Earticle

A rice cell suspension culture system with the RAmy3D promoter, which is induced by sucrose starvation, has been previously utilized to produce large quantities of recombinant proteins. However, using rice as production system for therapeutic proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plantspecific sugar residues. In this study, for generating glyco-engineered rice as production host for therapeutic glycoproteins, an intron-containing self-complementary hairpin RNA-mediated post transcriptional gene silencing was used to obtain a targeted down-regulation of the endogenous glycosyltransferase genes in rice cell suspension cultures. N-linked glycans from the generated RNAi lines were identified and their structures were compared with those isolated from the wild type suspension cells. The Δ3FT/XT glyco-engineered line with significantly reduced xylosylated and/or core α1,3-fucosylated glycan structures. In case of the Δ4FT/GT glyco-engineered cell lines, showed that completely suppressed of Lewis a epitopes (This work was supported by the grant from JBMI).

Bovine trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 24kDa trypsin molecule. It is widely used for commercial purposes to digest or process other proteins. Plant production system of recombinant proteins offers a way to replace animal-derived proteins with a safe and economical alternative. Recombinant bovine trypsin was produced in transgenic rice cell suspension culture with the rice amylase 3D promoter. It was purified from transgenic rice cell suspension culture by hydrophobic interaction chromatography and size exclusion chromatography. The enzyme activity of purified recombinant bovine trypsin was generally characterized by zymogram. The results demonstrate the use of recombinant bovine trypsin derived from plant cell suspension culture as protease.This work was supported by the grant from Korean Ministry of Knowledge Economy.

The N-glycans of human granulocyte-macrophage colony-stimulating factor produced in genetically engineered rice cell suspension culture were analyzed. In previous study, immunoblot assay detection of secreted hGM-CSF revealed the presence of multiple glycoforms with molecular weights higher than observed for E.coli-derived hGM-CSF. In this study, N-glycan patterns of total secreted proteins were analyzed and then N-glycan patterns of secreted hGM-CSF were examined using HPLC and MALDI-TOF mass spectrometry. From the rice-derived hGM-CSF, seven types of N-glycns were MMXF3 (30.96%), GnMXF3 (44.76%), GnMX (1.84%), GnGnXF3 (8.73%), AF4MXF3 (4.76%), AGnXF3 (7.20%) and AF4GnXF3 (1.75%). The ratios of paucimannosidic-type (MMXF3) and complex-type glycoforms were 30%, 70%, respectively and nearly 20% of complex N-glycans showed Lewis a structure (This work was supported by the grant from Korean Ministry of Knowledge Economy)..

Stirred-tank bioreactor technologies have been used in animal cell culture systems. However, the high cost of stirred-tank bioreactor operation limits their availability (1). In contrast to stirred-tank bioreactor, disposable bag bioreactor reduces operation costs since it can eliminate the need for cleaning, sterilization, and validation. (2, 3). But the main problems confronted in using disposable bag bioreactor are agitation and oxygen mass transfer (4). The volumetric mass transfer coefficient (KLa) is a critical parameter for oxygen mass transfer. Adequate oxygen supply for animal cell culture is essential for the maximum cell proliferation and useful protein production. In this study, various prototype disposable bags were designed and its KLa was investigated. To increase KLa values, we used a twist shaking motion rather than tilting motion. In addition, a novel structure which can be installed at inside of disposable bag bioreactor had been developed for further increase of KLa. The novel structure showed enhanced KLa values in all types of disposable bag bioreactors and agitations.

Plastic cell culture systems have a useful tool in research and biotechnlogy process, and a novel pneumatic bioteactor system (PBS) has been recently developed by PBS Biotech, Inc. In this study, to investigate the effects of agation speeds in 2L scale PBS, various rpm settings (10, 20 and 30) were used for cultures of rCHO cells producing monoclonal antibody (MAb). The maximum cell density in PBS at 20 rpm reached 3.4 X 106 cells/ml, and final MAb concentration was 141 mg/L. Also, when culture of commercially available suspension cell line CHO-S (Invitrogen) was evaluated in PBS, the maximum cell density reached 10.5 X 106 cells/ml. similar result was obtained in stirred tank reactor (STR, 9.6 X 106 cells/ml), which was better than in Erlenmeyer flask (5.4 X 106 cells/ml). These results revealed that the novel PBS is a strong candidate with variety of advantages such as low cost and easy scale-up up to a few thousands liters in cell cultures compated to comventional STR. Reference. 1. Sing V. (1999) Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnoloty. 30:149-158

