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Volume 51, Issue 6
Displaying 1-19 of 19 articles from this issue
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Toshimasa TOYO'OKA2002 Volume 51 Issue 6 Pages 339-358
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSBenzofurazan-bearing chiral derivatization reagents for the resolution of biologically important compounds, such as chiral drugs, by HPLC, based upon pre-column derivatization and diastereomer formation, are reviewed. The chiral reagents developed for various functional groups, i.e. amine (NBD-PyNCS, DBD-PyNCS), carboxyl (NBD-APy, DBD-APy, ABD-APy), carboxyl (NBD-ProCZ, DBD-ProCZ), hydroxyl (NBD-Pro-COCl, DBD-Pro-COCl) and thiol, are evaluated in terms of the reactivity, stability, wavelength, handling, versatility, sensitivity and selectivity. Some applications utilizing the reagents for the analyses of drugs and bioactive compounds are also included in the text.View full abstractDownload PDF (1701K) -
Hiroshi TSUKUBE, Tomoko NISHIMURA, Satoshi SHINODA2002 Volume 51 Issue 6 Pages 359-366
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSSupramolecular recognition provides two effective approaches, which offer chirality sensing of biological substrates using circular dichroism (CD) spectroscopy: (1) sophisticated receptors having highly complementary binding sites to targeted guests, and (2) programmed aggregates integrating the chirality up to the supramolecular level. The conjugation of 18-crown-6 and characteristic binding sites has typically attained chirality sensing of amino acids via synergistic binding, while highly structured polymers or amphiphiles have led to chirality sensing upon ordered aggregation. Since these supramolecular systems have broad structural variations, intelligent chirality sensing can be built up via further molecular architecture.View full abstractDownload PDF (3481K) -
Youji SHIMAZAKI, Yoko HIRAKA, Hiromi OHNISHI, Takashi MANABE2002 Volume 51 Issue 6 Pages 367-371
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSNon-denaturing two-dimensional electrophoresis (2-DE) can be applied to the separation of soluble proteins with high resolution. The separated proteins retain their physiological functions. The present study indicates that soluble proteins of the bovine retina were separated by non-denaturing 2-DE, and the enzyme activities of esterase, dehydrogenase, superoxide dismutase and transferase were detected in the presence of each enzyme-specific substrate and chromophore. While the activities of esterase and dehydrogenase were inhibited by a cholinesterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), the transferase activity was not affected. The enzymes separated by non-denaturing 2-DE were analyzed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS). These results indicate that non-denaturing 2-DE is applicable to analyzing of the functions and structures of soluble proteins.View full abstractDownload PDF (2702K) -
Yasunobu AOKI, Hiromi SATO, Kimiko AMANUMA2002 Volume 51 Issue 6 Pages 373-380
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA bio-assay for detecting adverse effects caused by chemicals contaminated in water and ambient air is a novel methodology for systematically evaluating the health risk of environmental chemicals. We intend to evaluate the mutagenicity of chemicals, and have developed a method to detect mutations induced in the body by environmental chemicals using transgenic animals, in which the monitor genes for detecting the mutation are intergrated into genomic DNA. We evaluated the mutagenicity of diesel exhaust (DE) using a transgenic rat harboring the lac I gene as a monitor gene (Big Blue rat). DE exposure at a concentration of 6 mg suspended particular matter/m3 caused mutations in Big Blue rat lung. We have established a transgenic zebrafish line harboring rpsL gene for detecting mutagens in the aquatic environment. Using the embryos of this transgenic fish, mutations induced by chemicals, such as ethyl nitrosourea and benzo[a]pyrene, were detected. The possibility of environmental monitoring for hazardous chemicals using transgenic animals and other experimental animals is discussed.View full abstractDownload PDF (7394K)
Research Papers
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Kazuyuki WATANABE, Hidetoshi ARAKAWA, Masako MAEDA2002 Volume 51 Issue 6 Pages 381-387
