (PI) by confocal microscopy. The fluorescent imaging data based on confocal microscopy was analysed with intensity of fluorescence for quantification of apoptotic cells.

Erythropoietin (EPO) is a glycoprotein hormone primarily synthesized in kidney and regulates mammalian erythropoiesis1). We participated in the European Biological Standardisation Programme and carried out biological and physicochemical assays on the candidate Biological Reference Preparation batch 3 (cBRP3), using BRP batch 2 (BRP2) and where necessary the 2nd World Health Organization (WHO) International Standard (IS) for recombinant EPO as the reference standards2). We performed the physico-chemical assays to investigate the suitability of cBRP3 as a reference substance. Physico-chemical assays using the cBRP3 as the test preparation and where appropriate, BRP2 as the reference preparation were performed as follows; SDS-PAGE, immunoblotting, capillary zone electrophoresis, peptide mapping, size- exclusion chromatography. In all of the physico-chemical assays, BRP2 and cBRP3 showed equivalent results. We performed the normocythaemic mouse bioassays to assign the potency unitage of cBRP3. The mouse bioassays were performed using as test samples the current and the candidate Ph. Eur. (European Pharmacopoeia) BRPs and as reference standard the 2nd WHO IS. In our results, BRP2 has an estimated potency of 34640 IU/vial and the cBRP3 has an estimated potency of 36857 IU/vial. Sixteen laboratories in 11 countries participated in the international collaborative study and the overall study mean gave a value of 35280 IU/vial to cBRP3.

It is well-known that certain mixtures of surfactants can provide better performance than pure surfactants for a wide variety of applications. A stable water-in-oil(W/O) nano-emulsions was reported to be prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), decane oil and water1). The w/o emulsion was prepared by spontaneous emulsification2). The organic phase was used to mix the n-decane and varying ratio (v/v) between Span80 and Tween80 surfactants. In this work, we investigated that a partial physical property of the emulsion such as partial molar volume of water, emulsion stability by size distribution along with time. Solubilization of fibroin and its hydrolysate was performed by using the emulsions of varying water fraction. Excess peptide precipitates were removed by filtration. The level of solubilization of peptide was between 0.1 - 1.1 mg/ml of transparent emulsion. Fibroin was solubilized upto 16 mg/L of transparent emulsion.

Aging is characterized by combination of progressive and degenerative changes in many physiological conditions with time. In addition, aging is an endogenous process distinguished by the progressive loss of function with decreased fertility, increased mortality and augmented susceptibility to the onset of age-related diseases. In most cases, aging and age-related diseases are caused by abnormal expression, modification and deposition of certain proteins. Thus proteomics research is expected to provide significant insights into the cellular and molecular mechanisms of aging and age-related disease processes. We focused on rat serum for proteome analysis because proteins in serum are tightly controlled to balance their physiological functions, circulating through the body. In this study, to screen the age-related proteins in rat serum, the comparative proteomics analysis was applied to get the two-dimensional electrophoresis (2-DE) profiles with high resolution and reproducibility from rat serum of 2 and 12 weeks. Differential proteins between the rat serum of two age groups were found with ImageMaster 2 D Platinum 6.0 software. As the results, several proteins were detected to be significantly changed in expression level.

Extracellular glutathione peroxidase (GPx3) is a secreted selenoenzyme with GPx activity. GPx3 in plasma is predominantly derived from the proximal tubules of kidneys in humans. Previous studies have shown differences in the expression level of GPx3 in serum from patients with diabetic nephropathy. In this study, the regulation of GPx3 under a high glucose condition in human proximal tubular (HK-2) cells was studied. When HK-2 cells were treated with different concentrations for 72 h, glucose did not show any cytotoxic effects as judged by MTS assay, but only decreased HK-2 metabolic activity. The glucose concentration of the cultured medium in which the cells were grown showed only 5 mM glucose. Cells exposed to hyperglycemia was provided the same osmotic stress. Both GPx3 mRNA expression of HK-2 and GPx3 protein were increased in osmotic condition as well as hyperglycemia condition. In results, hyperglycemia condition damaged the cell by providing osmotic shock as a hyperosmotic condition. Under hyperglycemia condition, GPx3 as a antioxidant enzyme was increased in HK-2 cells for protection from osmotic stress.

Neuronal cell death in various models of neurodegenerative diseases is esponsible by oxidative stress. In this study, we have investigated the effects of melatonin, the main secretory product of pineal gland, against hydrogen peroxide-induced oxidative stress. Hydrogen peroxide treatment caused up-regulation in the levels of phosphorylated c-Jun N-terminal kinase (JNK) in cultured noradrenergic cells. It also caused down-regulation of tyrosine hydroxylase, the rate limiting enzyme for norepinephrine biosynthesis, and eduction of p38 MAP kinase and Caspase-3. Melatonin prevented the activation of cell death signaling cascade under hydrogen peroxide mediation. Melatonin significantly prevented hydrogen peroxide-induced loss of cell viability and induction of JNK/Caspase-3 activation. In addition, melatonin pretreatment attenuated down-regulation of tyrosine hydroxylase. These results suggest that melatonin has some protective properties against noradrenergic neuronal degeneration.