Recombinant bovine trypsin was produced by genetically engineered transgenic rice cells using the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Batch culture was employed to investigate the effects of several alternative carbon sources on the trypsin production. In this study, succinic acid, malic acid and fumaric acid were used as substitutive carbon source to increase trypsin productivity. We have found that the productivity of recombinant trypsin increased 3.2- to 4.3-fold in medium containing an alternative carbon source compared with the control medium without carbon source. The accumulated recombinant trypsin in the medium containing fumaric acid reached 92.3 mg/L at day 7 after sugar starvation. Acknowledgments this work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry, and Energy

A strong tendency is emerging to remove not only serum but also any product of animal origin from animal cell culture media during production for recombinant proteins. Plant protein hydrolysates have been considered promising substitutes for serum in mammalian cell cultures. The effects of individual plant protein hydrolysates on cell proliferation have been studied, but no attempt has been made to study the effects of mixtures of fractionated plant protein hydrolysates. We selected a few growth promoting protein hydrolysates (Broadbean, Soy F, and Soy P) and made mixture compositions with those hydrolysates, which considerably improved cell growth and viability compared with those seen in media containing each protein hydrolysate. We also compared the effects of individual fractionated protein hydrolysates with those of mixture composition with fractionated hydrolysates, and found that some of the mixture compositions containing a small fraction of a high–molecular-weight hydrolysate supported cell longevity greater than that seen in the control media containing unfractionated hydrolysates. We conclude that cell growth and viability may be improved in media containing a mixture of unfractionated or fractionated plant protein hydrolysates compared with media containing a single hydrolysate.

Recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobuline (hCTLA4Ig) was produced by recombinant Chinese hamster ovary (rCHO) cells using 125-mL erlenmyler flasks. Mammalian cells produce relatively low level of recombinant proteins compared with microbial cells. The use of sodium butyrate in cell cultures has been known to enhance specific genes controlled by several promoters. Many reports have described significant increase of recombinant proteins in the presence of 0.5-4 mM sodium butyrate. Lowering temperature below 37℃ has also been considered as an effective tool for increasing specific productivity of recombinant protein. Unlike the specific growth rate, effect of low temperature on specific productivity still remains unclear. We investigated the combination effects of the addition of optimum concentration of sodium butyrate and optimal temperature shift on hCTLA4Ig specific productivity in rCHO cells. When viable cell density was reached at maximum level, 1 mM sodium butyrate was added to the culture medium and culture temperature was shifted to 34℃. Simultaneous application of sodium butyrate and temperature shift enhanced specific productivity up to 1.5-fold. Therefore, these results suggest that the specific productivity of hCTLA4Ig became higher because of the simultaneous application of sodium butyrate and temperature shift.

Abnormal glycosylations are closely associated with human cancers. And certain types of glycan structures are well-known markers for tumor progression. Therefore, the highly sensitive and high-throughput analysis of glycans from human body fluids is a growing issue in glycomics. In this study, we present a rapid and high-throughput human serum N-glycan release and purification technology using 96-well plate based procedures. The N-glycans released from a polyvinylidene fluoride membrane filter plate were subsequently loaded onto porous graphitic carbon containing 96-well plate to remove the salts and other contaminants. The purified N-glycans were characterized by qualitative and quantitative analysis using matrix-assisted laser desorption/ionization time-offlight mass spectrometry and carboxymethyl trimethylammonium hydrazide derivatization that was introduced to generate a permanent cationic charge to the reducing end of neutral oligosaccharides. Finally, this robust N-glycan preparation technology was applied to ovarian cancer diagnosis using 5 ul of patient serum. It is believed that the established 96-well plate platform described here may contribute to the rapid screening of potential N-glycan biomarkers in a large number of human patient serum sample sets.

