Biomedical subjects
Publications and source records attributed to Isao Saito.
We have devised novel pyrene-labeled BDF nucleosides, (Py)U, (Py)C, (Py)deazaA, and (Py)A. BDF probes containing these fluorescent nucleosides selectively emit fluorescence only when the base opposite the BDF base is a target base. Oligonucleotides containing these BDF nucleosides act as effective reporter probes for homogeneous SNP typing of DNA samples.
A novel uracil derivative tethering a solvatochromic chromophore, 6-dimethyl-2-acylnaphthalene (DAN), has been synthesized, and local dielectric constant in the recognition site of Klenow fragment (KF) has been investigated by steady-state fluorescence measurement using DNA substrates containing the solvatochromic uridine. Steady-state fluorescence measurements suggest that KF covers about 10 base pairs of the duplex DNA upstream of the primer terminus, and the vicinity of the incorporation site of triphosphate is the most hydrophobic environment.
We investigated electrochemical characteristics of gold electrodes modified with photosensitizer-tethered DNA. We synthesized 3'-thiol-modified DNA strands (ODN-SH) and 3'-anthraquinone-modified DNA strands (AQ-ODN). We prepared an AQ-ODN/ODN-SH duplex-modified electrode through hybridization of AQ-ODN with ODN-SH on the gold electrode. We used an amperometric technique for its photocurrent measurement. The large photocurrent was observed by irradiation of a DNA modified electrode at 365 nm. We also measured photocurrent with different DNA sequences on gold electrodes.
Two pyrene-labeled bases, (py)C and (8py)G, have been synthesized and incorporated into d(CG)n. In the CD spectra, structural transition of a modified oligodeoxynucleotide (ODN-Py) from B-form to Z-form has been observed under different salt concentrations. The conformational transition of ODN-Py was monitored by the change of emission wavelengths based on exciplex and monomer fluorescence of (py)C and (8py)G.
An efficient and reversible Template-directed Photoligation of oligodeoxynucleotides (ODNs) using alpha-5-cyanovinyldeoxyuridine (alpha-(CNV)U) was described. When ODN containing alpha-(CNV)U at the 3' end was photoirradiated with ODN containing thymidine at the 5' end in the presence of a template ODN, an efficient photoligation was observed. The ligated ODN can be split site-selectively to regenerate parent ODN by photoirradition at the 312 nm.
The photophysical properties of DNA oligomers containing novel uracil derivatives tethering a solvatochromic pyrene-dimethylaniline (Py-DMA) chromophore were investigated. In the steady-state fluorescence measurements of the modified DNA oligomer, dual fluorescence, originating from a locally excited (LE) and a twisted intramolecular charge transfer (TICT) state, was observed.
A new strategy for the detection of a single base alteration utilizing the fluorescence quenching by guanine base is described. We have devised a novel base-discrimination fluorescent (BDF) nucleoside, PyT, which contains pyrene carboxamide fluorophore at sugar 4' position. PyT-containing oligodeoxynucleotides (ODNs) exhibit an intense fluorescence only when PyT forms a complementary base pair with adenine.
PURPOSE: To report the clinical and immunohistochemical findings of a rapidly growing uveal melanoma. METHODS: A 71-year-old Japanese woman was diagnosed with a juxtapapillary uveal melanoma by clinical examination. The tumor grew rapidly with a 31.5-day doubling time. An enucleation was performed, and immunohistochemical examination for cell cycle proteins was performed in addition to routine histopathologic examination. RESULTS: The melanoma cells were classified as spindle B-cells. The tumor cells were positive for HMB-45 protein, but negative for S-100 protein. The tumor showed immunohistochemical staining for p16 and Bcl-2, although p53 was not detected. Immunoreactivity for pRb was found in normal retinal and choroidal cells but not in the tumor cells. CONCLUSION: This report describes a unique case of rapidly growing uveal melanoma, which showed low histopathologic grade while the cell cycle protein immunohistochemical profile suggests a more aggressive phenotype. The evaluation of cell cycle proteins may be useful adjunctive method to enhance the understanding of uveal melanoma growth patterns.
This study aims at clarifying the semen indices of insecticide sprayers who are exposed mainly to organophosphorus and pyrethroid insecticides. Eighteen male sprayers out of 54 working for 9 companies in central Japan and 18 age-matched students or medical doctors as unexposed controls participated in detailed reproductive check-ups conducted in summer and the following winter. The sprayers were exposed to insecticides more in summer, the busiest season, than winter, the off-season (p<0.05). Erythrocyte true cholinesterase activities in the sprayers were lower than in the controls in summer (p<0.05), and decreased in significant association with the increase in exposure frequency. Testicular volumes in the sprayers tended to be smaller than in the controls (p=0.06). The serum testosterone concentration in winter in the sprayers was higher than in the controls (p<0.05), though luteinizing hormone and follicle stimulating hormone concentrations were not significantly different. The sperm counts and vitality were comparable between the groups, but detailed sperm motility analysis in summer revealed that the percentages of slow progressive and nonprogressive motile sperm were twice as high in the sprayers (p<0.05), and that of rapid progressive sperm tended to be lower (p=0.06). Such differences were not observed in winter. Differential sperm morphology counts showed that interaction of group and abstinence effects were significant in sperm with normal morphology and with head deformity only in the summer check-up. Despite possible inherent differences between the groups, the above season-dependent differences suggested that the observed lower semen quality in the sprayers was associated with pesticide spraying work.
