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Biomedical subjects

I Karube

Publications and source records attributed to I Karube.

At least 55 records · Page 3Linked to original sources
PubMed1998

Light dose and time dependency of photodynamic cell membrane damage.

We have investigated the light dose and time dependency of photodynamic cell membrane damage using electrophysiological methods. This study controls the level of cell membrane damage by precisely administration of the light dose. The photosensitizer used was 5',5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride (BAT). A confocal laser scanning microscope was used to provide rapid light activation (< 1 s) and the subsequent membrane damage was monitored using standard patch clamp techniques. In the presence of 49 microM BAT, light levels less than 0.94 J/cm2 led to a reversible depolarization (approximately 20 mV) and reduction of resistance (approximately 10%) within 3 s of illumination. Higher intensities of illumination (> 1.57 J/cm2) caused a complete and irreversible loss of membrane potential and cell membrane resistance within 8 s illumination. The threshold dose of light required to induce cell death by illumination in the presence of BAT was increased in the presence of the antioxidant Trolox-C.

Animals↗
PubMed1998

Isoprenoid synthesis gene for geranylgeranyl diphosphate synthase from a hyperthermophile, Pyrococcus sp. strain OT3.

Pyrococcus sp. strain OT3, a hyperthermophilic archaeon that was isolated by the authors was found to contain tetraether lipid mainly in the membrane lipid, which was quite different from the other hyperthermophiles (Masuchi et al. 1997). Those isoprenoids are synthesized by a family of isoprenyl diphosphate synthases from isopentenyl diphosphate to allylic diphosphates. The gene that encodes one of these families, geranylgeranyl diphosphate synthase (GGPPSase), from this strain was cloned and sequenced. This coding gene has a 960-bp (320aa) sequence. The putative Shine-Dalgarno sequence was six bases upper of start codon, exactly the same as Methanobacterium thermoautotrophicum, a methnogenic thermophile. Comparison of the amino acid sequence of 13 organisms including Eukarya, Bacteria, and Archaea showed that Archaea strains including Pyrococcus sp. strain OT3 consisted of a separate group from the others, but five conservative regions are very homologous.

Journal Article↗
PubMed1997

Dielectric measurements of lysozyme and tri-N-acetyl-D-glucosamine association at radio and microwave frequencies.

Time domain dielectric measurements were applied to the monitoring of molecular recognition by proteins. Lysozyme and tri-N-acetyl-D-glucosamine((NAG)3) were selected as a typical lock and key type recognition system. After association of (NAG)3, relaxation related to lysozyme itself was increased and depended on the pH of the solution. No change was detected in hydration of the enzyme before and after association.

Electrochemistry↗
PubMed1997

A chemiluminescent FIA biosensor for phosphate ion monitoring using pyruvate oxidase.

We have constructed an automatic phosphate ion sensing system for the quality control of drinking water. The analyte was detected using the phosphate ion-dependent pyruvate oxidase reaction and the hydrogen peroxide produced was detected by luminol chemiluminescence catalyzed by Arthromyces ramosus peroxidase. We obtained a detection limit of 0.16 microM phosphate ion (5 ppb phosphorus) and it was possible to detect 0.32 microM phosphate ion for 48 days using pyruvate oxidase immobilized on Chitopearl BCW-2601 beads. An excellent correlation (r2 = 1.00) was obtained between the results obtained using our phosphate ion sensor and those using a modified Molybdenum Blue method.

Automation↗
PubMed1997

[Rapid detection of the Escherichia cori verotoxin gene using fluorescence polarization].

The effects of NaCl concentration, temperature and base-pair mismatches on the hybridization of two complementary single-stranded DNA 24-mers were investigated using a fluorescence polarization method. Over a temperature range of 46 degrees C to 56 degrees C in 0.8 M NaCl it was found that hybridization was essentially complete in under 10 minutes and that rapid in vitro determination of the target DNA was possible when the sample DNA had three or less base-pair mismatches in the 24-mer sequence. Polarization measurements on positive and negative samples showed excellent agreement with results obtained from electrophoresis. Under our optimized conditions, a 23 base-pair single-stranded DNA sequence of the Verotoxin gene (VT2) of Escherichia coli, previously multiplied using PCR (40 cycles), could be detected within 10 minutes.

Bacterial Toxins↗
PubMed1997

Novel detection system of flow injection analysis (1). The existence of significant relation between secondary structure of DNA and sensitivity in signal detection.

Polymerase Chain Reaction (PCR) products was detected quantitatively using a flow injection type sensor, based on Surface Plasmon Resonance (SPR). We used asymmetric PCR to amplify the two kinds of products; their DNA lengths are different. This novel design permitted us not only to detect PCR products with high-sensitivity, but also to develop a rapid DNA detection system for the sense of the genetic pathogen.

Bacterial Toxins↗
PubMed1997

Thrombin-binding properties of thrombin aptamer derivatives.

