[show abstract][hide abstract] ABSTRACT: Postmortem degradation of DNA was quantitatively estimated. Brain, liver, kidney and muscle samples were obtained from sacrificed rats left at 20℃ or 4℃. The quantity of DNA was measured by real-time PCR using a primer set for a sequence in the Rsrc 1 gene. When the quantity of amplified DNA using 10ng Human Genomic DNA was defined as 100 RFU, the quantities in the brain, liver, kidney and skeletal muscle (each 2μg of dry weight) on the day of sacrifice were 253±11, 338±22, 556±14 and 531±12 Relative Fluorescence Units (RFU), respectively (mean±S.E., n＝5). The quantity of amplified DNA decreased to below 10 RFU in 1-3 weeks in the liver, kidney and skeletal muscle at 20℃, while that in the brain was more than 10 RFU for six weeks, demonstrating the usefulness of the brain as a sample for DNA analysis of decaying corpses. It was suggested that quantifying the amplified DNA in the brain at 20℃ and in the liver at 4℃ as well as the ratio of the quantity of amplified DNA in the liver to the brain at 4℃ might be useful for diagnosing time of death. This study provides the first quantitative analysis of the postmortem progress of DNA degradation in the corpse.
[show abstract][hide abstract] ABSTRACT: Postmortem changes in myoglobin concentrations in blood and organs were investigated using an enzyme immunoassay by animal experiments in combination with immunohistochemical staining of human cases. Blood myoglobin concentrations were found to increase drastically within a very short time after death. Those in striated muscle, however, did not change by day 14 postmortem. Myoglobin content in the liver and kidney increased slightly by day 5 postmortem, and more obviously by day 7 or later. However, almost no change was observed by day 5 in the kidney when the renal artery and vein had been ligated just after death. In the thyroid gland and the lung, the myoglobin content markedly increased by day 7 postmortem, with the logarithmical values rising nearly linearly as the time after death passed. In the thyroid gland, concentrations reached the level of the striated muscle. The mechanisms of postmortem myoglobin increase in organs are thought to be direct diffusion from the striated muscle and/or distribution through the blood. To estimate the postmortem interval, the determination of myoglobin content in the thyroid gland or the lung appears to be useful.
[show abstract][hide abstract] ABSTRACT: We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y), STS (steroid sulfatase) and amelogenin (AMELX and AMELY) gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.
[show abstract][hide abstract] ABSTRACT: Methamphetamine (MAP) and stress both cause a variety of cardiovascular problems. Stress also increases stimulant drug-seeking or drug-taking behavior by both humans and animals. In addition to the physiological effects on circulation, metabolism, and excretion, stress affects subject's responses to stimulant drugs such as MAP. However, the mechanisms underlying the drug-stress interactions remain unknown. In the present study, we assessed the effects of stress on myocardial responses to MAP in mice. Mice were injected with MAP (30mg/kg) immediately before exposure to water-restraint stress (WRS), which has often been used as a stressor in animal experiments. The combination of MAP with WRS produced a significant increase (p<0.01) in the leakage of proteins specific to myocardial damage and the levels of cytokines IL-6, TNF-α, and IL-10. The histological findings indicated the possibility that a combination of MAP with WRS induced cardiac myocytolysis. We also examined the expression of heat shock proteins (Hsps), which have cardioprotective effects. Administration of MAP alone significantly stimulated the RNA expressions of Hsp32, 60, 70, and 90 and the protein Hsp70 in cardiac muscles, whereas the expressions due to WRS or MAP plus WRS were not increased. These results reveal the fact that exposure to WRS depresses the induction of Hsps, in particular Hsp70, due to MAP injection, following to enhance MAP-induced myocardial damage. We believe that interactions between MAP and severe stress, including environmental temperature, affect the induction of Hsps, following to susceptibility of hosts to cardiotoxicity due to the stimulant drug.
