• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
• /
• 2010.06a
• /
• pp.280-280
• /
• 2010

Alzheimer disease is a progressive neurodegenerative disease of the aged, characterized by memory loss and dementia. For diagnosis of Alzheimer disease we have simply modified macrophage with amyloid beta bonded with different molecules. Modified Macrophage was observed with microscope for co-localization of amyloid beta molecule. For this experiment we used fluoroscene labeling substances. The macrophage was modified also with cell staining method. For cell staining method was used avidin-biotin reaction principles. All experiments were carried out on poly-L-lysine coated and sterilized glass substrates. In the presentation we will show the further investigations and applications with modified macrophage.

• Proceedings of the Korean Vacuum Society Conference
• /
• 2012.02a
• /
• pp.529-529
• /
• 2012

Dispersion of carbon nanotubes in biocompatible media are of particular interest for diverse biomedical and nanomedicine applications. Various biomolecules and biopolymers such as DNA, proteins, poly L-lysine, starch, gelatin, steroid biosurfactants, and chitosan have shown capability for the effective dispersion of carbon nanotubes in water. Chitosan has demonstrated capacity for effective dispersion of single-walled carbon nanotubes (SWCNTs) in acidic medium and it also showed tendency to preferentially disperse smaller diameter nanotubes. Chemical functionalizations of chitosan enable its solubility in neutral pH water by reducing the intra and inter molecular hydrogen bonding. Herein, we present a neutral pH water soluble chitosan derivative, chitosan-hydroxyphenyl acetamide (CHPA), obtained by functionalizing the amino groups of chitosan with 4-hydroxyphenyl acetic acid, as an efficient biocompatible dispersant for debundling and solubilization of SWNTs in neutral aqueous solutions. Various process conditions for individual dispersion of SWCNTs are analyzed based on optical absorption and Raman spectroscopy.

• Fisheries and Aquatic Sciences
• /
• v.8 no.3
• /
• pp.122-127
• /
• 2005

A DNA chip that rapidly and accurately detects the viral genes in rhabdovirus-infected fish was developed. The N, Ml, and G proteins of three rhabdovirus strains, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and flounder rhabdovirus (HIRRV), were selected for use as probes. The sequences of the corresponding genes were obtained, and probes were prepared by PCR using specific primer sets. The specificity of the probes was confirmed by cross PCR. The prepared probes were spotted on poly-L-lysine- or aminosilane-coated glass slides and hybridized with target DNA under several different conditions in order to determine the optimal hybridization temperature, glass-slide coating, and target cDNA concentration.

• Macromolecular Research
• /
• v.11 no.1
• /
• pp.19-24
• /
• 2003

Polymeric gene delivery system attracts profound attention as it shows less toxicity, versatility, and reasonable gene expression efficiency. Terplex system, a synthetic biopolymeric gene delivery system consisting of stearyl poly-L-lysine (stearyl-PLL) and low density lipoprotein (LDL) was evaluated for its body distribution of gene expression of exogenously administered pDNA after tail-vein injection in mice. Kidney and spleen are two major organs with highest gene expression, whereas liver and heart showed marginal gene expression among the organs examined. Hemolytic effect of the terplex system was evaluated using human red blood cells, where terplex system did not cause significant hemolysis at the concentrations above the experimental ranges, although unmodified PLL or stearyl-PLL without LDL did. Serum stability of terplex system against enzymatic degradation was also significantly enhanced, presumably due to the steric stabilization from the polymers. Based on these findings and along with its high in vitro transfection efficiency, terplex system could serve as a safe and efficient polymeric gene delivery system with many applications for the in vivo gene therapy.

• Journal of Photoscience
• /
• v.7 no.3
• /
• pp.123-128
• /
• 2000

Phosphorylation of cellular proteins is a key regulatory mehanism for signal transduction pathway in living cells. Phytochrome, a red/far-red light photoreceptor in plants, is known to employ protein phosphorylation for its light signaling, although its detauked mechanism is still ambiguous. This study is intended to identify the phosphoproteins and protein kinases that are regulated by phytochrome, by employing transgenic rice seedlings that overexpress Arabidopsis phytochrome A. Red light stimulated phsophorylation of a 70 kDa protein and far-red light negated the effect. The red light induced phosphotylation of the 70 kDa protein was strongly activated by heparin and inhibited by poly-L-lysine, suggesting that the 70 kDa protein phosphorylating kinase belongs to GSK-3/SHAGGY protein kinase that has functional roles in establishing cell fate and pattern formation in Drosophila. Taken together with the fact that phytochrome controls plant development, these results may suggest that a GSK-3/SHAGGY-related protein kinase in plant(ASK) is likely to be involved in phytochrome signal transduction.