The surface carbohydrate antigens on pig endothelial cells are causing immune responses in transplantation of pig organs into humans. Therefore, analysis of the carbohydrates expressed on the pig organs is of importance to predict and overcome the potential immune rejection in xenotransplantation. In this study, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney are relatively quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and normal-phase high performance liquid chromatography (NP-HPLC) profiling after exoglycosidase digestion. The quantification results obtained fromMALDI-TOF MS and NP-HPLC indicated that the major alpha-Gal epitopes were over 30% of the total neutral N-glycans. Electrospray ionization (ESI) MS/MS was employed for the structural analysis of N-glycans from pig kidney cell membrane glycoproteins. We were able to identify various non-Gal carbohydrate antigens, including Hangenutziu-Deicher (H-D) antigens (NeuGc-Gal-GlcNAc) as well as Gal1-3LewisX (Gal-Gal-(Fuc)GlcNAc) epitopes. Over 100 N-glycans, including sialylated and neutral types, were identified. This quantitative and qualitative structural information on carbohydrate antigens from pig kidney will be critical in overcoming the immunological barriers in clinical alpha1,3-galactosyltransferase gene-knockout pig-to-primate xenotransplantation.

Cisplatin (cis-diammindichloroplatinum Ⅱ), a commonly used chemotherapeutic agent for the treatment of solid tumors, has a major limitation as a cancer drug because of its nephrotoxicity. Decursin isolated from the root of Angelica gigas is known to be a PKC activator. Our previous studies showed decursin mediated protection of cisplatin-induced nephrotoxicity in rats. Nephrotoxicity by cisplatin involves necrosis as well as apoptosis of renal tubular cells. In this study, we examined whether decursin ameliorates cisplatin-induced nephrotoxicity in cell viability and oxidative stress in renal mesangial cells (RMCs) and primary mouse kidney cell cultures, including proximal tubular cells. We also compared the effects of decursin with N-acetylcysteine (NAC) which is known as a strong antioxidant. Cisplatin-treated cells showed significant increase in cellular ROS levels and cell death. In contrast, pretreatment of cells with decursin recovered the rise of ROS levels and influenced cisplatin-induced apoptosis. Taken together, our data indicate that decursin has a protective effect against cisplatin-induced nephrotoxicity in renal mesangial cells and proximal tubular cells.

PEI used as an effective non-viral gene delivery vector in vitro and in vivo1). Although it has high transfection efficiency, it makes a damage to normal cell membrane because of its low targeting efficiency to target cell or tissue2,3). In order to overcome the problem, vitamin as a targeting moiety was introduced into PEI. Vitamin can act as a ligand for targeting of nucleus membrane4). The conjugation of PEI/vitamin were confirmed by UV-spectrophotometer and TNBSmethod, and characterized by gel electrophoresis, particle size and zeta potential to estimate a possibility as a gene carrier. In the case of PRA/DNA complex, a completed complexation was observed above 3 of N/P ratio, while PEI/DNA complex without PRA showed above 2. The size was appropriate to the cell transfection. The zeta potential of the conjugates decreased with increasing vitamin concentration. The transfection efficiency of PRA in HeLa cell was up to 10 times higher than that of PEI only. The cytotoxicity of PRA was also measured by MTT assay. The conjugates showed lower cytotoxicity compared with the cationic polymer system. Therefore, the PRA conjugate may be good candidate for non-viral gene carrier.

Hyperglycemia is recognized to be the key factor driving renal functional and pathological changes in diabetic nephropathy (DN). Most studies to date have focused on the glomerular abnormalities found in DN. However, nephromegaly in the early stages of diabetes and the correlation of tubulointerstitial pathology rather than glomerular pathology with declining renal function in DN suggest the involvement of the tubulointerstitium. The etiology of the tubulointerstitial pathology in DN is not fully understood. In this study, to overcome the more completely DN pathways, we constructed an initial 2-DE reference map for primitively cultured human proximal tubule (HK-2) cell in the presence of 5 mM and 30 mM glucose, which correspond to blood glucose concentrations during the early and late stages of diabetes, respectively. The differentially expressed HK-2 cell cellular proteins were identified by ESI-Q-TOF MS/MS. The changed protein expression will provide greater understanding of DN pathways in HK-2 cell at the high concentration of glucose.

