Biomedical subjects

Jun Miyake

Publications and source records attributed to Jun Miyake.

35 records · Page 2Linked to original sources

PubMed2004

Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis.

OBJECTIVE: Biodegradable materials with autologous cell seeding have attracted much interest as potential cardiovascular grafts. However, pretreatment of these materials requires a complicated and invasive procedure that carries the risk of infection. To avoid these problems, we sought to develop a biodegradable graft material containing collagen microsponge that would permit the regeneration of autologous vessel tissue. The ability of this material to accelerate in situ cellularization with autologous endothelial and smooth muscle cells was tested with and without precellularization. METHODS: Poly(lactic-co-glycolic acid) as a biodegradable scaffold was compounded with collagen microsponge to form a vascular patch material. These poly(lactic-co-glycolic acid)-collagen patches with (n = 10) or without (n = 10) autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Histologic and biochemical assessments were performed 2 and 6 months after the implantation. RESULTS: There was no thrombus formation in either group, and the poly(lactic-co-glycolic acid) scaffold was almost completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth muscle cells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extracellular components in the patch had increased to levels similar to those in native tissue at 6 months. CONCLUSIONS: The poly(lactic-co-glycolic acid)-collagen microsponge patch with and without precellularization showed good histologic findings and durability. This patch shows promise as a bioengineered material for promoting in situ cellularization and the regeneration of autologous tissue in cardiovascular surgery.

Animals↗

PubMed2004

Transfection microarray of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified glass slide.

Cell-based microarrays are emerging as a tool for analyzing the functions of genes in cells. However, partly due to the difficulty of cell immobilization, the application of this method has been limited to adherent cells. We previously reported a method that rapidly and strongly attached living nonadherent cells to glass slides modified with a cell membrane anchoring reagent, designated a biocompatible anchor for membrane (BAM). Here we demonstrate that plasmid DNA deposited in a defined area on BAM-modified glass slides was transfected into nonadherent K562 cells immobilized on the DNA-deposited and BAM-modified slides. This method allowed the transfection of K562 cells not only with plasmid cDNA expression vectors but also with small interfering RNA (siRNA) at a defined location on the BAM-modified slides. We expect this methodology to greatly expand the scope of current cell microarray technology.

Biocompatible Materials↗

PubMed2003

Novel method of preparing acellular cardiovascular grafts by decellularization with poly(ethylene glycol).

We have developed a new method of preparing acellular vascular grafts. Cellular components, including cell membranes and proteins in cytosol, were efficiently extracted from the vessels in a concentrated aqueous solution of poly(ethylene glycol), an amphiphilic biocompatible polymer. The residual DNA was digested by deoxyribonuclease I treatment after extraction with poly(ethylene glycol). The two-step extraction process proved quite effective at removing the cellular components while causing little damage to the extracellular matrices. We did not use any detergent that would damage the extracellular matrices. Therefore, vascular endothelial cells grew well on the acellular vessels after recellularization, promising longi-patent cardiovascular grafts.

Animals↗

PubMed2003

Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column.

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.

Antibodies↗

PubMed2003

Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip.

In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed2003

Conventional detection of 2,4-dinitrophenol using quartz crystal microbalance.

We present conventional detection of 2,4-dinitrophenol (DNP) for using the competitive reaction between DNP and DNP-conjugated albumin onto DNP antibody immobilized quartz crystal microbalance (QCM). This QCM method allows detection of DNP concentration in the range of 0.01 to 100 ng/ml; linear correlation obtains DNP concentration from 1 to 100 ng/ml.

2,4-Dinitrophenol↗

PubMed2003

Immobilized culture of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified surface.

Microarrays of living cells are an emerging tool in systems such as reverse transfection. These studies are limited to adherent cells partly because of the difficulty of cell immobilization. Using a newly developed reagent, the biocompatible anchor for membrane (BAM), we show herein the rapid and strong attachment of living nonadherent cells and adherent cells on BAM-modified surfaces. Normal cellular growth was observed for over 7 days on BAM-modified surfaces. We expect this methodology to greatly expand the scope of current cell microarray technology.

Animals↗

PubMed2003

Mesenchymal stem cells and bioceramics: strategies to regenerate the skeleton.

Bone is formed by cells called osteoblasts, which arise from mesenchymal stem cells (MSCs). The cells are known to exist in thin tissues surrounding bone (periosteum) and bone marrow, but the population is extremely small. The number of marrow-derived MSCs can be expanded using tissue culture techniques. These culture-expanded MSCs have the in vitro capacity to differentiate into osteoblasts. Importantly, the cultured osteoblasts can form extracellular matrix in culture. This matrix consists of fine crystals of hydroxyapatite comparable to natural bone mineral, as evidenced by X-ray diffraction and Fourier-transform infrared spectroscopy. It is possible to fabricate the osteoblasts/ bone matrix on the surface of bioceramics. Thus in vitro cultured bone can show further bone-forming capability after in vivo implantation. We have begun studying this tissue-engineering approach in patients with skeletal problems. This paper describes this and other approaches using MSCs to regenerate skeletal tissue.

Biocompatible Materials↗

PubMed2002

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity.

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2'-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.

Adenosine Triphosphate↗

PubMed2002

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans.

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.

