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Biomedical subjects

O H Kwon

Publications and source records attributed to O H Kwon.

At least 19 recordsLinked to original sources
PubMed2001

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces.

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.

Acrylic Resins↗
PubMed2000

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion.

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.

Adenosine Triphosphate↗
PubMed2000

Rapid cell sheet detachment from poly(N-isopropylacrylamide)-grafted porous cell culture membranes.

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.

Acrylic Resins↗
PubMed2000

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces.

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.

Acrylamides↗
PubMed2000

Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface.

We developed a novel method to obtain designed shape cell sheets for tissue engineering. Shaping of cell sheets were achieved by the use of poly(N-isopropylacrylamide) (PIPAAm) and poly(N,N'-dimethylacrylamide) (PDMAAm) for temperature-responsive cell adhesive and cell nonadhesive domains, respectively. These polymers were covalently grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation with mask patterns. At 37 degrees C, human aortic endothelial cells (HAECs) attached, spread, and proliferated to make a monolayer only on PIPAAm-grafted domains. HAECs did not adhere on PDMAAm-grafted domains for more than 1 month even under the serum-supplemented condition. By reducing the culture temperature below 32 degrees C, PIPAAm changed to hydrophilic and HAEC sheets were detached from PIPAAm-grafted surfaces without any need of an enzyme such as trypsin. Cell-cell junctions were retained in the recovered cell sheets and easily moved to virgin TCPS dishes with the aid of hydrophilically modified polyvinylidenefluoride membranes as a supporter during the transfer. Moved cell sheets rapidly adhered onto the dish surfaces, and the supporter was easily peeled off from the cell layers. HAEC sheets transferred to new dishes revealed the identical shape and size to those before transfer. This novel technique is the only way to create, harvest, and transfer designed shape cell sheets and would have promising applications in tissue engineering.

Acrylamides↗
PubMed2000

Radiation-induced grafting of methylmethacrylate onto ultrahigh molecular weight polyethylene and its adhesive characteristics.

The surface of ultrahigh molecular weight polyethylene (UHMWPE) was modified by radiation-grafting methylmethacrylate (MMA) in the presence of sulfuric acid and metallic salt to increase bonding strength with polymethylmethacrylate. The effect of the addition of metallic salts and sulfuric acid on the radiation grafting reaction was investigated when MMA was grafted to the irradiated UHMWPE. The adhesive characteristics with the grafting yield were investigated using conventional acrylic bone cement based on poly(methyl methacrylate) [PMMA]. The results showed that the inclusion of an FeSO4 . 7H2O and sulfuric acid in MMA grafting solutions was extremely beneficial and led to a most unusual synergistic effect, while CuSO4 . 5H2O led to a detrimental effect. The tensile bonding strength between UHMWPE and PMMA sheet increased remarkably with an increased grafting yield on UHMWPE surfaces.

Journal Article↗
PubMed2000

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures.

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.

Acrylic Resins↗
PubMed1999

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media.

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.

Bacillus↗
PubMed1999

Enzyme modification by polymers with solubilities that change in response to photoirradiation in organic media.

We have synthesized a hybrid subtilisin the solubility of which can be regulated by photoirradiation through coupling with a photoresponsive copolymer that carries spiropyran groups in its side chains. The copolymer was synthesized by polymerization of methacrylate, methacrylic acid, and spiropyran-carrying methacrylate. It was then covalently bonded to the amino groups of subtilisin Carlsberg via its carboxyl groups using a carbodiimide coupling agent. The hybrid subtilisin was perfectly soluble in toluene and efficiently catalyzed transesterification. After ultraviolet irradiation, the hybrid subtilisin precipitated and was easily and quantitatively recovered by centrifugation. Recovered hybrid subtilisin, resolubilized by visible light irradiation, retained its original transesterification activity even after several cycles of precipitation and solubilization.

Benzopyrans↗
PubMed1998

Hydrophobilization of esterase by genetic combination with polyproline or polytyrosine at the carboxyl terminal.

Polyproline (Poly-Pro) that is an amphiphilic polypeptide, or polytyrosine (Poly-Tyr) that is a hydrophobic polypeptide, were connected to the carboxy terminus of Pseudomonas sp. esterase by the recombinant DNA technique. The hydrophobicity of the esterase was enhanced by the introduction of Poly-Pro or Poly-Tyr, and also by increasing chain length of the polypeptides. Poly-Tyr increased the hydrophobicity of esterase more than Poly-Pro. Poly-Tyr induced significant conformational change of fusion esterase, but Poly-Pro did not. Consequently, the introduction of Poly-Tyr led to loss of activity of the fusion enzyme to a negligible level. On the other hand, the Poly-Pro-fusion-esterase retained enzymatic activity and the hydrolytic activity (kcat/Km) of the fusion esterase carrying 40 proline residues (esterase-Pro40) relative to that of the wild-type esterase with the substrates p-nitrophenyl-propionate, -pentanoate, and -hexanoate was 1.76, 1.95, and 4.7, respectively. The results could be explained in terms of easier access of long-chain carboxylate to the fusion esterase compared to the wild-type esterase in aqueous solution.