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSThe infection of Enterohaemorrhagic Escherichia coli (EHEC), especially serotype O157 : H7, causes hemorrhagic colitis, and has been associated with hemolytic uremic syndrome or encephalopathy, which may lead to death. Consequently, the prevention of serious illness due to infection via early stage diagnosis is of the utmost importance. To this end, the development of detection methods that are simple, rapid and highly sensitive is necessary for EHEC. O157 produces two vero toxins, VT1 and VT2. VT1 is identical to the Shiga toxin, while the other type, VT2, is different from VT1. The conventional diagnosis of O157 is conducted after isolating bacteria from clinical specimens, followed by a serological determination and the identification of VTs. This method is both complicated and time-consuming. Recently, rapid, direct immunological methods for the identification of O157, i.e., immunochromatography and latex agglutination, have been developed. However, these techniques can not simultaneously measure these two toxins. On the other hand, the use of fluorescence lanthanide ions as a labeling material of antibodies and antigens makes it is possible to develop an immunoassay that can measure two or more subjects simultaneously. After excitation by short-pulse light, a fluorescence lanthanide ion, such as europium and terbium, exhibits a large Stokes shift and very narrow emission spectra that has no overlap with other lanthanide's emission spectra. In addition, their long fluorescence lifetimes allow the use of time-resolved fluorescence detection, which has led to the development of ultra sensitive time-resolved fluorometry (TRF). In this paper, we describe a sensitive and specific method based on time-resolved fluoroimmunoassay (TRFIA) for the simultaneous determinations of two verotoxins.View full abstractDownload PDF (643K) -
Kazunobu ASANO, Atsushi ONO, Setsuko HASHIMOTO, Tohru INOUE, Jun KANNO2002 Volume 51 Issue 6 Pages 389-396
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSBecause concern over endocrine disrupting reactions caused by chemicals to human and animals is growing, a rapid and reliable screening assay for endocrine disrupting chemicals is required. We have developed an in vitro screening assay based on a hormone receptor mechanism using a surface plasmon resonance (SPR) sensor. When a DNA fragment containing a sequence of the estrogen response element (ERE) is immobilized on a sensor chip of a SPR sensor and an estrogen receptor α (ER) is injected over the sensor chip, the interaction of ER and ERE can be monitored in real time. In the presence of a chemical with estrogenic activity, the ER-ERE interaction is enhanced and the kinetic parameters are altered. We have validated the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. It has been shown that the assay is very reliable as a rapid and quantitative screening method to judge the estrogenic activities of chemicals.View full abstractDownload PDF (3451K) -
Hideki SHIBATA, Takao TSUDA2002 Volume 51 Issue 6 Pages 397-401
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSParabens sampled from human skin on forefinger was determined by GC/MS for 10 young subjects. The sampling of parabens from forefinger was performed with 75 μl of a 70% ethanol-aqueous solution in a 1.5 ml vial. After the solution was contacted with the skin for 2 min, a the sample was dried under a vacuum, followed by GC/MS analysis after adding methyl benzoate as an internal standard. The amounts of methyl-, ethyl-, propyl-, and buthyl-parabens in human skin of forefinger were 3 to 1350, 0.3 to 196, 0.3∼516, and 0∼52.1 pmol/cm2, respectively. High amounts of parabens were found for subjects who used a cosmetic liquid for hair and body make up. The increment of parabens during 1 hour was also studied. After several steps of a washing procedure (washing with tap water, immersed in 43°C hot water, washing with ethanol, and finally with distilled water), the first sampling was performed as a blank test; a second sampling was performed after one hour. The value of the increment of paraben was 2.0∼6.9 pmol/cm2, which was estimated from the difference in the amount of parabens at the first and second sampling. These increments are supposed to be derived by the migration of paraben from inside of the body. There is not much difference in the stability of parabens in the skin on the short term (1 hour period). The present sampling method is very simple and was easily adopted for collecting chemical substrates in human skin.View full abstractDownload PDF (507K) -
Iwao SUZUKI, Yuya EGAWA, Jun-ichi ANZAI2002 Volume 51 Issue 6 Pages 403-407
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSElectrode reactions of catecholamines were investigated by employing a gold electrode covered with a monolayer of a cyclodextrin derivative (1), which was prepared from bis (ω-carboxyundecyl) disulfide and 6-amino-6-O-deoxy-β-cyclodextrin. The electrode reaction of L-DOPA was mostly prevented by a 1-monolayer at the surface of a gold electrode. On the other hand, the electrode reactions of dopamine, norepinephrine, epinephrin still maintained about 50∼30% of their original values when compared with corresponding anodic peak currents. Combining the results on the electrode reactions of ferrocene derivatives at the 1-monolayer modified electrode, we considered that the 1-monolayer would recognize the presence or absence of the carboxyl group in catecholamines.View full abstractDownload PDF (537K) -