• 한국생물공학회:학술대회논문집
• /
• 2000.11a
• /
• pp.703-706
• /
• 2000

We constructed a biospecific affinity surface using hyper-branched dendrimers on gold for biospecific recognition, and characterized the resulting surfaces by using confocal fluorescence microscopy. The dendrimer monolayer was firstly constructed on the mercaptoundecanoic acid SAM/Au with pentafluorophenyl ester activation and further functionalized with sulfo-NHS-biotin, an activated ester of biotin. To confirm the formation of biospecific affinity surface, FITC(fluorescein isothiocyanate)-labeled avidin was loaded onto the biotinylated dendrimer monolayer, and fluorescence images of the bound avidins were investigated with a confocal microscope. The constructed biospecific affinity surface showed a much more dense and uniform fluorescence compared to those from poly-L-lysine- and cystamine SAM-based affinity surfaces. For the dependency on the concentration of added FITC-labeled avidin on the affinity surface, derived fluorescence could be detectable from as low as $1{\mu}g/ml$, and intensified up to $50{\mu}g/ml$. Further reaction of FITC-labeled avidin layer with TMR(tetramethylrhodamine)-biocytins resulted in the efficient FRET(fluorescence resonance energy transfer) phenomenon. As an extension of the study, we attempted a patterning of the affinity surfaces on gold by microcontact printing. Fluorescence of the patterned surface demonstrated that FITC-labeled avidin molecules were specifically bound to the biotinylated patches.

• Biomolecules & Therapeutics
• /
• v.10 no.3
• /
• pp.156-164
• /
• 2002

In the present study, we tried to investigate whether polymerized basic amino acid e.g. poly-L-lysine (PLL) which has the degree of polymerization under 50mer significantly affects the physiological and stimulated mucin release from cultured hamster tracheal surface epithelial cells. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with $^3{H}$-glucosamine for 24 hr and chased for 30 min in the presence of either PLLs or adenosine triphosphate (ATP) and PLL to assess the effects on basic or ATP-stimulated $^3{H}$-mucin release. Possible cytotoxicities of PLLs were assessed by measuring lactate dehydrogenase (LDH) release from HTSE cel1s during treatment. The results were as follows: PLLs significantly inhibited basic mucin release from cultured HTSE cells in a dose-dependent manner from the range of 46mer to 14mer; PLL 46mer significantly inhibited the stimulated mucin release by ATP from cultured HTSE cells; there was no significant release of LDH from cultured HTSE cells during treatment. We conclude that PLLs inhibit both physiological and stimulated mucin release from airway epithelial cells without significant cytotoxicity and PLL lost its activity under the range of 14mer. This finding suggests that polymer of basic amino acid like PLL might function as a regulator for hypersecretion of mucus manifested in various respiratory diseases.

• Molecules and Cells
• /
• v.19 no.1
• /
• pp.124-130
• /
• 2005

Protein kinase CKII is composed of two catalytic (${\alpha}$ or ${\alpha}^{\prime}$) subunits and two regulatory (${\beta}$) subunits. The ${\beta}$ subunit mediates tetramer formation through ${\beta}-{\beta}$ homodimerization and ${\alpha}-{\beta}$ heterodimerization. In a previous study R26 and R75, point mutants of $CKII{\beta}$ defective in ${\beta}-{\beta}$ dimerization, were isolated. In the present work we characterized these $CKII{\beta}$ mutants in vitro. Purified R26 and R75 bound to $CKII{\alpha}$ but were defective in binding to $CKII{\beta}$. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with $CKII{\beta}$ and to activate $CKII{\alpha}$.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.5
• /
• pp.783-790
• /
• 2007

The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prismtype dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor® 488, Qdot® 525 and Alexa Fluor® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.

• IMMUNE NETWORK
• /
• v.1 no.1
• /
• pp.87-94
• /
• 2001

Background: Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of cord blood (CB) hematopoietic stem cells for transplantation. As well as stem cell number, stromal cells are necessary for functional maturation of hematopoiesis. The purpose of this study was to analyze the development of stromal cells during ex vivo expansion of CB $CD34^+$ cells. Methods : $CD34^+$ cells were purified from CB by magnetic bead selection. The levels of of interleukin-3, interleukin-$1{\beta}$, interleukin-6, granulocyte macrophagecolony stimulating factor and tumor necrosis factor-${\alpha}$ were measured in culture supernatants on 0, 1, 2, and 3 weeks, using ELISA techniques. CB $CD34^+$ cells were expanded in Iscoves modified Dulbeccos medium in the presence of several cytokines. The expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet/endothelial cell adhesion molecule-1, von Willebrand factor, vimentin, and CD14 in newly developed stromal cells was examined by immunocytochemical method. Relevant extracellular matrix (ECM) proteins and proper cytokines were also assayed for the most suitable condition for expansion of stromal cells. Results: Several cytokines were found to have been produced by CB $CD34^+$ cells as well as bone marrow-derived $CD34^+$ cells. During ex vivo expansion of CB $CD34^+$ cells, stromal cells appeared in the culture by day 4 and expanded over the following 7-10 days before being confluent by day 2 1. These cells expressed surface markers characteristic of cells of endothelial lineage. Furthermore, these stroaml cells also expanded effectively when treated with thrombopoietin+flt-3 ligand+stem cell factor+leukemia inhibitory factor or 0.1% poly-L-lysine-coated wells. Conclusion: Stromal cells were developed during ex vivo expansion of CB $CD34^+$ cells and that this development could be enhanced further by treating the stromal cells with cytokines or ECM.