Base Sequence↗
PubMed1998

Benzylamine-based selective and orally bioavailable inhibitors of thrombin.

A series of p-aminomethylphenylalanine derivatives were investigated as novel thrombin inhibitors. This study led to potent inhibitors of thrombin (Ki up to 3.3 nM) that are trypsin-selective, highly orally bioavailable in rats, and highly permeable across Caco-2 cells. The P1 benzylamine binding mode in the thrombin active site was identified by X-ray crystallographic analysis.

Animals↗
PubMed1997

Bactericidal activity of human lysozymes carrying various lengths of polyproline chain at the C-terminus.

The amphiphilic polypeptide polyproline having different chain lengths was connected to the C-terminus of human lysozyme by the recombinant DNA technique. The hydrophobicity of human lysozyme increased with increasing length of the polyproline chain. Although the bactericidal activity of wild-type lysozyme is limited to gram-positive bacteria and the hydrolytic activity of the mutant lysozyme decreased with increasing chain length of polyproline, the mutant lysozymes showed bactericidal activity to gram-negative bacteria and the activity increased with increasing hydrophobicity of the mutant enzyme. Experiments with Escherichia coli phospholipid liposomes revealed that the mutant human lysozymes dissipated the valinomycin-induced transmembrane electrochemical potential, and the dissipation increased with increasing hydrophobicity. The increased hydrophobicity of the mutant enzyme may induce interaction of lysozyme with the outer membrane and subsequent penetration into the inner membrane of E. coli, resulting in an increase of bactericidal activity.

Anti-Bacterial Agents↗
PubMed1997

Apolipoprotein E genotyping and phenotyping in type II diabetes mellitus patients with hypertriglyceridemia.

OBJECTIVES: We investigated the possible effect of apolipoprotein E (apo E) polymorphism on hypertriglyceridemia in type II diabetes mellitus (DM) patients by both apo E genotyping and phenotyping methods. DESIGN AND METHODS: Eighty Korean type II DM patients were evaluated. Restriction isotyping after DNA amplification was used for apo E genotyping. The isoelectric focusing of neuraminidase-treated sera followed by immunoblotting was used for apo E phenotyping. RESULTS: The concordant rate between apo E genotyping and phenotyping was 96.3%. Apo E genotype frequencies for all 77 concordant cases were as follows: 72.7% for epsilon 3/3; 16.9% for epsilon 3/4; 7.8% for epsilon 2/3; 1.3% for epsilon 2/4, 1.3% for epsilon 4/4; and 0% for epsilon 2/2. There were no significant differences in apo E genotype frequencies between hypertriglyceridemic (n = 42) and normotriglyceridemic (n = 35) groups. CONCLUSIONS: Our findings could not support the association between apo E polymorphism and hypertriglyceridemia among type II DM patients.

Aged↗
PubMed1997

In vitro blood compatibility of functional group-grafted and heparin-immobilized polyurethanes prepared by plasma glow discharge.

Blood compatibilities of functional group-grafted and heparin-immobilized polyurethanes (PUs) were investigated using in vitro thrombus formation, plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) activation. In the experiment with plasma proteins, PRT was shortened on amine group-grafted PU (PU-NH2) but prolonged on heparin-immobilized polyurethane (PU-Hep) when compared to PU control. APTT was significantly prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion was slightly increased by the introduction of functional groups such as carboxylic acid and primary amine on PU surfaces, but significantly decreased by the immobilization of heparin on the same substrate. The percentage of serotonin released from platelets adhered on surface-modified PUs was increased with increase of platelet adhesion. In the PBMC experiment, cells adhered less on heparin-immobilized PUs than on functional group-grafted PUs, and the production levels of tumour necrosis factor mRNAs from the cells stimulated by heparin-immobilized PU (PU-N-Hep) were smaller than those by the other substrates.

Amines↗
PubMed1997

Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.

Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.

Arginine↗
PubMed1996

Preparation and surface characterization of functional group-grafted and heparin-immobilized polyurethanes by plasma glow discharge.

Functional group-grafted polyurethanes were prepared by oxygen plasma discharge treatment, followed by graft polymerization of 1-acryloylbenzotriazole (AB) and a subsequent substitution reaction of AB with sodium hydroxide and ethylene diamine. The primary amine or carboxylic acid groups grafted on the surfaces were coupled with heparin using water-soluble carbodiimide. The modified surfaces were characterized by measuring the water contact angle, electron spectroscopy for chemical analysis and attenuated total reflection Fourier-transform infrared spectroscopy. The amount of heparin covalently immobilized on the primary amine- and carboxylic acid group-grafted polyurethanes were 2.0 and 1.4 micrograms cm-2, respectively, as determined by the toluidine blue method. The water contact angle of the polyurethanes was decreased by AB grafting, and further decreased by the introduction of functional groups such as carboxylic acid and primary amine and immobilization of heparin, showing increased hydrophilicity of the modified surfaces. Heparin was almost not released from the immobilized surfaces in the physiological solution for 100 h, indicating good stability of immobilized heparin.

Amines↗