Humans↗

PubMed2002

A novel ISFET-type biosensor based on P450 monooxygenases.

We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase. ISFETs are electrical devices and have been used as pH sensors. We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity. The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22. Yeast microsomal membranes were immobilized in an agarose layer on the ISFET. o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH). Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence. The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase. There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity. This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds.

Biosensing Techniques↗

PubMed2002

A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode.

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.

Aluminum Silicates↗

PubMed2002

Design and activity of antimicrobial peptides against sporogonic-stage parasites causing murine malarias.

Insects produce several types of peptides to combat a broad spectrum of invasive pathogenic microbes, including protozoans. However, despite this defense response, infections are often established. Our aim was to design novel peptides that produce high rates of mortality among protozoa of the genus Plasmodium, the malaria parasites. Using existing antimicrobial peptide sequences as templates, we designed and synthesized three short novel hybrids, designated Vida1 to Vida3. Each has a slightly different predicted secondary structure. The peptides were tested against sporogonic stages of the rodent malaria parasites Plasmodium berghei (in vitro and in vivo) and P. yoelii nigeriensis (in vitro). The level of activity varied for each peptide and according to the parasite stage targeted. Vida3 (which is predicted to have large numbers of beta sheets and coils but no alpha helices) showed the highest level of activity, killing the early sporogonic stages in culture and causing highly significant reductions in the prevalence and intensity of infection of P. berghei after oral administration or injection in Anopheles gambiae mosquitoes. The secondary structures of these peptides may play a crucial role in their ability to interact with and kill sporogonic forms of the malaria parasite.

Animals↗

PubMed2002

Enhancement of hydrogen production by a photosynthetic bacterium mutant with reduced pigment.

A novel mutant MTP4 was created from the wild-type strain Rhodobacter sphaeroides RV by UV irradiation for the enhancement of hydrogen production. The amount of light absorbed by MTP4 was lower than that by the wild-type strain at any wavelengths ranging from 350 to 1000 nm. This nature enables the illumination of cells in the deeper parts of a reactor. The contents of bacteriochlorophylls and carotenoids of the chromatophores prepared from MTP4 under the conditions for hydrogen production were reduced to 41 and 49% of those from the wild-type strain RV, respectively. Analysis of the light-harvesting (LH) complexes by SDS-PAGE showed that the amounts of LH1s and reaction centers (RCs) in MTP4 were retained, whereas that of LH2s was much less than that in RV. Although MTP4 had less pigments, its growth rate was equivalent to that of RV over a wide range of light intensities. MTP4 produces hydrogen with a stable manner. Using a plate-type reactor, it produced 50% more hydrogen than RV. A novel method of pigment reduction was found to be effective for the enhancement of hydrogen production per unit reactor.

Journal Article↗

PubMed2002

Spectral properties of phthalocyanines incorporated into resting and stimulated human peripheral blood cells.

Human peripheral blood cells stimulated by phytohemagglutinin (which serve as a model of cancerous cells) and resting cells were incubated in dimethyl sulfoxide solutions of various phthalocyanines. In order to diminish the influence of atmospheric oxygen the cells were embedded in a polymer (polyvinyl alcohol) film. Fluorescence spectra of the samples were measured over two regions of excitation wavelengths: at 405 nm (predominant absorption of the cell material) and in the regions of strong absorption of phthalocyanines (at about 605 nm and 337 nm). The intrinsic emission of cell material became changed as a result both of cells' stimulation and of incubation of cells in dye solution. In most cases the stimulated cells when stained by dye exhibited higher long wavelength fluorescence intensity than resting cells. This suggests higher efficiency of dye incorporation into cancerous cells than into healthy cells. The absorption spectra of samples were also measured. The spectra of various phthalocyanines in incubation solvent, in polymer and in the cells embedded in polymer, were compared. The comparison of properties of the cells stimulated for different time periods enabled to establish the conditions of stimulation creating a population of cells incorporating a large number of sensitizing molecules.

Humans↗

PubMed2001

Effect of Thermozeaxanthins on Liposome Membranes in Acidic and Basic Conditions.

Thermozeaxanthins (TZS), a group of carotenoid-glucoside esters extracted from the lipids fraction of thermophilic bacterium, are amphiphilic compounds. The effects of TZS on membrane permeability were examined on large unilamellar liposomes (LUVs) in different pH buffers. The LUVs were composed of 0.01 molar of TZS and phosphatidylcholine (PC) of various lengths and saturation degrees of hydrocarbon chains. The results showed that the LUVs containing TZS were more stable than that liposomes without TZS in pH 5.0, and no significant effects in pH 8.2 and 9.0 buffer solutions. The liposomes composed of TZS and egg PC or E.coli PE were better than those DPPC or DOPC LUVs containing TZS at same experimental conditions. There was no observed difference in stability between the liposomes with or without TZS in pH 6.5 solution. Conclusions (1) Matching of rigid TZS in the lipid bilayer was important in stabilization effects on liposomes, (2)The TZS showed some stabilization effects on liposome membranes in acidic conditions and no significant effects in basic environment.

Journal Article↗

PubMedDate not supplied

PCR-based ribosomal DNA detection technique for microalga (Heterosigma carterae) causing red tide and its application to a biosensor using labeled probe.

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.

Base Sequence↗