Whole-body vibration exercise is a new type of exercise currently being tested in rehabilitation and that recently been developed for the prevention and treatment of osteoporosis[1, 2]. But most research in this field has been limited to osteogenesis and has focused on intervertebral disc degeneration. The aim of this study is to investigate the influence of lipolysis on matured 3T3-L1 cells by sonic vibration for 6 days. Cultured 3T3-L1 cells were subjected to sonic vibration 1 h treatment 3 times per day on an apparatus which was used sonic vibration with 3.5 and 5.1 volt and the frequency was the 50 Hz. The total TG (triglyceride) content of cells was similar compared with the control. But in the experiments of lipolytic activity, glycerol secretion was increased with the rates of approximately 59% and 60% at the intensity of 3.5 and 5.1 volt respectively. HSL (hormone-sensitive lipase) has traditionally been considered the key lipolytic enzyme in adipocytes[3].The gene expression levels of HSL was increased by using RT-PCR. In conclusion, treatment of sonic vibration inhibits adipogenesis through recduction in lipid accumulation.

Recombinant protein production in plant cell suspension culture is limited by secreted proteases with high level. To overcome this problem, we have been previously identified secreted protease and reported that the cysteine proteinase (CysP) was one of the main secreted protease after sugar starvation in the rice suspension culture. It was investigated whether the production of trypsin in rice cell line (pMYT111-04) which stably produced trypsin could be improved by suppressing expression of CysP gene. The suspension culture of rice cell transformed with both the trypsin and a dsRNA of CysP contained reduced total protease and CysP activities, and increased trypsin production levels compared those of cell lines harboring the trypsin only. The production of trypsin increased by up to 2 folds at 5 days using RNAi technology Acknowledgements This work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry and Energy.

Gaucher disease caused by deficiency of glucocerebrosidase (GCD) also known as β-glucosidase, is currently treated by enzyme replacement therapy.1) For GCD to be effective in this therapy, terminal mannose residues must be presented on the glycan chains to permit binding to the mannose receptors on macrophage which is target cell. In this study, GCD was expressed in transgenic rice cell suspension cultures and its characteristics were investigated. Plant-based systems were commonly used the production of glycoprotein, together with its targeting strategies generate GCD with terminal mannose structures for biological activity.2) The expression vector with rice RAmy3D promoter was constructed to express GCD and the production could be induced by sucrose starvation in medium.3) GCD cell lines were screened to confirm the fundamental characteristics of growth and production in callus form, respectively. After the induction, intracellular GCD extracted from the callus was characterized using Western blot. The calli expressing high levels of GCD was used to establish suspension cell lines. Growth properties of suspension cell lines were comparable to that of the other rice systems. The GCD was produced in suspension cultures after induction for 12~24 hours with sugar-free medium. The productivity of intracellular and extracellular GCD increased until 60~84 hours. Therefore, we can conclude that GCD could be successfully produced in transgenic rice suspension cells.

Calcium is a critical parameter required for influencing the interaction of both the cell-cell and the cell-matrix. Extracellular calcium has strong potentiating effects on cell aggregation for diverse cell types in vitro. Cell aggregation during cell culture causes obstruction to nutrient and oxygen transfer, leading to cease of cell growth and eventually cell death. It has been reported that low calcium levels in serum-free media reduces aggregation of HEK293 cells and chondrocytes. In preceding studies, we observed the effect of high calcium concentrations on cell aggregation occurred with various recombinant Chinese Hamster Ovary-DG44 (rCHO-DG44) cell lines expressing different protein in suspension culture and found each rCHO-DG44 cell lines had different optimum calcium concentration on cell growth. In this study, we observed how the phenomenon of different aggregation characteristics by calcium levels with CHO-DG44 host cell line and various rCHO-DG44 cell lines recombination by same host cell line and expressing identical proteins but selected differently.

Increasing interest is now set in clinical applications of human mesenchymal stem cells (MSC). In order to explore there usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of cord blooddrived mesenchymal stem cells using Hellex microcarrier. Hillex microcarrier showed the results for adherence with over 80% adherent cells after 3 h of incubation. We also cultivated the MSCs in 12 well plate to comparison with two dimensional monolayer cultivation. Results shown that the three dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two dimensional monolayer cultivation method.References 1. M. Du¨ rrschmid, K. Landauer, G. Simic, G. Blu¨ml and O. Doblhoff Dier Strategies for Microcarrier Based Animal Cell Bioprocesses BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 6, SEPTEMBER 20, 2003 681-686S. Tielens, H. Declercq, T. Gorski, E. Lippens, E. Schacht and M. Cornelissen Gelatin-Based Microcarriers as Embryonic Stem Cell Delivery System in Bone Tissue Engineering: An in-Vitro Study BIOMACROMOLECULES 2007, 8, 825-832.

