Okayama University Medical SchoolActa Medica Okayama0386-300X6552011Quantitative Analysis of DNA Degradation in the Dead Body299306ENMikiItaniYujiYamamotoYusukeDoiSatoruMiyaishiOriginal Article10.18926/AMO/47011Postmortem 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.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6522011A New Method for Sex Determination Based on Detection of SRY, STS and Amelogenin Gene Regions with Simultaneous Amplification of Their Homologous Sequences by a Multiplex PCR113122ENToshioMorikawaYujiYamamotoSatoruMiyaishiOriginal Article10.18926/AMO/45270We 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.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5722003Analysis of short tandem repeat (STR) polymorphisms by the powerplex 16 system and capillary electrophoresis: application to forensic practice.5971ENOsamuOkamotoYujiYamamotoSachiyoInagakiKeiYoshitomeTakakiishikawaKiyomiImabayashiSatoruMiyaishiHideoIshizuArticle10.18926/AMO/32819<p>Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5722003Role of adenohypophyseal mixed cell-follicles in age estimation.8389ENTakakiIshikawaSatoruMiyaishiToshiakiTachibanaYujiYamamotoHideoIshizuArticle10.18926/AMO/32816<p>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.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5532001Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.175184ENToshiakiOnoSatoruMiyaishiYujiYamamotoKeiYoshitomeTakakiIshikawaHideoIshizuArticle10.18926/AMO/32024<p>We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we were able to distinguish human samples from those of 21 kinds of animals: the crab-eating monkey, horse, cow, sheep, goat, pig, wild boar, dog, raccoon dog, cat, rabbit, guinea pig, hamster, rat, mouse, whale, chicken, pigeon, turtle, frog, and tuna. However, we were unable to distinguish between human and gorilla samples. This method enabled us to detect the target sequence from 25 pg of human DNA, and the target DNA fragment from blood stored at 37 degrees C for 6 months, and from bloodstains heated at 150 degrees C for 4 h or stored at room temperature for 26 years. Herein we also report a practical application of the method for human identification of a bone fragment.</P></p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5952005A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples.179194ENKiyomiImabayashiYujiYamamotoSachiyoInagakiYusukeDoiKeiYoshitomeSatoruMiyaishiHideoIshizuArticle10.18926/AMO/31971<p>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.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6342009Factors affecting the choice of suicide method in Okayama: a database analysis from a forensic perspective177186ENEigoKamizatoKeiYoshitomeYujiYamamotoToshihideIwaseToshihideTsudaSatoruMiyaishiHiroyukiDoiOriginal Article10.18926/AMO/31815<p>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 (<-24, 2544, 4564, and >-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.</p>No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5612002A case of suffocation by an advertising balloon filled with pure helium gas.5355ENKeiYoshitomeTakakiIshikawaSachiyoInagakiYujiYamamotoSatoruMiyaishiHideoIshizuArticle10.18926/AMO/31726<p>We encountered a rare case of suffocation by an advertising balloon filled with pure helium gas. Suffocation caused by inhalation of atmosphere lacking in oxygen is not exceptional, but reports of death by suffocation due to a pure inert gas such as helium are very rare. In this case, the balloon mooring on the ground was enclosed, warning signs were displayed, and it was clear that entering the balloon filled with an atmosphere lacking in oxygen was extremely dangerous and should not be done; the accident did, however, occur. Accidents of this kind may occur in the future unless appropriate education and countermeasures are taken.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5652002Evaluation of a method for typing the microsatellite D12S391 locus using a new primer pair and capillary electrophoresis.229236ENYoshiakiShigetaYujiYamamotoYusukeDoiSatoruMiyaishiHideoIshizuArticle10.18926/AMO/31707<p>We describe a modified method for typing a polymorphic microsatellite D12S391 locus by PCR using a newly designed primer pair. This primer pair produces shorter D12S391 amplified fragments (104-156 bp) than the primer pair originally described by Lareu et al. (209-261 bp). The detection system for the D12S391 locus using the new primer pair and capillary electrophoresis (CE) analysis was evaluated using various forensic samples. The typing results from 70 DNA samples using the new primer pair and the original primer pair were completely identical. One hundred twenty-five amplified fragments from D12S391 alleles were sized correctly within +/- 0.25 bp of the D12S391 allelic ladder. A rare allele, 19.3, previously found only in Caucasians, was found for the first time in a Japanese subject, and it was clearly distinguished from allele 20 by the CE analysis. This detection system was sensitive and could detect D12S391 types from 16 pg of genomic DNA, and from a minor component at a ratio of 1:10 in mixed samples. This system was more useful for the analysis of degraded DNA than was the method using the original primer pair, and could detect D12S391 types from bloodstains that had been stored for 26 years. In addition, the specificity of the method was demonstrated using nonhuman DNA.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5261998Improvement of sensitivity in HLA-DRB1 typing by semi-nested PCR-RFLP.289296ENSeiichiInoueYujiYamamotoOsamuOkamotoHirokiMurakamiSatoruMiyaishiHideoIsizuArticle10.18926/AMO/31305<p>A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5241998Haptoglobin genotyping by allele-specific polymerase chain reaction amplification173181ENAkemiYanoYujiYamamotoSatoruMiyaishiHideoIshizuArticle10.18926/AMO/31301<p>We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.</p>
