Yoshihiro Yamada

Novel detection of plankton from lung tissue by enzymatic digestion method

Studies are reported on the enzymatic digestion method for detection of plankton from lung tissue by using proteinase K with sodium dodecyl sulphate. This method is simple, safe and effective for detection of not only phytoplankton including diatoms but zooplankton which are destroyed by the acid digestion method. The present method is, therefore, much more advantageous for diagnosis of drowning than the disorganization method using strong acids.

Transport characteristics of paraquat across rat intestinal brush-border membrane

The mechanism of absorption of paraquat, which is a type of quaternary ammonium compound (QAC), was studied using rat intestinal loops and brushborder membrane vesicles. Approximately 47% and 37% of radioactively labeled paraquat injected into jejunal and ileal loops disappeared, respectively, after 60 min. Since only a small amount of radioactivity was detected in the mucosal fraction, most of the paraquat that disappeared from the intestinal lumen was considered to have been carried away by the bloodstream, indicating that paraquat absorption was greater than expected. In spite of its low lipid solubility, the uptake of paraquat by brush-border membrane vesicles reflected smooth penetration into the intravesicular space rather than binding to the membrane. According to the increase in extravesicular paraquat concentration, paraquat uptake in the early stage was saturable. Moreover, early paraquat uptake was significantly inhibited by structurally-related QACs such as tetramethylammonium and choline, but not by an endogenous dicationic amine (putrescine). On the other hand, inside-negative membrane potential had no significant effect on the time course of paraquat uptake. From these results, it is suggested that paraquat is absorbed through a specialized mechanism associated with the carrier-mediated transport system for choline on the brush-border membrane.

Death due to a methane gas explosion in a tunnel on urban reclaimed land

Studies of four male victims who were killed in an accidental tunnel gas explosion on urban reclaimed land are described. The studies were judicial autopsy examinations to determine the precise causes of death. Two men died of carbon monoxide intoxication, one died of massive brain damage, and the fourth died of drowning. The concentrations of methane in several organs were much lower than the lethal level, whereas those in adipose tissue were relatively high. These findings indicated that a low concentration of methane was almost always present in the atmosphere at the construction site. Recently, coal mine accidents have been decreasing in Japan. However, there is still a possibility of underground explosions or gas leaks in confined spaces other than coal mines. To determine the precise cause of death in such cases, careful autopsies and other examinations should be performed using methods similar to those used in coal mine accidents.

Immunohistochemical dynamics of leukotoxin (9.10-eooxv-12-octadecenoic acid) in lungs of rats

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24. 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1′-di-methyl-4,4′-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.ZusammenfassungDieser Artikel untersucht die immunhistochemische Dynamik von Leukotoxin (9,10-Epoxy-12-octadecensäure, LTx) in den Lungen von Ratten, die einer Hyperoxie mit oder ohne Paraquat ausgesetzt waren. Die Ratten wurden behandelt mit 100% Sauerstoff oder Umgebungsluft für 24, 48, 72 und 96 Stunden mit oder ohne Injektion einer niedrigen oder hohen Dose Paraquat (1,1′-Dimethyl-4,4′-bipyridium, PQ). Die Immunfärbung für LTx zeigte positive Reaktionen in den Neutrophilen. Diese zeigten eine zunehmende Verstärkung der Färbungsintensität in Abhängigkeit der Zeit in allen Gruppen mit Exposition von 100% Sauerstoff und in der Gruppe mit hoher Dose PQ, jedoch waren die positiven Befunde in der Gruppe mit Injektion nur niedriger Dose PQ schwach. Wir fanden die positive Immunfärbungsreaktion nicht nur in Neutrophilen, sondern auch in Alveolarmakrophagen. Dies zeigt, daß LTx sowohl von den Alveolarmakrophagen als auch von den Neutrophilen, abhängig von der Behandlungszeitdauer unter hyperoxischen Bedingungen, hergestellt wird, was dafür spricht, daß LTx ein wichtiger chemischer Mediator in Lungenerkrankungen ist.

Advantages of dental mitochondrial DNA for detection and classification of the sequence variation using restriction fragment length polymorphisms.

After amplification by polymerase chain reaction (PCR), the nucleotide sequences of a 452-bp section of the D-loop region of human mitochondrial DNA (mtDNA) were determined in 40 teeth extracted from patients living in Kanagawa prefecture, Japan. Dental DNA was extracted separately from the dental pulp and dentin (i.e., the attached pulp cells from the most superficial layer of the pulp cavity wall) of the same tooth. Comparison of the nucleotide sequences of the 452-bp region of the D-loop demonstrated that nucleotide substitutions and insertion/deletion events were identical in material from both sources. Thus, dentin produces equivalent results when the dental pulp of a tooth is unsuitable for mtDNA analysis. To establish the reliability of the screening procedure for the sequence analysis, we identified restriction sites for the enzymes KpnI and MnlI in the 452-bp region of the D-loop. Thirteen of 14 patterns of four polymorphisms analyzed using the mtDNA from the 40 tooth samples were identifiable by an initial screening procedure involving restriction fragment length polymorphism (RFLP) analysis. Combined use of sequence analysis and RFLP analysis proved extremely efficient in analyzing mtDNA polymorphisms, allowing identification of individuals.