Mucosal vaccination has been considered as an attractive immunization principle based on the consideration that mucosal surfaces are vulnerable for invasion by pathogen. In that sense, transgenic plant is noticed as promising carrier offering attractive option for production and delivery of mucosal vaccine. Classical swine fever (CSF) is a highly contagious viral disease of pigs and results in devastating financial losses. Because CSFV E2 glycoprotein mediates the viral attachment and penetration to susceptible cells, it is considered as a target for inducing protective immunity. In this study, in order to develop the vaccine materials for mucosal immunization against the CSFV infection, rice calli expressing E2 were established. Among them, p15B, not expressing KDEL, and p16B, expressing KDEL, express E2 protein of about 5 mg/mg dry weight. In addition, the proteins expressed in the transgenicrice were immunogenic in mice. More importantly, recombinant E2 conjugated with M cell ligand targeting ligand, Co1, effectively induced immune response after oral immunization of the mice. Currently, we are analyzing immune responses induced by oral administration of E2-expressing rice cells. (This work was supported by the grant from ARPC.)

Currently, obesity has become the risk factor to lead many metabolic diseases including hypertension, type 2 diabetes mellitus, cancer and osteoarthritis. Capsaicin as a potential thermogenic agent has been reported as a tool for obesity management by increasing energy expenditure and fatty acid oxidation on skeletal muscle. In the present study, effects of capsaicin on expression of skeletal muscle proteins in rats fed with high-fat diet (HFD) were investigated. Rats were fed with the HFD for a period of 8 week and/or were treated with capsaicin. After HFD feeding, capsaicin-treated group weighed an average of 8% less than HF-diet group. Capsaicin treatment increased phosphorylation of AMPK and ACC compared with HFD control group. HFD fed rats led to increase of UCP3 protein expression, whereas capsaicin treatment reduced UCP3 contents. These data suggest that effect of capsaicin on energy expenditure and fatty acid oxidation in muscle might be mediated through activating AMPK-ACC-malonyl-CoA metabolic signaling pathway. The proteomic analysis demonstrated that 33 spots were differentially significantly expressed from a total of about 500 matched spots, of which 27 spots were identified as muscle proteins altered in response to capsaicin feeding, and 6 spots were found to be unidentified by mass fingerprinting.

It is well recognized that capsaicin increases thermogenesis by enhancing catecholamine secretion from the adrenal medulla. In the present study, rats (5-week old) were administrated with capsaicin (10 mg/kg) with high fat diet for 2 months. The body weights of capsaicin-treated group were decreased by 8% compared to saline-treated rats. We also performed a differential proteomic analysis using 2-DE combined with MS to clarify the molecular mechanism for the thermogenic and anti-obesity effect of capsaicin in white adipose tissue (WAT). Protein mapping of WAT homogenates by 2-DE revealed that numerous proteins showing significant alterations: 11 spots were significantly up-regulated and 10 spots were remarkably down-regulated by capsaicin treatment in comparison to normal diet group. Among them, significant downregulation of heat shock protein 27 (Hsp27), Steap3 protein and up-regulation of olfactory receptor (Olr1434) in obese WAT were reported for the first time in relation to obesity. Most of the identified proteins are associated with lipid metabolism, cytoskeleton, and redox regulation, in which the levels of fatty acid binding protein 4 (FABP4), vimentin, peroxiredoxin were significantly reduced (two-fold), whereas aldo-keto reductase, NAD(P)H dehydrogenase, flavoprotein were increased with capsaicin treatment. These data imply that nonshivering thermogenesis-related proteins as well as anti-obesity related proteins were altered upon capsaicin treatment in WAT, suggesting that capsaicin may be useful phytochemical for attenuating obesity.

Transgenic plant cell culture is an attractive alternative for the production of recombinant proteins such as pharmaceutical proteins due to the absence of contaminants associated with mammalian or other culture systems. Plant cells are routinely maintained by periodic subculture that is a major drawback of the stable maintenance of high-producing cell lines1). To conform to existing regulatory requirements, plant cell cultures expressing biopharmaceuticals should be derived from a consistent and stable master stock. Cryopreservation would be an ideal method for retaining the original morphological forms and characteristics2). However, cryopreservation of plant cells has not been established yet because of the enormous difficulties for cell banking such as osmotic pretreatment, cryoprotection, freezing, thawing and regeneration. It is known that rice is resistant to anoxia, and sugar availability plays an important role3). In this study, transgenic suspension cells of Oryza sativa L. utilized as a host for producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) were preserved in anoxia condition for 2 months. Cell viability was stably maintained at 76% for 2 months in the media containing 174 mM glucose under anoxia condition, and the cells were successfully recovered. There were no significant differences in the cell growth and hCTLA4Ig production between non-preserved and 2-monthpreserved cells.