[show abstract][hide abstract] ABSTRACT: Postmortem acidification of blood and the contribution of this phenomenon to increased flecainide concentrations in cardiac blood were evaluated in rabbits. Flecainide was administered intravenously, antemortem peripheral blood was collected 15 min after administration, then rabbits were sacrificed. Blood and organs were collected immediately or 24 h after death, or immediately or 24 h after performance of cardiac massage. Postmortem left/right cardiac blood and organs showed lower pH than antemortem blood, and flecainide concentrations in all postmortem blood samples were higher than those in antemortem blood. Increased flecainide concentrations in cardiac blood were enhanced by postmortem cardiac massage and postmortem interval. In perfusion experiments using rabbit lung and heart, even if the flecainide concentration in inflow was kept constant, outflow concentrations were 2- to 3-fold higher than in inflow when inflow pH changed from 7.4 to 5.5. In contrast, flecainide concentration in outflow decreased immediately and then remained low when pH of perfusate changed from 5.5 to 7.4. These results demonstrate that flecainide accumulates in the lungs before death, and this accumulated flecainide releases into blood following postmortem acidification of blood/organs.
Journal of analytical toxicology 01/2010; 34(1):26-31. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: The annual number of suicides in Japan increased sharply in 1998, and since that time it has consistently exceeded 30,000 per year. In this study, we analyze a database of personal and background characteristics of 824 cases (605 men, 219 women) who completed suicide in Okayama Prefecture in 2002 and 2003. The data were obtained with cooperation from the police. Using the methodologies in a previous European study as a model, we classified the suicide methods into 8 categories. To examine the generational and regional differences in the choice of methods, we stratified the sample into 4 age groups (< or =24, 25 approximately 44, 45 approximately 64, and > or =65) and 2 regional groups (Okayama/Kurashiki vs. other areas). Our results on gender differences in 7 of the suicide methods were mostly similar to the European data. However, our data showed a remarkably higher proportionate male-to-female mortality ratio for poisoning by other substances (ICD-10, X65-X69 codes) (1.83, 1.15-2.92). In terms of generational differences in the choice of suicide methods, the Mantel-Haenszel test of homogeneity was significant for most of the categories in our study, suggesting an impact of age on how people commit suicide. There were no remarkable regional differences in our sample. An epidemic curve for suicides via carbon monoxide poisoning using charcoal briquets revealed a trend of time clustering not observed in the other 6 means. The database constructed and used in this study contains richer information than conventional death statistics and is expected to provide helpful knowledge and insights for future epidemiological studies.
[show abstract][hide abstract] ABSTRACT: We encountered a case of fatal congestive heart failure that occurred under the influence of flecainide. In this case, an extreme postmortem increase in the flecainide level was identified in cardiac blood. The patient had been administered 400 mg/day of flecainide for seven days before death. Antemortem plasma obtained 13 h before death showed a flecainide concentration of 2.5 mg/L and a pH of 7.4. In comparison, centrifuged supernatants of postmortem right and left cardiac blood contained flecainide concentrations of 13.8 and 44.2 mg/L, respectively, with pH of 5.5 in both samples. This increase in blood flecainide concentration was attributed to postmortem redistribution, as about 18 h had passed between the last intake of flecainide and death.
Journal of analytical toxicology 01/2008; 32(6):451-3. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: The hypothalamic-pituitary-adrenocortical (HPA) system in drug abusers may be affected due to disorders of the hypothalamic dopaminergic system. The present study investigated alterations in the adenohypophysis of middle-aged drug abusers (40-60 years of age), using clusterin-containing mixed cell-follicles as the indicator, in which clusterin (apolipoprotein J) is a multifunctional glycoprotein related to neurodegeneration. The paraffin-embedded adenohypophyses of methamphetamine and psychotropic drug abusers (n = 76) were compared with those of non-abusers (n = 82). The number of follicles was larger in drug abusers independent of the immediate cause of death, although the size was not significantly different. When cell types forming the follicles were immunohistochemically examined, drug abusers showed an increase of prolactin (PRL) cells and gonadotroph cells and a reciprocal decrease of growth hormone cells, suggesting hypofunction of dopaminergic neurons in the hypothalamus, while there was no change in the adrenocorticotropic hormone and thyroid-stimulating hormone cells. These increases of the clusterin-containing follicles and PRL cells in the follicles may be related to the dysfunction of dopaminergic neurons in the hypothalamus of chronic drug abusers and may be useful for investigating drug abuse in forensic casework.