A multiresidue method that enables determination of many pesticides in agricultural products by GC-ECD was studied. First, 63 pesticides were selected as agrochemicals commonly used in crop protection in this country, and/or found in agricultural products over the past 6 years (April 1996-March 2002) in Aichi Prefecture. A sample was extracted with acetonitrile and the acetonitrile layer was separated by salting-out. The extract was purified on a GPC system with a graphitized carbon columns, and then by a Florisil mini-column fractionation. The test solution was subjected to one-injection, dual-column GC with dual ECD (column: Stx-CLPesticides and Stx-CLPesticides2). The detection limits of the pesticides were in a suitable range (0.0001-0.01 microg/g) for monitoring pesticide residues in agricultural products. The method was applied to 203 commercial agricultural products to demonstrate its suitability for routine analysis.
A study was conducted to evaluate the applicability of ICP-MS techniques for determination of metals in brown rice and fish. Cadmium, lead, mercury, arsenic, selenium, manganese, copper and zinc were determined by this method. An open digestion with nitric acid (Method A) and a rapid open digestion with nitric acid and hydrochloric acid (Method B) were used to solubilize analytes in samples, and these procedures were followed by ICP-MS analysis. Recovery of certified elements from standard reference materials by Method A and Method B ranged from 92 to 110% except for mercury (70 to 100%). Analytical results of brown rice and fish samples obtained by this ICP-MS agreed with those obtained by atomic absorption spectrometry (AAS). The results of this study demonstrate that quadrupole ICP-MS provides precise and accurate measurements of the elements tested in brown rice and fish samples.
We evaluated simultaneous analytical methods for organophosphorus pesticides in cruciferous vegetables by gas chromatography (GC). Firstly, 36 pesticides were selected on the basis of monitoring data (April 1996-March 2003) in Aichi Prefecture. A sample was extracted with acetonitrile and the acetonitrile layer was separated by salting-out. The extract was cleaned up with gel permeation chromatography (GPC), and then with a tandem silica-gel/PSA mini-column. The test solution was subjected to dual-column GC equipped with a pulsed FPD (P mode, Rtx-OPPesticides column) and a FTD (Rtx-OPPesticides 2 column). Organophosphorus pesticides in such sulfur-rich matrices as cabbage and radish were determined without any serious interfering peaks on the pulsed FPD chromatograms after diluting the extracts 10- to 20-fold (0.25-0.5 mg/L of sample). The method was applied to cruciferous vegetables to demonstrate its usefulness in routine analysis.
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Vital statistics for coronary heart disease (CHD) were dramatically influenced by the tenth revision of the International Classification of Diseases (ICD-10) in 1995. To better understand the accuracy of death certificate diagnosis of CHD and heart failure, validation studies in Japan were reviewed. Positive predictive values and sensitivity, calculated as validation measures, varied widely between studies, differing with regard to autopsy rates, amount of information on medical records, and period investigated. However, heart failure, which has been frequently assigned on death certificates in Japan, was validated in some studies. Half of these were evaluated to be sudden deaths, including coronary deaths. Because autopsy-based studies on sudden deaths indicated that 30-50% of these were accounted for by CHD deaths, deaths assigned to heart failure should be taken into consideration in order to determine the actual number of CHD deaths in Japan. Focusing on changes in vital statistics after the 1995 ICD revision, the Oita Cardiac Death Surveys (OCDS) allowed interpretation of its effects on CHD and heart failure. Much of the increase in CHD deaths on vital statistics reflects more false positive cases, particularly for out-of-hospital deaths. Considering the Japanese features of vital statistics for CHD, further epidemiological validation studies are needed in order to confirm the accuracy of CHD death certificate diagnoses and to monitor actual CHD trends in Japan.
The site-directed incorporation of bis-pyrenyl fluorophore into anti-ATP DNA aptamer results in a creation of an intelligent fluorescent sensor with high signal intensity and specificity for detecting the target ligand in a homogeneous system.
Recent studies predict that adenine radical cation (A*+) contributes to the hole-trapping process through long A/T sequences and exists as a real chemical intermediate. However, the experimental evidence for the existence of A*+ has not been observed in the DNA-mediated hole transport reaction. To examine the direct contribution of A*+, we have developed a novel hole-trapping nucleobase N6-cyclopropyldeoxyadenosine (dCPA) which possesses a cyclopropyl group as a radical trapping device. One-electron oxidation of dCPA revealed that dCPA radical cation undergoes a rapid cyclopropane ring opening. With the use of the dCPA-containing DNA, we have demonstrated that the migrating hole was trapped at CPA incorporated into a long A/T bridge between two GG sites. The present results indicate that nucleobases possessing ionization potential higher than that of dG, such as dA, are able to participate directly in the multistep hopping mechanism.
We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.
We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.