Transgenic plants are one of the promising protein production systems because they have several advantages including the capacity of post-translational modifications (PTMs). N-glycosylation is one of the most important PTMs related with the protein stability, serum half-life, and immune responses. Even though there are similarity in N-glycosylation mechanism between plant and mammalian cells, a little difference makes it difficult to develop therapeutic proteins in transgenic plant cells. Mammalianderived glycoproteins have penultimate β1,4-galactose and terminal sialic acid residues. On the other hand, plant-derived glycoproteins contain plant specific terminal β1,3-galactose, β1,2-xylose and α1,3-fucose residues.1) In this study, transgenic rice cells were transformed with human β1,4-galactosyltransferase (hGalT) gene for the production of more humanized galactose-extended human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4IgP).2) To investigate the effect of hGalT expression on the glycan structure of the recombinant protein, purified hGalT-CTLA4IgP was analyzed by Western blot and lectin affinoblot method using antibodies and glycan-specific lectins.3) We confirmed that hGalT in transgenic rice cells is not only functionally very active but also efficiently able to change the glycan structure of the recombinant protein into a more human-compatible type. Moreover, it was possible to interact with sialic acid residues and terminal galactose residues from hGalT-CTLA4IgP by in vitro sialylation. From these results, it is expected that enhanced in vivo halflife and protein stability of hCTLA4IgP with humanized glycan structures as well as reduced potential of immune response from much lower contents of xylose and fucose.

Newcastle disease virus (NDV) V protein is well known for its interferon antagonistic activity (1-4). The addition of anti-interferon antibody for blocking the binding between interferons secreted from NDV-infected cells and interferon receptors increases NDV virus productivity in cell-based production system. To block the host cellular interferon signaling efficiently, the stable cell lines expressing either lentogenic or velogenic NDV V protein were established. The overexpression of either lentogenic or velogenic V protein has an effect on enhancing NDV production kinetics but no effect on Japanese encephalitis virus (JEV) production. The overexpression of NDV V protein functions as an interferon antagonist through inhibiting NDV-induced increase of type-I interferons rather than incorporating virus particles as a structural protein or degrading STAT proteins (5-9). And the interferon signals in NDV V-expressing cell lines were weakened by decreased activation or expression of dsRNAactivated enzymes, PKR and OAS. These interferon antagonist activities can help rapid virus replication and spread in the early phase of virus production.

Consumption of spicy foods has been reported that it has thermogenic properties. Thus, those ingredients could be considered as potential agents which help preventing a positive energy balance and obesity. Specially, capsaicin (Cap) is the main capsaicinoids, which works pungent principle in red pepper, and is also utilized as a medicine. ated aboved In this study, twodimensional gel electrophoresis (2-DE) was carried out to identify the changes and/or differential expression of liver proteins in rats fed with highfat diet (HFD) in response to capsaicin treatments. In addition, immunoblot analysis of some liver proteins was examined for validation of proteomic analysis and suggestion of molecular action of capsaicin. Weight gain of rats in the HFD+Cap group was decreased by 8% compare to HFD control group. We analyzed the differential expression patterns in rat’s liver using 2-DE in order to search for other proteins associated with thermogenesis and obesity. The proteomic analysis of liver sample demonstrated that about 120 spots were differentially expressed from a total 950 matched spots, of which 23 spots have been identified by peptide mass finger printing using MALDITOF mass spectrometry. The protein levels of UCP2 and FAS in HFD+Cap group were decreased compared to HFD-Cap group, whereas those of p-AMPK, p-ACC and CPT-1 in HFD+Cap group were increased compared to HFD-Cap group. These data suggest that effect of capsaicin on energy expenditure and fatty acid oxidation in liver might be mediated through activating AMPK-ACC-malonyl-CoA metabolic signaling pathway.