Koji NAKANO, Sachiko TANAKA, Makoto TAKAGI, Seiji SHINKAI2002 Volume 51 Issue 6 Pages 409-413
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSSelf-assembled monolayer (SAM) electrodes were prepared using calix[6]arene provided with thiols, and electrode reactions of catecholamines on bare gold electrodes and the SAM electrodes were investigated. It was found that the calixarene adlayer partly blocked the electrode reaction of dopamine in the solution phase. This blocking effect was also observed in the case of 3,4-dihydroxy-DL-alanine (DOPA), but only slightly. In contrast, the electrode reaction of 3,4-dihydroxyphenylacetic acid (DOPAC) was suppressed almost completely. These results suggested that cationic substrates (dopamine and DOPA) bound with the calixarene molecules on the electrode surface and made Faradaic reaction with the underlying gold electrode. The calixarene molecular-pore was expected to contribute as a spatial pass for electron transfer, leading to a feasible electrode reaction. Mathematical simulations of the cyclic voltammetric i-E responses were made, and the parameters for the electrode reaction were considered. A possible application to an electrochemical dopamine sensor is also described.View full abstractDownload PDF (513K) -
Hiroko KAWAMOTO, Osamu NOZAKI2002 Volume 51 Issue 6 Pages 415-421
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA rapid and highly sensitive determination method for a total free catecholamines assay using a minicolumn as an off-line reactor was developed. Although separation methods are widely used for the diagnosis and follow-up of diseases related to catecholamine disturbances, they have the problem of a time-consuming for assay. Therefore, a rapid and highly specific assay method for the total free catecholamines (free adrenaline + noradrenaline + dopamine) is needed to solve this problem. The newly developed method employed the principle of recognizing both the two functional groups of catecholamines (amino group and catechol group). The gels in of off-line reactor had a sulfuric acid group on the surface, and bound to amino groups of catecholamines. Then, the phenolic group of catecholamine on the gel reacted to produce hydrogen peroxide by heating at 60°C for 30 min. Glass beads or polystyrene beads were chosen as a gel support. The hydrogen peroxide produced in the off-line reactor was eluted with an imidazole solution (1.0 mol/l), and finally assayed by the micro-flow injection horseradish peroxidase catalyzed luminol chemiluminescence. The change in the rate of dopamine to hydrogen peroxide with the off-line reactor was 47∼69% (average 56.8%). A calibration curve was produced by an external standard method with dopamine, and the regression formula was Y = 2.04 X2 + 12.4 X - 16.8, where Y is the light intensity and X is dopamine (1.63∼8.15 nmol). This is a rapid method applicable to clinical assays of total free catecholamines.View full abstractDownload PDF (3233K) -
Touru SHINOHARA, Akira KOTANI, Fumiyo KUSU2002 Volume 51 Issue 6 Pages 423-427
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA flow injection analysis with electrochemiluminescence (ECL-FIA) with high-sensitivity and manipulation was developed for determining hydrogen peroxide. The peak height of the flow signal was proportional to the amount of hydrogen peroxide, ranging from 59 fmol to 59 pmol (r>0.999). Standard hydrogen peroxide at 4.76 pmol was determined ten times with a relative standard deviation (RSD) of 1.89%. The detection limit (S/N=3) was 59 fmol. Glucose and uric acid were determined using the ECL-FIA connected with a column filled with immobilized glucose oxidase and uricase, respectively. The calibration graphs were linear for 111 fmol∼111 pmol for glucose and 119 fmol∼59.4 pmol for uric acid. The results of glucose and uric acid in control serum by the presented method coincided with those by commercially available glucose and uric acid measuring kits.View full abstractDownload PDF (486K) -
Sadaharu SUGASE, Takao TSUDA2002 Volume 51 Issue 6 Pages 429-435