Diabetes mellitus has reached epidemic proportions and affects more than 170 million individuals worldwide [1]. Cardiovascular events and microvascular complications are major cause for morbidity and mortality of the patients with diabetes [2]. In this study, we compared the protein expression of human serum in healthy controls (n = 30) and type 2 diabetic patients without complications (n = 30) using two-dimensional electrophoresis (2-DE). Differentially-expressed proteins were identified with ESI-Q-TOF MS/MS. In patients, three proteins were up-regulated more than 2-fold (serum amyloid P component, apolipoprotein CIII, and ficolin-3) and three proteins (adiponectin precursor, complement factor I light chain, and histidine-rich glycoprotein) were down-regulated less than 50% compared to those in control. Ficolin-3 was higher in the serum of type 2 diabetic patients than in healthy subjects, and decreased during the development of diabetic nephropathy. These results imply that differentially-expressed proteins in type 2 diabetic patients may provide further information on the risk of developing diabetic complications.

Antibiotics are commonly used in livestock for preventing diseases and promote growth. However, the presence of antibiotics in the wastewater from livestock farm can cause negative effect on the various biological treatment processes such as activated sludge, anaerobic digestion In this study, we aimed to evaluate the effect of some common used antibiotics in the Korea livestock farm such as bezathine penicillin G, procaine penicillin G and sulfadimethoxine sodium to nitrification efficiency as well as organic compounds removal rate in biological system for treating swine wastewater. The experiment was conducted in aeration batch reactor and using activated sludge collecting from municipal wastewater treatment plant. For batch test, diluted piggery wastewater was used, two commercial antibiotics products, PPS (mainly consisted of bezathine/procaine penicillin G) and SULFA-40 (sulfadimethoxine sodium) were selected for subsequent experiment. The control and each antibiotic experiment were conducted identically in 1.0 L batch reactor. The results showed that the nitrification rate is reduced when the addition of PPS increase in range of 50 - 500 ppm. The profile of NOx - during the batch test indicated that the decrease of ammonium oxidation rate is mostly due to the decline of ammonia oxidizing bacteria (AOB) activity. In the different way, the nitrification rate was not significantly affected by the SULFA-40 under the antibiotic concentration up to 700 ppm. COD removal rate was also examined, it showed that the oxidation of biodegradable organic matters were not inhibited by such commercial antibiotic products. However, it seemed that those antibiotics are hardly degraded biologically after short acclimation time using activated sludge. Further work for evaluating the long-term effect of common used antibiotics should be done in order to stabilize the biological treatment system of livestock wastewater.

Prion diseases are transmissible neurodegenerative disorders of protein conformation where the posttranslational modification of host-encoded prion protein PrPc yields a high -sheet content β modified protein PrPsc which further polymerizes into amyloid fibrils. PrP106-126 is the key region for initiating conformational changes that leads the conversion of PrPc to PrPsc.1) Molecules which can destabilize and defunctionalize such proteins can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules are formed to prevent protein denaturation and maintain protein stability and function.2) Hence they can prevent abnormal protein folding like amyloid formation. This work explores the effect of such small stress molecules on PrP106-126 amyloid formation. The characterization tools used for this study include turbidity, atomic force microscopy and cell viability assay. According to our results ectoine and mannosylglyceramide exhibited inhibitory effects against prion peptide aggregation and toxicity to human neuroblastoma cells. Our findings conclude that small stress molecules could be potential inhibitors for prion diseases.

This study was designed to examine the effects of Hydrangea macrophylla var. thunbergii extract (HMTE) on the blood glucose in diabetic rats. Diabetes mellitus was induced in male Institute of Cancer Research (ICR) rats by injection of streptozotocin (STZ) dissolved in a citratebuffer (pH 4.5) into abdominal of 45mg/kg of body weight. The rats' body weights were measured along with plasma level of glucose, total cholesterol (TC), HDL-cholesterol, triglyceride (TG) and free fatty acids (FFA). The experimental groups were divided into 6 groups which consisted of Control (n=12); Normal rats group, C1 (n=12); STZ-induced diabetic rats group, C2 (n=12); STZ-induced diabetic rats with Hydrangea macrophylla var. thunbergii group, C3 (n=12); STZ-induced diabetic rats with hydrangea macrophylla var. thunbergii (50%) + polygonatum odoratum group (50%), C4 (n=12); STZ-induced diabetic rats with hydrangea macrophylla var. thunbergii (50%)+Cassia tora group (50%), C5 (n=12); STZ-induced diabetic rats with hydrangea macrophylla var. thunbergii (50%) + polygonatum odoratum (25%) + Cassia tora group (25%). Body weight, food intake, HDL-cholesterol, and blood glucose were recorded 6 weeks. The blood glucose concentration of C2 and C5 group were lower than the other groups. There was no significant difference of cholesterol level among diabetic group. The C2 group in weight, FFA and TG levels significant change among all groups. This study shows that HMTE beneficially modulate blood glucose concentration in diabetic animals.