Carbon-based composite electrodes were heralded in biosensor applications as a new paradigm for an engineered electrode material because of their high electronic conductivity and useful mechanical properties. In particular, the exfoliated graphite can be an inexpensive alternative to carbon nanotubes for the fabrication of biosensor devices due to its high sensitivity and fast responsibility [1, 2]. Here we report about the exfoliated graphene/Nafion electrode (EGNE) for detection of glucose. The morphology of EGNE was investigated by SEM imaging. The electrochemical properties of EGNE were characterized by cyclic voltammetry and amperometric responses. The exfoliated graphene/Nafion hybrids have the potential use of an excellent electrode material in the biosensor application due to biocompatibility and excellent bioelectrocatalytic properties. [This work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through KEIT and by the KOSEF through the Center for Ultramicrochemical Process Systems.]

Transparent conducting carbon nanotube (CNT) films were fabricated on the glass, which have the electrical conductivity and the optical transparency [1]. The singlewalled CNT (SWNT) film was investigated with gold-hybridization by a simple and cheap process, which electroless reduction of gold ions on the SWNT networks by the vacuum filtration can enhance their electrical conductivity without a substantial change in their transmittance of visible light. The gold-fabricated CNT film as a highly conductive electric device was also utilized for the efficient immobilization of the gold binding polypeptide (GBP) for detecting the biomolecular sensing [2]. The organophosphorus hydrolase (OPH) was used as a model protein for the specific immobilization of GBP-fusion protein onto the gold surface of the CNT film. As subsequent binding of GBP-OPH fusion protein and target molecules, electric conductance was reduced to compare with the value before binding. This phenomenon is suggesting that electrical signals represent the specific detection of organophosphorus pesticides [This work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through KEIT].

Localized surface plasmon resonance (LSPR) has been considered as a useful optical property for medical applications because it measures the local refractive index, which changes with composition, size, shape and local electric environments [1,2]. This method detects an immediate increase in the thickness of a biomolecular layer on the surface of a sensitive element caused by a reaction between the biomolecule in the solution and the receptor layer immobilized on the surface. In this study, we report LSPRbased detection of the BIGH3 gene mutations by using multi-spot goldcapped nanoparticle array (MG-NPA) biochip. The analytical range and sensitivity of the MG-NPA biochip were determined by measuring different concentrations of each Corneal Dystrophy (CD) target DNA in the range of 1 fM to 1 μM. The selective discrimination against a single-base mismatch DNA sequence was also achieved by using both homozygous and heterozygous CD samples. This MG-NPA platform would be useful for selective and sensitive detection of various DNA point mutations [This work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through KEIT].

Prostate-specific antigen (PSA) is a protein produced from the prostate gland. PSA is present in small quantities in the serum of normal men and is not present in the serum of normal women. However, the concentration of PSA is often elevated in the presence of prostate cancer and in other prostate disorders. Detecting abnormal concentration of PSA in the body is the main way for early discovery of the prostate cancer. There are two types of particles to detect pathogen in the enzyme amplification based bioassay, a magnetic microparticle(MMP) and an Au nanoparticle(AuNP). A MMP is functionalized with a monoclonal antibody which is attached by an affinity to a target of interest such as PSA, and an AuNP is funcionalized with an polyclonal antibody which is attached by an affinity to the same target of interest. The DIG-labeled Oligonucleotides with thiol terminal group was covalently attached on the AuNP, and then HRP labeled anti-DIG was bound to DIG labeled DNA on AuNP. By this process, the number of HRP on AuNP could be enhanced and effectively act as reporter groups for the target of interest. The immobilized enzyme on AuNP probes is reacted with tetramethylbenzidine (TMB) that is a substrate of HRP, followed by stopping the reaction with 2M H2SO4. The resulting end products were analyzed by UV-vis spectroscopy. Acknowledgments: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2009-0069113) , and by Nuclear R&D program through the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. M20706010003-08M0601-00310), and by the Ministry of Knowledge Economy(MKE) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Strategic Technology.