We synthesized a single-stranded DNA with triple G-quartet structure and found that its thrombin-binding activity at 37 degrees C is as much as that of thrombin aptamer with double G-quartet structure which is a product of in vitro selection and amplification. This result suggests that the sequence motifs obtained by in vitro selection and amplification method might not be the only answer.

Base Sequence↗
PubMed1997

Novel DNA detection system of flow injection analysis (2). The distinctive properties of a novel system employing PNA (peptide nucleic acid) as a probe for specific DNA detection.

In order to realize immediate detection of a double stranded DNA amplified by Polymerase Chain Reaction (PCR), we applied Peptide Nucleic Acid (PNA) to the probe of DNA detection system using Surface Plasmon Resonance (SPR). We report our success in immediate detection of PCR products solution with high sequence-specificity.

Bacterial Toxins↗
PubMed1996

Optimization of the rate of DNA hybridization and rapid detection of methicillin resistant Staphylococcus aureus DNA using fluorescence polarization.

The hybridization rate of two complementary single-stranded DNA 24-mers was determined at various NaCl concentrations using a fluorescence polarization method. The rate was taken as the rate constant of the second order reaction, obtained by mathematical fitting of the time course curves of hybridization. The rate increased with the NaCl concentration, plateauing in the concentration range of about 1-2 M. Over the temperature range of 46 degrees C to 56 degrees C, it was found that in 0.8 M NaCl, hybridization was complete in under 10 min and that the difference in the polarization values between specific hybridization and non-specific binding was greater at lower temperatures. Under the optimized conditions of 0.8 M NaCl at 46 degrees C, the time for DNA hybridization to reach 90% completion decreased to less than 5 min. The assay time for one sample was 10 min and the detection limit was of the order of 10(-10) M (40 fmol per assay). Under the optimized conditions, the DNA of methicillin resistant Staphylococcus aureus (MRSA), which had been multiplied by PCR, could be detected within 10 min.

Base Sequence↗
PubMed1996

Microbial cyanide sensor for monitoring river water.

A microbial cyanide sensor using Saccharomyces cerevisiae for monitoring a river water is described. This sensor is based on the inhibition of S. cerevisiae's respiration by cyanide. This sensor is a reactor type flow system and composed of two oxygen electrodes and a reactor which contains S. cerevisiae immobilized beads. The S. cerevisiae's respiration activity is measured using the oxygen electrodes. The sensor showed a linear response in the range from 0 to 15 microM and maintained stable response for 9 days at ambient temperature. The sensor was optimized for the monitoring of river water and was applied to river water analysis.

Biosensing Techniques↗
PubMed1996

A novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized films for immunosensors.

A novel method of immobilizing antibodies on quartz crystals for use in immunosensors was developed using an ethylenediamine plasma-polymerized film matrix. The films formed on the quartz crystals are extremely thin and homogeneous, and they incorporate amino groups. Sensors produced using this method are more reproducible from sample to sample and exhibit lower noise and higher sensitivity than sensors made using conventional immobilization methods, e.g., via polyethylenimine and (gamma-aminopropyl)trimethoxysilane. This is, to our knowledge, the first reported application of plasma-polymerized films to quartz crystal microbalance immunosensors. Results on orientation-controlled immobilization of anti-bodies, reusability and calibration tests are also presented.

Animals↗
PubMed1996

Electrical conductivity of tear fluid in healthy persons and keratoconjunctivitis sicca patients measured by a flexible conductimetric sensor.

BACKGROUND: It is known that the osmolarity of tears increases in keratoconjunctivitis sicca (KCS) patients and therefore could be a sensitive and specific indicator for the diagnosis of KCS. However, owing to the difficulties in using the current methods of tear fluid measurement these procedures have not been employed in clinical practice. A newly devised flexible conductimetric sensor fabricated using microelectronic techniques is small and flexible enough to be placed on the ocular surface to measure the electrical conductivity can be considered as an indirect function of electrolyte activity, or osmolarity. Therefore, we applied this new sensor to measure the tear fluid conductivity in KCS patients and healthy volunteers. METHODS: A flexible conductimetric sensor, consisting of a hydrophilic polytetrafluoroethylene membrane placed between two gold-coated layers, was placed directly into the temporal conjunctival cul-de-sac. The tear fluid conductivity was monitored graphically on a computer display. The sodium chloride concentration of tear fluids was calculated from the calibration curve and converted to the equivalent electrolyte concentration. RESULTS: The electrolyte concentrations were 324.8 +/- 41.0 mEq/1 in KCS patients (29 samples obtained from 16 KCS patients) and 296.4 +/- 30.1 mEq/1 in healthy persons (33 samples obtained from 17 healthy persons). The difference was significant (P < 0.01). A positive correlation was found between the electrolyte concentrations in KCS and the rose bengal score (coefficient = 0.36). CONCLUSION: The tear fluid conductivity in healthy persons and KCS patients could be monitored without ocular damage, and the measured values were consistent with previous reports. This method will be a new diagnostic tool for detecting tear abnormalities.