Cardiovascular disease is the single most common cause of death in developed world. The most general reason for blockage of blood vessels is a build-up of deposits, inflammation and atherosclerotic plaques on their inner walls. If it is possible to monitor the increase or decrease of protein concentrations in blood, which are related to the cardiovascular disease, the early diagnosis of cardiovascular disease could be possible. The protein profiles of human sera from 20 patients with non-diabetic cardiovascular disease were compared with those of 30 healthy controls by using the two-dimensional gel electrophoresis. As the results, the differentially expressed spots in cardiovascular disease patients were screened and identified with ESI-Q-TOF mass spectrometry. Among the identified proteins, several proteins were confirmed to be good candidates as biomarkers for cardiovascular disease. These results suggest that proteins identified can be used as diagnostic and monitoring biomarkers of cardiovascular disease.

Foodwastes can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of polysaccharides in the foodwastes to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from foodwastes is relatively high based on current technologies, and the main challenges are the low yield and high cost of hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of foodwastes. Pretreatment of foodwastes to remove starch and polysaccharides can significantly enhance the hydrolysis of sugars. Foodwastes were hydrolyzed using various concentration of glucoamylase, carbohydrases and both mixing enzymes in order to obtain high glucose yield for ethanol fermentation; mixing enzymes revealed higher performance(2.53 g-glucose/g-dry foodwastes) than each glucoamylase (2.32) and carbohydrases (2.01). As a result, our results suggest that foodwastes can be a high potential substrate for ethanol production.

The thiol-disulfide oxidoreductase thioredoxin-1 (TRX) is known to be secreted by leukocytes and to exhibit cytokine-like properties. However, specific cell surface proteins and pathways coupling extracellular TRX redox activity to cellular responses have not been identified so far. We first found that exogenous TRX not only scavenger but also effected intracellular proteins using proteomic approaches. Mechanism-based Histidine pull down and immunoprecipitation technique used to identify TRX binding partner proteins. We found that up/down regulated intracellular proteins were identified under the condition of LPS-treated A375 cells and co-treat with exogenous TRX using in vitro 2-DE proteomic approaches. As well as, confirmed in vivo mouse inflammation models used to quantitatively measure IL-6, IL-10, MCP-1 and TNF protein levels in serum samples. Our studies demonstrate that exogenous TRX has anti-inflammatory properties and intracellular regulator activity in vivo and in vitro. These results have a therapeutic role in skin inflammation therapy.

Frontal affinity chromatography equipped with mass spectrometry (FAC-MS) has been used to screen ligands binding to immobilized target proteins and measure their dissociation constants [1]. Herein, FAC-MS system has been developed and utilized for monitoring protein-carbohydrate interactions, which play significant roles in biological events such as cell-cell interactions, invasions, metastasis, and so on. Complex glycans expressed on porcine endothelial cell are responsible for immune rejections upon pig-to-human xenotransplantation [2]. Except terminal alpha-linked galactose of porcine glycan structures, called alpha-Gal epitope, mediating hyperacute rejection, other carbohydrate moieties, called non-Gal epitopes, could be potential xeno-antigens because alpha-Gal knock-out method could not prevent immune rejection completely. Recently, we found that human CD154 was bound to porcine glycans immobilized on solid support with/without terminal alpha-Gal epitope in vitro. Therefore, we tried to reveal which glycan structures would be specific for human soluble CD154 using FAC-MS. After FAC-MS experiment, various glycan structures were categorized by their relative affinit ies for human souluble CD154.

Extremolytes are osmolytes accumulated by extremophiles as a response to external stress and are known to act as chemical chaperones by stabilizing cellular structures1). Extremolytes have been shown to prevent protein misfolding by stabilizing its structure and can reach high intracellular concentrations without interference in metabolism. ß-Amyloid peptide (Aß) is the major constituent of senile plaques, the key pathological feature of Alzheimer´s disease. Aß is physiologically produced as a soluble form, but the aggregation of Aß monomers causes neurotoxicity. Ectoine, an extremolyte widespread in halophiles has been shown to interfere with the formation of amyloid aggregates in vitro and amyloid-induced cytotoxicity2,3). Using AFM and an assay based on thioflavin T fluorescence, we have tested synthetic ectoine analogs and other osmolytes for their ability to interfere with Aß amyloid formation in vitro. We show that a synthetic analog of ectoine with widened ring size interferes effectively with amyloid fibril formation. The results indicate the possibility of designing synthetic compounds as potential inhibitors of amyloid formation associated with neurodegenerative diseases.