No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5011996IgA2 genotyping by polymerase chain reaction (PCR) using allele-specific amplification primers.19ENShingoTakataYujiYamamotoHideoIshizuArticle10.18926/AMO/30512<p>A method of genotyping IgA2 alleles in the human immunoglobulin alpha 2 heavy chain constant region (C alpha 2 gene) was developed by using the polymerase chain reaction (PCR). By this method, the genotype was determined by discriminating base substitution in the 3'-flanking region of alleles, A2m*1 and A2m*2, which manifest A2m serum types, by nested PCR using allele-specific primers. Three types, IgA2*1/IgA2*1, IgA2*2/IgA2*1, and IgA2*2/IgA2*2, were detected from DNA extracted from lymphocytes. Genotyping was possible from 100 pg of DNA by this method. The estimated allele frequency in 318 Japanese subjects was 0.561 for IgA2*1 and 0.439 for IgA2*2. Analysis of 29 cases of paternity tests suggested that the data follow Mendel's law of inheritance. This genotype could also be detected in whole blood, blood stains, saliva stains, and various organs and tissues. These results suggest the usefulness of the present method for paternity testing and individual identification in forensic medicine.</p>
No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-1558979-101985頭蓋外―頭蓋内動脈吻合術における脳循環動態に関する実験的研究783803ENYujiYamamotoIn order to evaluate extracranial-intracranial arterial anastomosis in acute ischemic stroke, changes in the local cerebral blood flow (lCBF) in the cortical and subcortical areas were investigated by hydrogen clearance during left middle cerebral artery (MCA) occlusion and maxillary artery (MA)-to-MCA anastomosis in 24 mongrel dogs. Following MCA occlusion, the lCBF was maintained at more than 25ml/100g/min in 17 dogs because of effective leptomeningeal collaterals. In seven dogs, cortical ischemia was shown with progressive lCBF reduction to below 25ml/100g/min. Severe persistent ischemia of less than 10ml/100g/min was not observed. Subcortical ischemia with lCBF reductions to below 25ml/100g/min in the gray matter and below 10ml/100g/min in the white matter occurred more frequently than cortical ischemia. Following MA-MCA anastomosis, the cortical lCBF returned to nearly the preocclusion value, and the subcortical lCBF showed a slight or moderate increase. Reactive hyperemia was noted in two dogs (at three sites), but marked hyperemia was not abserved in any of the animals. The increase in the lCBF after the anastomsis showed a linear relationship to the donor arterial supply. Postmortem and histological examinations revealed no cerebral edema or hemorrhagic infarct. Comparative analyses of these results with the concept of "ischemic penumbra" led to the conclusion that cerebral ischemia with the cortical lCBF ranging from 15 to 25ml/100g/min (30 to 50% of the preocclusion value) after MCA occlusion cannot be reversed even by the leptomeningeal collaterals and may produce progessive neurological dysfunction. In such an ischemic condition, rapid restoration of the lCBF by extracranial-intracranial arterial anastomosis might be effective in the prevention of cerebral infarction as well as in the recovery of neuronal function.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-15581039-101991外傷事例及び軽度運動負荷時の血中ミオグロビン濃度11031107ENSatoruMiyaishiTakashiKitaoFumioMoriyaYujiYamamotoHideoIshizuHiroyukiIshiiThe blood myoglobin level was determined in 18 trauma cases of long bone fracture and the changes in the blood myoglobin level and activities of enzymes in the blood exuded from the muscle tissues after a slight exercise of a 2-km marathon of healthy persons were determined. The blood myoglobin level of the trauma cases increased while there were no changes in the blood myoglobin level and activities of the enzymes in the blood after a 2-km marathon. The blood myoglobin level may be of help to determine whether a man has been injured or not even if he had been exercising at the time of injury.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-15581059-101993トロンビン点滴静注による事故死の一剖検例839845ENHideoIshizuSatoruMiyaishiYujiYamamotoShingoTakataA 63-year-old male died in the hospital from erroneous drip infusion of thrombin, a local hemostatic, after polypectomy for intestinal polyposis. At autopsy, thromboses were observed in the cutaneous veins of the right arm, the right axillary vein and the right subclavian vein, and soft fibrin clots in the superior vena cava, the right atrium, the right ventricle and the pulmonary arteries. Thrombi were also found in the rami of the arteries in both lungs under the microscope. Leucocyte infiltration was recognized in the thrombus, but no organization. This finding shows that the thrombi were fresh. The remainder (150ml) of the drip solution was analyzed for thrombin activity and 8 to 16 units of thrombin activity per ml were demonstrated. From these results, the cause of death was considered pulmonary thrombosis due to intravenous administration of thrombin.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155811332001外因死の疑いで司法解剖となった内因性急死の2例241245ENTakakiIshikawaSatoruMiyaishiYujiYamamotoKeiYoshitomeSachiyoInagakiMichihikoOkamuraHideoIshizuTwo judicial autopsy cases of sudden unexpected natural death were reported. In both cases it was suspected that the victims had died of a traffic accident or violent injury. It became clear, however, that both the cause of death had been myocardial infarction by the forensic autopsies and that the suspects had not been criminally responsible for the death of the victims. It is expected that sudden unexpected natural death whose cause and manner are unclear at first may occur more frequently following the increase of the number of death from ischemic heart disease in the future. In these circumstances the forensic autopsy of such cases will become more important socially.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155811432003岡山県下で起きた早春室内での凍死例303308ENTakakiIshikawaSatoruMiyaishiYusukeDoiTomoyoTakataKiyomiImabayashiSachiyoInagakiKeiYoshitomeYujiYamamotoHideoIshizuWe encountered two cases of unnatural death occurring indoors in early spring in Okayama Prefecture. The two cases were both females aged 31yearsold and 88yearsold. Autopsies revealed death from hypothermia as the cause of death. The diagnosis of death from cold was not based solely on the characteristic findings of the dead body. After confirming that there were no other accidents and diseases that may cause death, an overall evaluation should be made considering the conditions surrounding occurrence of death from cold. We describe these procedures using these two autopsy cases of death from hypothermia.No potential conflict of interest relevant to this article was reported.