Adult↗
PubMed1996

A novel synthesis method for cyclodextrins from maltose in water-organic solvent systems.

A novel enzymatic synthesis method of cyclodextrin (CD) from low-mol-wt maltose using cyclomaltodextrin glucanotransferase (CGTase) from Bacillus macerans has been developed in various water-organic solvent systems. A beta-CD was synthesized in a two-phase system consisting of water and cyclohexane. However, no CDs could be synthesized in an aqueous buffer solution. A maximal yield of beta-CD has been obtained at a cyclohexane content volume of 44%. This synthesis has been obtained only at low temperatures, i.e., 7 degrees C, and did not take place at 50 degrees C. In addition, various organic solvents have been used for the enzymatic synthesis of CD from maltose. Consequently, beta-CD could be synthesized in various water-organic solvent systems, e.g., cyclohexane, benzene, xylene, and chloroform, but no enzymatic reaction occurred using aliphatic n-hydrocarbon solvents such as hexane, dodecane, and hexadecane. Furthermore, alpha- and beta-CD could be synthesized in water mixture solutions using organic solvents having an alcoholic group (e.g., ethanol, propanol, butanol, and pentanol) in a wide range of the reaction temperatures, typically 7-50 degrees C. In this temperature range, alpha- and beta-CD were also formed and the maximal yield from maltose to beta-CD of approx 13% was reached in 60 h.

Chromatography, High Pressure Liquid↗
PubMed1996

Determination of cyanide using a microbial sensor.

A microbial cyanide sensor was prepared, consisting of immobilized Saccharomyces cerevisiae and an oxygen electrode. When the electrode was inserted into a solution containing glucose, the respiration activity of the microorganisms increased. The change in the respiration activity is monitored with the oxygen electrode. When cyanide is added to the sample solution, the electron transport chain reaction of the respiration system in the mitochondria is inhibited, resulting in a decrease in respiration. The inhibition is caused by cyanide binding with respiration enzymes such as the cytochrome oxidase complex in the mitochondrial inner membrane. Therefore, the cyanide concentration can be measured from the change in the respiration rate. When the sensor was applied to a batch system at pH 8.0 and 30 degrees C, the cyanide calibration curve showed linearity in the concentration range between 0.3 microM and 150 microM CN-.

Cyanides↗
PubMed1996

Disposable sensor for biochemical oxygen demand.

Disposable-type microbial sensors were prepared for the determination of biochemical oxygen demand (BOD). The yeast, Trichosporon cutaneum, was directly immobilized on the surface of miniature oxygen electrodes using an ultraviolet crosslinking resin (ENT-3400). The oxygen electrodes (15 mm x 2 mm x 0.4 mm) were made on silicon substrates using micromachining techniques. They were Clark-type two-electrode systems with-1021 mV applied to the working electrode. Typical response times of the BOD sensors were in the range of 7-20 min. At 20 degrees C, the sensors' dynamic range was from 0 to 18 mg/1 BOD when a glucose/glutamate BOD standard solution was used. The lower limit of detection was 0.2 mg/l BOD. This value was one order of magnitude lower than that of sensors previously reported. The sensors' operational lifetime of 3 days was satisfactory for a disposable type. The sensors' responses were reproducible to within 8% relative standard deviation. The BOD sensors' were applied to untreated and treated waste waters from industrial effluents and municipal sewage. BOD values determined using these sensors correlated well with those determined by the conventional 5-day BOD determination method.

Biosensing Techniques↗
PubMed1996

Development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine.

A simple immunosensor based on a conductivity method was developed for determination of methamphetamine (MA, a stimulant drug) in urine. Anti-MA antibody was immobilized onto the surface of a pair of platinum electrodes. The reaction of MA with the antibody causes a decrease in the conductivity of the anti-MA immobilized layer between the electrodes. A linear relationship was obtained between the conductivity and MA concentration in the range of 1-10 micrograms/ml. The method requires the sample to be rinsed with water on the electrodes after the immunoreaction. This detection system was applied to the determination of MA in urine and proved to be a useful and a simple detection technique of MA in forensic science in comparison with a gas chromatography-mass spectrometry method.

Biosensing Techniques↗
PubMed1996

[Micro fabricated enzyme battery].

Although various work has been done in the field of implantable micro actuators such as artificial organs and micro surgery robots, a suitable electric power supply for these is yet to be developed. For this purpose a micro fabricated enzyme fuel cell was developed which uses glucose contained in the human body as a fuel. In order to obtain enough voltage each cell was formed as part of a serial array on a silicon wafer. Glucose solution enters the cells by a capillary effect. In this article fuel cells already developed using biocatalysts are described, and the future possibility of a micro fabricated enzyme battery is discussed.

Biosensing Techniques↗