Antimicrobial peptides (AMPs) play an important role to cope with invasion of microorganisms. Based on the antimicrobial mechanism of AMPs, there have been considerable efforts to improve the antimicrobial activities of a-helical AMPs in association with structure-activity relationship (SAR) study. In this study, to demonstrate the relationship of distribution of cationic amino acids and antimicrobial activity of AMP, we designed pertinent KL (lysine and leucine) model AMPs to have same hydrophobic arc, net charge, and overall hydrophobicity and investigated effect of cationic amino acid distribution. Next, we confirmed it using natural AMP analogue, CRAMP18, and its derivatives. In results, we observed enhancement of AMP activity by modulation of dispersity for cationic distribution. Thus, we proposed that distribution of cationic amino acids on the hydrophilic surface of helical wheel structure is a determining factor for antimicrobial activity of AMP. It is expected that antimicrobial activities of natural AMPs can be improved by rational redesign of amino acid sequences through rearrangement of positively-charged amino acids on the hydrophilic surface region of helical wheel to have more uniform dispersity as well as parallel regulations of other known structural parameters.

Optical fiber sensors have been developed to determine the concentrations of dissolved oxygen(DO) and pH value in chemical and bioprocesses. In this work, Rudpp(tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II)) as a DO sensitive fluorescent dye and HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) as a pH sensitive fluorescent dye were attached separately on the tip of a bundle optical fiber for fabricating the DO/pH optical fiber sensor by using MTMS (methyl trimethoxy silane) and the mixture of GPTMS(3-glycidoxypropyl trimethoxy silane) and APTMS (3-aminopropyl trimethoxy silane) as their support materials respectively. This sensor could analyze the concentrations of DO and pH in simulation processes in the DO range 0-100% and pH range of 4.0-8.0.

Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological situations such as tumor tissue, osteoarthritis (OA) and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important information for diagnosis, prognosis, and therapy monitoring of diseases relating with MMP. A number of attempts have been made to detect activities of MMPs in biological samples and to evaluate the effects of MMP inhibitors. Previously, we have demonstrated that a highly efficient MMP fluorogenic probe, which is a pair consisting of a fluorophore (Cy5.5) and a quencher (BHQ-3), can detect MMP-13 in OA-induced rat model with a low background and significant signal recovery, enabling a highly sensitive visual detection in vitro and in vivo. In this study, we developed a new high-throughput MMP detection kit based on a 96-well microplate by immobilizing a rationale designed chitosan-conjugated fluorogenic probe and investigated whether this new MMP detection kit can monitor MMP activity. Glycol chitosan (GC), natural polymer, was used as a linker to immobilize and amplify MMP fluorogenic probe on a 96-well microplate for a highly sensitive detection. When MMP enzymes were simply added and incubated in a 96-well microplate immobilized with GCconjugated MMP probe, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through Kodak Image Station. Furthermore, recovery of fluorescent signals in the MMP detection kit was proportional to concentrations of immobilized GCconjugated MMP probe and, importantly, treated concentrations of pure MMP enzymes. The detection kit is most specific for MMPs, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the detection kit was used to detect MMP activity in biological samples such as synovial fluids from OA patients and showed the fluorescence intensity to correlate with the stage of OA development. The detection kit may be applicable in detecting MMP activity in other various biological samples and evaluate the effects of MMP inhibitors in a rapid and easy fashion.

In this work a microplate-based bioreactor equipped with the optical online monitoring system(MABOOMS) has been investigated for high-throughput screening of microorganisms and medium optimization in biotecnological processes for online monitoring of optical density (OD). An optical density sensing system has been made with 630 nm LED(light-emitting diode) and PMT(photomultiplier tube). It has been used to detect the reflect light intensity of a culture medium in 24-well microplate. A 24-well microplate has been employed to online monitor the optical density of fermentation processes with E.coli DH5α and B.cereus 318. The online monitoring data of the optical density have been compared with offline measurement data by using spectrophotometer. The tendency of the online data agreed with offline data, that is, the 24-well microplate based bioreactor was validated to be very useful to online monitor the cell growth characteristics.

Single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines and other agents. There are many SNP detection methods such as taqman method, invader method , MALDI-TOF method and Gene chip. But these methods need expensive equipments. For the cost-effective SNP detection, we used DNA duplex consisting of a reporter strand labeled with FAM or HEX and a sensor strand labeled with BHQ1. The sensor strand has a 6-nucleotide overhang that serves as a nucleation site. Depending on the SNP in a target oligonucleotide, the nucleation step is kinetically controlled. This leads to differential fluorescence changes, which can be used to identify the nucleotide at the SNP site. Because the FRETbased SNP detection method needs a conventional PCR cycler and a fluorometer, it enables SNP detection with a cost lower than the established procedures. AcknowledgementThis work was supported by BK21 program from the Korean Ministry of Education and Seoul R&BD Program NT080612, KU080657).