Immunohistochemical localization and dynamics of paraquat in the stomach and esophagus of rats

SummaryThe dynamics of paraquat in the stomach and esophagus of rats were demonstrated using immunohistochemical techniques. The Rats were killed 3 h, 12 h, 24 h, 3 days, 7 days and 10 days after intravenous administration of paraquat. In the stomach, paraquat was localized in the epithelial cells between 24h and 10 days after injection, whereas in the esophagus, paraquat was localized in epithelial cells and the lamina propria mucosa between 12 h and 10 days after administration. Although these findings were similar to those observed in the intestine of rats, no clear changes in the distribution of paraquat with time were observed; suggesting that the stomach and esophagus are important reservoirs for the redistribution of paraquat.ZusammenfassungDie Verteilung von Paraquat im Gewebe des Magens und des Ösophagus von Ratten wurde mittels immunhistochemischer Verfahren bestimmt. Nach intravenöser Applikation von Paraquat wurden Ratten nach Zeitspannen von 3 h, 12 h, 24 h, 3 Tagen und 10 Tagen getötet. Im Magen wurde Paraquat in den Epithelzellen in einem Zeitraum von 24h bis 10 Tagen nach Applikation gemessen, wohingegen im Ösophagus sowohl in den Epithelzellen als auch in der Lamina propria mucosa Paraquat 12 h bis 10 Tage nach Injektion gemessen werden konnte. Obwohl diese Ergebnisse mit Paraquat-Befunden im Intestinum von Ratten korrelieren, konnte keine eindeutige Zeitabhängigkeit der Verteilung beobachtet werden. Dieses Ergebnis führt zu der Annahme, daß Magen und Ösophagus als wichtige Speichergewebe an einer Redistribution von Paraquat beteiligt sind.

Production of monoclonal antibody reactive with leukotoxin and its immunohistochemical application

A sensitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ALM-1) was developed for the determination of leukotoxin (9,10-epoxy-12- octadecenoic acid), which was reported to exist in human burned skin and neutrophils, and was regarded as a toxic and/or defensive substance in living beings. The leukotoxin was conjugated with bovine serum albumin (BSA) by means of the mixed anhydride technique as immunogen, and BALB/c mice were immunized over 6 months. The detection limit of leukotoxin was at least as low as 5 ng in this ELISA. This antibody had a strong specificity to leukotoxin and no cross-reactivity with the other analogs tested, and was usable for an immunohistochemical application. By using the antibody, leukotoxin was immunohistochemically observed not only in neutrophils, but also in alveolar macrophages in an oxygen-exposured rat lung.

DNA analysis of hairs in a suspected case of child abuse

Hairs are often used for DNA analysis in criminal investigations. DNA analysis of hairs with root sheaths is easy in many cases, but analyses using only the shaft or tip of the hair are often difficult. Here we describe a suspected case of child abuse in which we were commissioned to perform DNA analysis. Among 100 hairs, PCR amplification was succeeded in 99 samples, and as a result of direct sequencing, mitochondrial DNA (mtDNA) of the 99 hairs were classified into 6 types. The most common type was the 8-base substitution type of 16,168T-16,172C-16,183C-16,189C-16,217C-16,249C-16,325C-16,390A, which was observed in 86 hairs. This corresponded to the type of the victim. Total 736 STRs (75.5%) in 975 loci of 65 hairs could be typed, and only an amelogenin locus was typed in another hair. All 15 loci were typed in 10 hairs. STR types of 65 (98.5%) in 66 hairs were consistent with that of the victim. From 10 naturally-shed hair of a person, only 37 STRs (30.8%) in 120 loci of 8 hairs were typed, and all 15 loci could not be typed in these hairs. This difference in success rates of STR analysis suggested strongly that the hairs in this case were not shed naturally but forcibly, and the relevance to child abuse was suspected.

Transmission of dental data on a microdotted paper-based dental chart from a digital pen – trial operations at disaster exercise sites and at a landslide disaster at Hiroshima City

ABSTRACT In Japan, dental charting following a disaster is performed by handwriting the information on a paper-based dental chart before manual input into a computer database. Even if available, radiographic facial or intraoral images, regardless of whether they are analogue or digital, must be stored separately from the dental chart. To create a comprehensive and more permanent database accessible worldwide, we developed a system employing digital pens with image microprocessors. This system digitizes and records characters and drawings as they are being handwritten on a microdotted-paper-based post-mortem dental chart. Data stored in the built-in memory of the digital pen are transferred to a computer or tablet via Bluetooth. Using a cognitive wireless router, the information is relayed to a remote data centre. Radiographic and other images are also transferred into the same folder under a unique ID number. After several practical field tests, the system was employed at a landslide disaster in Hiroshima City. Data transfer was excellent, confirming the efficacy of the new system. This system enables data in any language to be quickly stored in the database, facilitating worldwide searches for missing persons by matching with ante-mortem dental records. The new system saves labour, helps prevent misidentification, and significantly expedites identification processes following a mass disaster.

Comparison of hard tissues that are useful for DNA analysis in forensic autopsy

Forensic analysis of DNA from hard tissues can be important when investigating a variety of cases resulting from mass disaster or criminal cases. This study was conducted to evaluate the most suitable tissues, method and sample size for processing of hard tissues prior to DNA isolation. We also evaluated the elapsed time after death in relation to the quantity of DNA extracted. Samples of hard tissues (37 teeth, 42 skull, 42 rib, and 39 nails) from 42 individuals aged between 50 and 83 years were used. The samples were taken from remains following forensic autopsy (from 2 days to 2 years after death). To evaluate the integrity of the nuclear DNA isolated, the percentage of allele calls for short tandem repeat profiles were compared between the hard tissues. DNA typing results indicated that until 1 month after death, any of the four hard tissue samples could be used as an alternative to teeth, allowing analysis of all of the loci. However, in terms of the sampling site, collection method and sample size adjustment, the rib appeared to be the best choice in view of the ease of specimen preparation. Our data suggest that the rib could be an alternative hard tissue sample for DNA analysis of human remains.