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSLactic acid, uric acid, xanthine and tyrosine in human sweat were determined by HPLC. These chemical substances are commonly used as important clinical makers. A handy method for sweat sampling was devised. After filling 40 μl of 1% ethanol aqueous solution in a 0.6 ml vial, the content was contacted with a finger and collected sweat for 5 min. Concurrently, sweat secretion was estimated by the identical location on the other hand by measuring with a perspiration rate meter. The average values for 8 young subjects were 154 mM, 150 μM, 225 μM and 2.25 mM for lactic acid, uric acid, xanthine and tyrosine, respectively. The variation of lactic acid in sweat after the intake of wine was studied. Although the amount of sweat secretion was almost constant before and after the intake of wine, the lactic acid concentration in sweat showed a maximum at around 25 to 45 min after its intake. We suppose that lactic acid in sweat is induced from blood capillaries.View full abstractDownload PDF (621K) -
Hiroyuki TAKANO, Yutaka ITABASHI2002 Volume 51 Issue 6 Pages 437-442
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA facile method for the determination of molecular species of regioisomeric 1,3-diacylglycerols (1,3-DG) in edible oils was developed. For this purpose, a DG-rich cooking oil (Econa), which can reduce fat deposits, was treated with 3,5-dinitrophenyl isocyanate in dry toluene under the presence of pyridine at room temperature. The resulting 1,3-DG 3,5-dinitrophenylurethanes were then separated by preparative thin-layer chromatography on silicic acid. The 1,3-DG was followed by reversed-phase HPLC on a C30 column (25 cm × 4.6 mm i.d.), which gave a clear resolution of the individual molecular species. Reversed-phase HPLC in conjunction with negative electrospray ionization MS (ESI-MS) showed a prominent [M-H]- ion, by which individual molecular species could be identified.View full abstractDownload PDF (452K) -
Tsuyoshi MURAI, Kana YAMAGUCHI, Teruki YOSHIMURA, Shu-Ping HUI, Takao ...2002 Volume 51 Issue 6 Pages 443-448
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA method for the analysis of bile alcohols and hydroxycholesterols as intermediates in mono-hydroxyl bile acid biosynthesis has been developed by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring in a high-resolution mode. The linearity of the calibration curves for the bile alcohols, hydroxycholesterol and bile acids ranged from 10∼250 pmol (r>0.999), and the detection limits were 50∼100 fmol (S/N = 2∼6). The recoveries of the bile alcohols, hydroxycholesterol and bile acids from the rat liver mitochondrial fraction ranged over 97.9∼111%, respectively, of the added amounts of their standard samples. The present method was made available for the determination of bile alcohols, hydroxycholesterol and related monohydroxy bile acids in incubation mixtures after only a simple extraction procedure using a solid-phase extraction cartridge.View full abstractDownload PDF (531K) -
Tetsuya KONDO, Kumiko KAWAI, Kyoden YASUMOTO, Tokuji IKEDA2002 Volume 51 Issue 6 Pages 449-454
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSThe effect of bactericides on bacteria was studied using an electrochemical method for measuring the glucose-oxidizing activity of bacterial whole cells. When Esherichia coli IFO3301 (E. coli) and Lactobacillus plantarum IFO14711 (L. plantarum) were exposed to ethanol, hydrogen peroxide, or hypochlorous acid, the glucose-oxidizing activity and the colony formation decreased with increasing bactericide concentration. The glucose-oxidizing activity of both E. coli and L. plantarum was more sensitive to bactericides than the colony formation. In contrast with the case of E. coli and L. plantarum, the glucose-oxidizing activity of Pseudomonas putida IFO14164 (P. putida) was resistant to bactericides. Exposure of P. putida cells to 26% ethanol or 0.5% hydrogen peroxide at 30°C for 10 min caused a loss of colony formation, while the glucose-oxidizing activity remained at approximately 20%, even after exposure to 95% ethanol or 3% hydrogen peroxide. In P. putida and E. coli, the colony formation and the glucose-oxidizing activity of cells grown to log the phase were more sensitive to hypochlorous acid than those of the cells grown to the stationary phase. When P. putida cells were exposed to a low concentration of hypochlorous acid, the bactericide-resistance of the colony formation increased, but the resistance of the glucose-oxidizing activity decreased. In the case of E. coli, the pretreatment with hypochlorous acid enhanced the bactericide-resistance of both the colony formation and the glucose-oxidizing activity.View full abstractDownload PDF (588K) -
Daisuke HOBARA, Yusuke UNO, Takashi KAKIUCHI2002 Volume 51 Issue 6 Pages 455-460
