Takuma Tokiyasu

Identification of animal species using the partial sequences in the mitochondrial 16S rRNA gene

This study investigated a PCR direct sequencing method for species identification by analyzing partial sequences in the mitochondrial 16S rRNA genes of many animal species amplified with universal primers. Samples from 182 vertebrates and 103 invertebrates were analyzed, and the sequences could be obtained in 182 and 72 species, respectively. The sequence divergence was sufficient to identify the species at the level of genus with the aid of the GenBank database and the BLAST tool. This method could be a powerful tool for animal species identification, especially in forensic cases in which many unknown biological samples should be analyzed.

Detection of green algae (Chlorophyceae) for the diagnosis of drowning.

The plankton test (generally, diatom test) is one of the methods available to diagnose the cause of death of submerged bodies. The solubilization method using tissue solubilizer Soluene-350 was used in this study to detect not only diatoms but also green algae, based on the fact that the solubilizer does not digest the cell walls of green algae which are made from cellulose. Detection of green algae from organs of submerged cadavers is very informative to determine drowning in fresh water, and also in cases where only few diatoms are detected in the organs.[/p]

Determination of paraquat and diquat by liquid chromatography-thermospray mass spectrometry

Abstract Thermospray liquid chromatography-mass spectrometry can be used for identifying and determining both paraquat (C 12 H 14 N 2+ 2 ) and diquat (C 12 H 12 N 2+ 2 ). Reversed-phase liquid chromatography was performed using a 15-cm Shim-pack CLCODS column, with methanol-water (80–20) + 0.1 M ammonium acetate (adjusted to pH 5 with trifluoroacetic acid) as buffers, at a flow-rate of 1.0 ml/min. The mass spectral sensitivity was best when the temperatures of the vaporizer, block and tip heater of the ion source block were set at paraquat and diquat, respectively. Detection limits by selected ion monitoring were of the order of 20 ng ( S / N = 3.5). The mass spectra are influenced by temperature and therefore, precise temperature contorl is essential.

PCR-based genotyping of MNSs blood group: Subtyping of M allele to MG and MT

SummaryPCR-based genotyping of MNSs blood group system was investigated in combination with restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and allele-specific PCR amplification (ASPA) techniques. M and N alleles are based on three nucleotide substitutions in exon 2 and one base change (G or T) in an intron of glycophorin A locus. The latter single base change was also found among M alleles analyzed in this study, so that M allele appeared to be subdivided into MG and MT. All three alleles, MG, MT and N were identified clearly by RFLP or SSCP analysis following a single amplification. S and s alleles are based on one nucleotide substitution in exon 3 of glycophorin B gene. Genotyping of Ss blood group system was also explored by PCR-SSCP or ASPA analysis, and problems in the methods were discussed.

Intralobular distribution of class I alcohol dehydrogenase and aldehyde dehydrogenase 2 activities in the hamster liver.

The hepatic lobular localization of class I alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) 2 activities was examined histochemically using livers of hamsters with high ethanol preferences. The activity of class I ADH detected by the nitro blue tetrazolium method using 5 mM ethanol as a substrate was extremely high and was almost homogeneously distributed throughout the lobule. The ALDH 2 activity (substrate, 8 μM acetaldehyde) was localized to the centrilobular zone, whereas low Km ALDH (ALDH 1 + ALDH 2) activity (substrate, 50 μM acetaldehyde) showed a gradient distribution in the lobule with high centrilobular to moderate periportal activity, suggesting that the ALDH 1 activity was distributed throughout the lobule.

Rapid detection of dihydrocodeine by thermospray mass spectrometry

Rapid assay of dihydrocodeine (DHC) by thermospray mass spectrometry is explored. Liquid-liquid extractions of blood, urine and gastric contents were injected into a thermospray mass spectrometer, to which there was no column connected, and DHC was assayed by the flow injection method. The mass spectra of DHC under thermospray ionization and filament-on ionization modes consist of the MH+ ion of mlz 302 alone, which was clearly detected in the samples. Although DHC should be quantitated by gas chromatography-mass spectrometry, this method is applicable for rapid identification of DHC in biological materials.

The proof of flat-line scalp EEGs of brain dead patients by an automatic EEG analysis system

Scalp electroencephalograms (EEGs) of brain dead patients are macroscopically flat under 7 or 10 microV/mm electroencephalograph sensitivity, but significant noises are detected in EEGs under 2 microV/mm sensitivity, interfering with the analysis. EEGs of 20 brain dead patients (17-76 years old) were therefore analyzed quantitatively as equivalent electric potentials in frequency bands delta, theta, alpha and beta using the automatic EEG analysis system developed in Kansai Medical University. The equivalent electric potentials in each band were about or less than 1 microV, which is used as a criterion of judgment of flat-line EEGs or brain death. Then, macroscopically flat EEGs of 12 comatose patients including infants (3-67 years old) were analyzed by the system, confirming their brain death. Thus, the automatic EEG analysis system could be used as a supporting tool to confirm flat-line EEGs of brain dead patients. ATAMAP II for Windows software was also evaluated.

ABO and Rh phenotyping by absorption–elution technique using cerebral dura maters

Phenotyping of ABO and Rh blood groups was performed by the absorption-elution technique using cerebral dura maters. For the ABO system, the cerebral dura maters extracted from nine autopsied cadavers including two burnt bodies, two putrefied corpses, and one half-mummified and one skeletal structure were tested with commercially available Wako antisera (animal polyclonal antibodies). At the same time, blood, fingernails or bones were sampled. In all the cases, the phenotypes could be typed correctly and more clearly with the use of 2 x 2 x 0.3 mm dura maters (0.6 mm thick dura maters were sliced to 0.3 mm thickness) than the phenotyping using 2 x 2 mm fingernails or 2 x 2 x 1 mm bones. For the Rh system, the cerebral dura maters extracted from eight autopsied cadavers within 2 days after death including two burnt bodies were tested with commercially available Ortho Bioclone anti-C, anti-c, anti-E and anti-e sera (human monoclonal antibodies), and Ortho anti-D serum (human polyclonal antibody). The eluate of anti-D antibody was needed to perform the indirect anti-globulin test (Ortho Coombs serum). At the same time, blood was sampled. In all the cases, the Rh blood groups of cerebral dura maters were in agreement with those of blood.