Masateru Kane

The detection of picoplankton 16S rDNA in cases of drowning

Picoplankton belonging to theSynechococcus genus in cyanobacteria (approximately 1 μm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria by microscopy. In this study we attempted to use picoplankton for the diagnosis of drowning by PCR analysis of the 16S ribosomal DNA (rDNA). We designed primers complementary to the variable regions of 16S rDNA of the picoplankton we had sequenced. A comparison was made of the PCR products from the three picoplanktons, five other cyanobacteria,Melosira (diatom),Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human lung tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The isolation of the picoplankton from human lung tissue increased the sensitivity of PCR more than a hundred-fold. The specific PCR products of the picoplankton were obtained from formalin-fixed drowning tissue. Molecular biological diagnosis of drowning was successful using picoplankton 16S rDNA.

Determination of ABO genotypes with DNA extracted from formalin-fixed, paraffin-embedded tissues.

SummaryThe gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 by and 200 by could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample.ZusammenfassungDas Gen wird für die spezifischen Glykosyltransferasen kodiert, welche die Konversion des H-Antigens in das A- oder B-Antigen katalysieren, weist eine leichte, aber definierte Variation seiner allelischen Nukleotid-Sequenz auf. Auf diese Weise lassen sich 6 Genotypen unterscheiden, nach Spaltung mit spezifischen Restriktionsenzymen. Wir haben DNA von formalinfixierten, paraffineingebetteten Geweben extrahiert, indem wir SDS-Proteinase-K-Behandlung mit nachfolgender Phenol-Chloroform-Extraktion anwandten. Die Sequenz der Nukleotide für die A-, B- und O-Gene wurde amplifiziert mit Hilfe der Polymerase-Kettenreaktion (PCR). DNA-Fragmente mit 128 Bp und 200 Bp konnten in der zweiten PCR-Phase amplifiziert werden, indem ein Aliquot der ersten PCR-Phase eingesetzt wurde. Degradierte DNA von Paraffin-Blöcken, welche bis zu 10,7 Jahren gelagert waren, konnte erfolgreich typisiert werden. Der ABO-Genotyp wurde dann abgeleitet von den Mustern nach Spaltung mit einer geeigneten Kombination von Restriktionsenzyneu und war kompatibel mit dem Phänotyp, der von der Blutprobe erhalten wurde.

ABH-related antigens in human male genital tract

SummaryThe localization of ABH related antigens in human male reproductive tract was examined using monoclonal antibodies and an avidin biotin complex method. No positive reaction with blood group antibodies on spermatozoa was observed in testis and ductus epididymidis apart from erythrocytes and endothelial cells. The expression of ABH and ABH related antigens in ductuli efferentes testis, ductus epididymidis, seminal vesicle and prostate was complexly coded by a combination of H, Se, Le and X genes. The results obtained in this study indicate that the ABH antigens detected on spermatozoa of seminal stains are coating antigens and not inherent to the cell membrane, and the ABO, H, Se, Le and X genes are subjected to a tissue-dependent differential expression.ZusammenfassungDie Lokalisation (and Verteilung) der Antigene des ABH-Komplexes im Bereich der inneren Geschlechtsorgane des Mannes wurde mittels monoklonaler Antikörper unter Benutzung einer Avidin-Biotin Technik untersucht. Dabei konnten positive Reaktionen im Hoden und im Ductus epididymidis lediglich an Erythrozyten und Endothelzellen beobachtet werden. Die Expression von ABH-Antigenen in den Ductuli efferentes testis, im Ductus epididymidis, in den Samenbläschen und der Prostata wird offensichtlich komplex durch H-, Se-, Le- und X-Gene kodiert. Die Resultate der vorliegenden Untersuchungen zeigen, daß die ABH-Antigene der Spermienoberfläche offensichtlich aus der Samenflüssigkeit stammen und die ABO-, H-, Se-, Le-und X-Gene gewebsabhängig unterschiedlich exprimiert werden.

Phylogenetic analysis of picoplankton in Lake Biwa and application to legal medicine

Three strains of picoplankton designated as brown, green, and pink belonging to the Synechococcus genus in cyanobacteria (approximately 1 μm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria such as Proteobacteria and Bacillus by microscopy. In this study, we attempted to use the polymerase chain reaction (PCR) analysis of the 16S ribosomal DNA (rDNA) from picoplankton for the diagnosis of death by drowning. A segment of 16S rDNA was sequenced in order to investigate their phylogenetic relationships and to design the specific primers. The PCR products from three picoplanktons were compared with those from five other cyanobacteria, Melosira (diatom), Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The efficiency of PCR was increased more than hundredfold by isolating the picoplankton from human lung tissue. The specific PCR products of the picoplankton were obtained from a formalin‐fixed drowning body (lung and liver) that was found in a downstream river and Lake Biwa. The PCR analysis of the picoplanktion 16S rDNA is considered useful for the diagnosis of death by drowning.