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSThe surface distribution and emzyme activity of horseradish peroxidase (HRP) selectively immobilized on binary self-assembled monolayers (SAMs) of dithiobis-N-succinimidyl propionate (DTSP) and 1-tetradecanethiol (TDT) have been studied. Scanning tunneling microscopic images of the phase-separated binary SAMs formed by coadsorption on Au (111) substrates show nanometer-scale domains which are 10∼40 nm in diameter and 0.3∼0.7 nm in height. HRP was covalently immobilized on the DTSP domains by immersing the SAM-modified substrate into an HRP solution, followed by washing with a 1 M KCl solution. A tapping-mode atomic force microscopic image of the HRP-immobilized substrate shows holes having a similar diameter to those of the binary SAMs and a depth of 3.6±1.9 nm, indicating the selective immobilization of HRP molecules on the DTSP domains. The enzymatic activity of the immobilized HRP has been confirmed using cyclic voltammetry in the presence of ferrocenecarboxylic acid as an electron-transfer mediator.View full abstractDownload PDF (3048K)
Technical Papers
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Junichi INAGAWA, Kaori MORIMOTO2002 Volume 51 Issue 6 Pages 461-468
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSA high resolution optical biosensor assay for screening of low molecular weight compounds using Biacore® S51 has been examined. Carbonic anhydrase(CA) and human serum albumin(HSA) were immobilized on the sensor surfaces respectively and 34 chemical compounds involved 10 of inhibitors and 24 of non-interacting reference compounds were injected directly to each sensor surface simultaneously. Sulfanilamide, CBSA (carboxybenzenesulfonamide), furosemide, azosulfamide, acetazolamide, dichlorphenamide, chlorothiazide, hydrochlorothiazide, methazolamide and indapamide were bound to immobilized CA but reference compounds were not bound at all. All compounds were bound to immobilized HSA at variable binding levels. This analysis was based on single concentration association: dissociation analysis, and provided a higher level of data than standard YES/NO analysis, enabling compounds to be prioritized based on their binding profile and stability as well as activity. Kinetics analysis of azosulfamide, furosemide and indapamide were estimated as ka(association rate constant) and kd(dissociation rate constant) from a series of sensorgram at different concentrations of inhibitors. The method is expected to be generally applicable to secondary screening and optimization of lead compounds of low molecular weight compound libraries with protein as targets.View full abstractDownload PDF (2935K)
Notes
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Toshio YAO, Chong-Ok HO2002 Volume 51 Issue 6 Pages 469-472
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSCatecholamine biosensors were prepared by cross-linking tyrosinase from mushroom with gelatin using glutaraldehyde and by holding a mushroom tissue slice using a dialysis membrane on the flat end of a glassy carbon electrode. A tyrosinase-based biosensor responded rapidly (ca. 8 s) to catecholamine with a selectivity ratio of 100 : 27.4 : 4.9 for dopamine, noradrenaline, and adrenaline; the current response to dopamine was linearly related to the concentration over a range of 1 × 10-7∼5 × 10-5 M; the detection limit was 2 × 10-8 M for dopamine. Similarly, a mushroom tissue-based biosensor responded to catecholamines with a similar selectivity ratio, though the sensitivity was only ca. 10% of that of the tyrosinase-based biosensor and the response time was somewhat slower (ca. 60 s). Both electrodes were based on the amperometric detection (-0.1 V vs. Ag/AgCl) of the quinone form of catecholamines produced by the same enzyme-catalyzed reaction, which involved amplification based on substrate recycling occuring on the surface of a glassy carbon electrode. The mushroom-based biosensor appeared to be effective for the measurement of dopamine and had advantages of ease of construction and low cost, though it was inferior to the tyrosinase-based biosensor in both sensitivity and long-term stability. Therefore, it was useful for the preparing a disposable biosensor for catechol amine.View full abstractDownload PDF (374K)
Technical Letters
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Yukiko NAKANO, Satoshi NOMURA, Tadafumi KAWAMOTO, Junji TAGAMI, Atsush ...2002 Volume 51 Issue 6 Pages 473-476
Published: June 05, 2002
Released on J-STAGE: March 13, 2009
JOURNAL FREE ACCESSAn attempt of pH mapping of biological samples was made on the whole body sections of fresh frozen rats by means of the pH-imaging microscope using a semiconductor silicon sensor. Fine tuning of experimental conditions for data acquisition and its digital processing successfully yielded pH images at the tissue and organ levels. Present data suggest usefulness of the application of the pH-imaging microscope for the analysis of local pH values of animal tissues that have a potential importance in regulation of biological activities.View full abstractDownload PDF (1961K)
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