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 10/2007; 121(5):395-402. · 2.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: The authors designed a questionnaire to investigate the differences in German and Japanese general practitioners? (GP) awareness of suicide and attitudes toward patients with suicidal ideation in their respective societies. The purpose of this study was to obtain insights leading to a better means of suicide prevention in primary care in Japan. The background for conducting the study was declining suicides in the past 20 years and the lower suicide rate in Germany compared with the present situation in Japan, where the number of suicides has in recent years continued to exceed 30,000, resulting in a suicide rate approximately 2 times higher than that in Germany. The questionnaire was randomly mailed to GPs in Okayama-Prefecture (western Japan) and Hamburg-State (northern Germany) and was collected in the same way. The patterns of answers were compared between the 2 countries, and the differences were statistically analyzed. Japanese GPs seem to have a lower will to prevent suicide in daily practice compared to German GPs and a great lack of knowledge about treatment of suicidal patients. These observations suggest that improving GPs? interest in the problem of suicide and providing training programs for the treatment of patients with suicidal intentions might be a means of achieving better suicide prevention in Japan.
[show abstract][hide abstract] ABSTRACT: The authors encountered a case of hydrochloric acid (HCl) poisoning, thought to be caused by oral ingestion of concentrated HCl. Coagulation of the surface of the tongue and the mucosa of the pharynx, esophagus, and stomach were observed at forensic autopsy. An overabundance of Cl- was found in the gastric contents, corresponding to 8.19 mL of concentrated HCl. This was suggested to be a lethal oral dose of concentrated HCl, and the cause of death was determined to be HCl poisoning. Measuring the pH and concentrations of various ions in body fluids and contents of the alimentary tract enabled postmortem diffusion of HCl to be determined.
Journal of analytical toxicology 06/2006; 30(4):278-80. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.
[show abstract][hide abstract] ABSTRACT: Major objectives in forensic gerontology are physical and mental disorders during aging, which can be caused by various factors involving nutrition and stress, often accompanied by dysfunction in the neuroendocrine systems including the hypophysis. The objective of the present study was to investigate the histopathological changes in the adenohypophysis in elderly subjects using autopsy materials. Hypophyses with a scaphoid shape (group S: 16 males and 4 females; mean age, 78.6 years) and a normal one (group C: 30 males and 20 females; mean age, 65.2 years) were compared. Incidence of the scaphoid-shaped hypophysis mildly increased with age, being 17% in the elderly over 65 years of age. The weight of the pituitary gland in group S (0.42 +/- 0.1 g) was lower than that of group C (0.65 +/- 0.2 g). The degree of fibrosis was higher in group S (31.6% +/- 5.4%) than in group C (18.3% +/- 6.3%). Immunohistochemical staining showed no significant differences in the proportion of the ACTH cells and the TSH cells between the two groups (p > 0.05). However, there was an increase in the proportion of gonadotrophs, prolactin cells, and S-100-containing cells in group S and a decrease in that of GH cells (p < 0.05). These findings may be associated with reduced anabolic, gonadal and hepatic functions due to malnutrition.
Human Cell 09/2005; 18(3):157-62. · 1.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: We developed a new method for ABO genotyping using a multiplex single-base primer extension reaction. The method allows for the simultaneous detection of six SNP sites in the ABO gene (nt 261, 297, 681, 703, 802, and 803) and the determination of ABO genotypes from their combinations. It enabled ABO genotyping of all samples of peripheral blood DNA extracted from 103 Japanese individuals, and had a highly satisfactory detection sensitivity being capable of genotyping 0.1 ng of genomic DNA. Using this method, we were able to determine ABO genotypes of minute stain samples, heated bloodstains, aged bloodstains and mixed samples. Experiments with samples from 26 animal species and bacterial samples to test the species-specificity of the method showed that genotyping was possible in the chimpanzee and gorilla, but their genotypes were extremely rare in humans. In addition, we applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal fluid in which ABO grouping by conventional serological techniques was not possible. This new method enables the sensitive, simultaneous detection of six SNP sites in the ABO gene by two specific reactions, i.e. PCR and a primer extension reaction. Therefore, it holds promise as an effective method of ABO genotyping particularly for forensic samples.