Electrophoretic phenotyping of erythrocyte enzymes

Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in pI values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provides a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood group typing by electrophoresis based on isoelectric focusing

The electrophoresis of proteins and enzymes is described with respect to its application and contribution to the blood group typing in the medico-legal field. Isoelectric focusing (IEF) has many advantages over conventional electrophoresis because of minimizing band diffusion, higher resolution, phenotyping under equilibrium and non-equilibrium conditions. The difference between the resolving capabilities of IEF and conventional electrophoresis is due to the pH environment within the gel. In IEF, protein separation is accomplished in a pH gradient across a gel and the net charge of a protein is variable, whereas with conventional electrophoresis the pH is essentially constant throughout the gel and net charge of a protein is also constant. A variable net charge difference along the gel plays an important role for good separation under non-equilibrium conditions. Moreover, the appropriate addition of chemical separators increases the net charge difference of proteins under non-equilibrium conditions and provides good resolution in shorter focusing times. The chemical separators also improve the resolution of isozymes and increase the protein solubility as a result of the buffering capacity. On the other hand, a shift of isozyme patterns from the same individual can be observed depending on the kind of sample. The shift is caused by the hemoglobin in phosphoglucomutase (PGM1) typing, the actin in group specific component (GC) typing and the different isoelectric points (pI) in α-1-antitrypsin (PI) typing. Therefore, the most suitable technique should be selected based on the system used and the sample conditions.

Species identification from tissue particles using lectin- and immuno-histochemical methods

Many kinds of forensic biological materials found at scenes of murder and traffic accidents are brought to forensic scientists for identification. Although various immunological methods and DNA techniques for the species identification from blood and bloodstains are now available, there are few investigations for blood grouping and species identification from tissue particles or tissue debris(Fechner et al 1989). In this study we report a method for species identification from tissue particles using lectin- and immuno-histochemical techniques.

A fatality following difluoroethane exposure with blood and tissue concentrations

1,1-Difluoroethane (DFE; HFC-152a) is a colorless and odorless gas often used as a propellant to remove dust from electronic equipment. Because DFE affects the central nervous system and produces a sense of euphoria, it is also often abused. We report a case of fatal, acute DFE poisoning in which the DFE concentrations in the blood and tissues were determined. A 30-year-old man with no known medical history was found dead in his room, lying on the floor and gripping an air-duster canister (AD-152A; Elecom, Osaka, Japan). Ten empty and two unused canisters were also found in the room. The man had been arrested in the past for substance use and abuse (paint thinner and Cannabis). Furthermore, he was known to have sold illicit substances for at least 2 years prior to his death and to lose consciousness at times after inhaling DFE in the restrooms of department stores. Because he had died of unnatural causes, a forensic autopsy was performed approximately 14 h after the body was discovered. The decedent was 171 cm tall and weighed 51.8 kg. Autopsy findings included fluid blood, petechial hemorrhage on the conjunctivas and epicardium, and congestive changes in the viscera that suggested sudden death. Neither injuries nor diseases were found macroscopically or microscopically. Body fluids and tissues were collected for DFE analysis. Quantitative analyses of DFE were conducted by gas chromatography/mass spectrometry (GC/MS) (Agilent 7890B and 5977A instruments; Agilent Technologies, Santa Clara, CA). Table 1 shows the concentrations of DFE detected in body fluids and tissues. Screening for toxicologic substances by GC/MS (GCMS-QP2010; Shimadzu, Kyoto, Japan) with the SHIMADZU GC/MS Forensic Toxicological Database revealed no other toxicologic substances in the blood. No alcohol was detected in the heart blood by GC (GC-14B; Shimadzu). Given the findings from the autopsy and toxicological analyses and the scene described by the police, the cause of death was determined to be acute DFE poisoning and the estimated the time of death to be 4–5 days prior to the autopsy. To our knowledge, 47 fatal DFE poisonings have been reported since 2004. Among the 15 cases in which DFE blood concentrations were noted, nine were DFE-only poisoning cases in which the DFE concentration in the heart blood ranged from 99.2 to 430.0 mg/ml (mean, 225.3 mg/ml) [1–5]. In our case, the DFE heart blood concentration was 81 mg/ml, a lower value than those previously reported in fatal DFE-only poisonings. According to the autopsy and histopathologic examination findings, the mechanism of death was determined to be fatal arrhythmia. The lethal mechanism was considered to involve DFE-induced sensitization of the myocardium to catecholamines, which may lead to fatal arrhythmia [1]. The measured DFE concentrations in the blood and various tissues may help in the evaluation of further fatal DFE poisoning cases.

ABH-Related Antigens Participate in the Spermatogenesis of Cats and Rats

The ABH antigens were expressed in the secretory cells of many mammals(1), including humans. Although the allelic cDNA of ABO blood group had been cloned and sequenced(2), no clear explanation of the biological significance of the ABH and related antigens has been proposed. In this study we examined the distribution of ABH related antigens in the urogenital organs from cats and rats in order to pro mote better understanding on the biological significance of the antigens, using monoclonal antibodies (MoAbs) against the ABH and related antigens, and lectins. The results obtained from the present study suggested that the ABH and related antigens may play certain roles in the processes of development of spermatogenic system. In view of forensic practice, the importance of species identification prior to ABO blood grouping from seminal, saliva and urinal stains are stressed on the basis of the present results.

Lectin- and Immuno-Histochemistry on Mucous Substances of the Taste Buds and Lingual Glands in Some Mammals

The ABO blood group antigens were discovered at the beginning of this century. Although the biochemical and molecular-biochemical nature of the ABH and related antigens is now quite well characterized and it is presumed that they are concerned with oncodevelopmental and/or cell recognition phenomena(1), no clear explanation of the biological significance of these antigens has been proposed. Characterization of cells with regard to their surface and secretory glycoconjugates is important in order to understand the basis of their physiological and interactive behaviour. The taste buds of vertebrates are covered by mucous substances and it has been suggested that the carbohydrate residues of the mucous substances are essential for chemoreceptor processes in the function of taste buds. Therefore, in the present study, we examined the distribution of different types of carbohydrate antigens in the taste buds cells and lingual glands using monoclonal antibodies(MoAb) against blood group antigens and lectins conjugated with horseradish peroxidase(HRP).