Legal Medicine 11/2004; 6(4):213-23. · 1.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-Möller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.
Forensic Science International 09/2004; 144(1):45-57. · 2.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: We developed a method for identifying human brain from a tissue-like fragment by detection of neurofilament protein (NF) using enzyme-linked immunosorbent assay (ELISA). NF was extracted from 0.1 g of organ/tissue homogenized with Tris-HCl buffer (pH 7.2) containing urea, phenylmethylsulfonyl fluoride (PMSF), EDTA and, EGTA. It was necessary to dilute the extract at more than 2(3)-fold to avoid immunosuppression by urea. Positive reaction was always obtained for NF-H in 2(3)-fold diluted extract of brain tissue, however, NF-L and NF-M were not always detected when a brain fragment contained gray matter. Human cerebral white matter could be easily distinguished from other organs/tissues by detecting any of the NF-subunits. Brains of human and some animals could be discriminated by detecting NF-L or NF-M, although the species specificity of NF-H was not good. Our findings suggested that detection of NF-H was more useful than NF-L and NF-M for identifying a brain from a tissue-like fragment. The present ELISA method for NF-H could identify human brain specimens under the following conditions: putrefied at 4 degrees C for up to 3 weeks, dried at 37 degrees C for at least 4 months, heated at 50 degrees C for at least 4 weeks. Our results showed that our method is useful for identification of brain tissue in forensic stain analysis. Two practical cases are described.
Forensic Science International 09/2004; 144(1):1-6. · 2.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: We found small cytoplasmic vacuoles in the hormone-producing cells of anterior pituitary in hypothermic death. The vacuoles were found in approximately 40% of anterior pituitary cells both in males and females that had died from cold (n=31) while the detection rate was lower than 1% (P <0.001) in the other causes of death (n=180: fire death, n=25; fatal injury, n=24; asphyxia, n=24; poisoning, n=8; natural diseases, n=103). The detection rate in hypothermic death was the highest in ACTH cells (about 65%), followed by gonadotrophs (about 43%), and the lowest in TSH cells (about 16%) (P <0.001). These findings suggest that the cytoplasmic vacuoles in the anterior pituitary cells may be the most closely related to cold exposure among the above-mentioned cause of death, providing a supplementary evidence for determining the causes of death.
Legal Medicine 07/2004; 6(3):157-63. · 1.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: We presented an unusual case of negligent homicide by thorax compression, which is the expanded concept of traumatic asphyxia. A 58-year-old man was restrained in the prone position by six prison officers. They were ordered by their superiors to continue restraining him for about 15 min and the victim died. At the forensic autopsy, typical findings of thorax compression with intramuscular hemorrhages on the back and multiple fractures of the ribs were observed. No evidence of neck compression/smothering or other fatal issues likely to occur by chest compression was found. The reconstruction of the scene corresponded exactly with the localization of the injuries found in the victim. This is the first case of death by pure thorax compression without other fatal factors during intentional restraint, in which the force causing the chest compression was distinctly determined by the autopsy and reconstruction.
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 05/2004; 118(2):106-10. · 2.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we used paraffin-embedded human pituitary obtained from 248 autopsy cases and identified mixed cell follicles by the immunohistochemical method. We examined the number and size of the mixed cell follicles, and the ratio of each component cell of these follicles, in the anterior pituitary at various age groups. The number of follicles increased with age, and the size of the follicles also tended to enlarge with age. Statistical analysis showed that a high correlation existed between age and the number or the size of the mixed cell-follicles formed by various adenohypophyseal cells. In addition, when the proportions of the different cell types that formed the follicles were examined, sex differences were observed with aging for the GH cells, the PRL cells, and the gonadotroph (GTH) cells, while no changes were observed with aging in both men and women for the ACTH cells and TSH cells. These results indicate that the number, size, and ratio of each component cell of follicles in the anterior pituitary are adequately applicable for the purpose of age estimation in routine forensic medicine.