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A 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 PCR
Acta Medica Okayama , 2011,
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.
Postmortem Changes in Myoglobin Content in Organs
Acta Medica Okayama , 2011,
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.
Increase of S-100 protein-positive stellate cells in the anterior pituitary of chronic alcoholic patients with fatty liver or fatty cirrhosis.
Ishikawa T,Tachibana T,Ishikawa H,Miyaishi S
Acta Medica Okayama , 2003,
Abstract: Healthy subjects 40 years old were used as controls in a study of stellate cells (S-100 protein-containing cells, or S-100 cells) in subjects with chronic alcoholism and fatty liver or fatty cirrhosis. S-100 cells were sparsely found in the adenohypophysis of control subjects, and these cells sometimes formed small clusters. However, in chronic alcoholics with fatty liver or fatty cirrhosis, the number of stellate cells in the anterior pituitary tended to be 17 times higher than it was in the control group. No increase in the number of S-100 positive cells that constitute the large and small follicles in the intermediate pituitary. The physiological function of the S-100 protein has not yet been identified. The fact that an increase in prolactin-secreting and growth hormone-secreting cells, as well as a decrease in gonadotrophs were observed in the hypophysis of alcoholics suggests that the function of stellate cells may be closely related to these phenomena. Our results also imply that the stellate cells found in the anterior and intermediate pituitary differ in function although they both produce S-100 proteins.
Comparison of German and Japanese general practitioners' awareness of suicide and attitudes toward patients with suicidal ideation.
Acta Medica Okayama , 2006,
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.
Quantitative Analysis of DNA Degradation in the Dead Body
Acta Medica Okayama , 2011,
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.
Evaluation of a method for typing the microsatellite D12S391 locus using a new primer pair and capillary electrophoresis.
Shigeta Y,Yamamoto Y,Doi Y,Miyaishi S
Acta Medica Okayama , 2002,
Abstract: 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.
Role of adenohypophyseal mixed cell-follicles in age estimation.
Ishikawa T,Miyaishi S,Tachibana T,Yamamoto Y
Acta Medica Okayama , 2003,
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.
Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.
Ono T,Miyaishi S,Yamamoto Y,Yoshitome K
Acta Medica Okayama , 2001,
Abstract: 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.
MUTYH Gln324His gene polymorphism and genetic susceptibility for lung cancer in a Japanese population
Aiko Miyaishi, Kayo Osawa, Yasunori Osawa, Natsuko Inoue, Kana Yoshida, Mayumi Kasahara, Akimitsu Tsutou, Yoshiki Tabuchi, Kazuo Sakamoto, Noriaki Tsubota, Juro Takahashi
Journal of Experimental & Clinical Cancer Research , 2009, DOI: 10.1186/1756-9966-28-10
Abstract: We analyzed associations among OGG1 Ser326Cys and MUTYH Gln324His gene polymorphisms in relation to lung cancer risk using PCR-RFLP. The study involved 108 lung cancer patients and 121 non-cancer controls divided into non-smokers, smokers according to pack-years smoked in Japanese.The results showed that the MUTYH His/His genotype compared with Gln/Gln genotype showed an increased risk for lung cancer (adjusted odds ratio [OR] 3.03, confidence interval [95%CI], 1.31–7.00, p = 0.010), whereas there was no significant increase for the Gln/His genotype (adjusted OR 1.35, 95%CI 0.70–2.61, p = 0.376). The MUTYH His/His genotype was at a borderline increased risk for both adenocarcinoma and squamous cell carcinoma (adjusted OR 2.50, 95%CI 0.95–6.62, p = 0.065 for adenocarcinoma; adjusted OR 3.20, 95%CI 0.89–11.49, p = 0.075 for squamous cell carcinoma, respectively). However, the OGG1 Ser/Cys or Cys/Cys genotypes compared with the Ser/Ser genotype did not have significantly increased risk for lung cancer, containing either adenocarcinoma or squamous cell carcinoma. The joint effect of tobacco exposure and the MUTYH His/His genotype compared with the Gln/Gln genotype showed a significant association with lung cancer risk in smokers, and there was not significantly increased in non-smokers (adjusted OR 3.82, 95%CI 1.22–12.00, p = 0.022 for smokers; adjusted OR 2.60, 95%CI 0.60–11.25, p = 0.200 for non-smokers, respectively). The effect of tobacco exposure and the OGG1 Ser326Cys showed also no significant risk for lung cancer.Our findings suggest that the MUTYH Gln324His polymorphism appear to play an important role in modifying the risk for lung cancer in the Japanese population.Lung cancer is a well-known cancer that is caused by carcinogens, such as those in tobacco smoke. Tobacco smoke contains many chemical carcinogens and reactive oxygen species, including polycyclic aromatic hydrocarbons. DNA damage induced by these carcinogens or by endogenous metabolic processes can
Association of MUTYH Gln324His and APEX1 Asp148Glu with colorectal cancer and smoking in a Japanese population
Mayumi Kasahara, Kayo Osawa, Kana Yoshida, Aiko Miyaishi, Yasunori Osawa, Natsuko Inoue, Akimitsu Tsutou, Yoshiki Tabuchi, Kenichi Tanaka, Masahiro Yamamoto, Etsuji Shimada, Juro Takahashi
Journal of Experimental & Clinical Cancer Research , 2008, DOI: 10.1186/1756-9966-27-49
Abstract: The case-control study involved sixty-eight colorectal cancer patients and 121 non-cancer controls divided into non-smokers and smokers according to pack-years of smoking. The genetic polymorphisms of DNA repair enzymes,OGG1 Ser326Cys, MUTYH Gln324His, APEX1 Asp148Glu and XRCC1 Arg399Gln, were examined using PCR-RFLP.The MUTYH Gln324His showed strong significant associations with a risk of colorectal cancer (crude odds ratio [OR] 3.30, 95% confidence interval [95%CI] 1.44–7.60, p = 0.005; adjusted OR3.53, 95%CI 1.44–8.70, p = 0.006). The ORs for the APEX1 Asp148Glu were statistically significant (crude OR 2.69, 95%CI 1.45–4.99, p = 0.002; adjusted OR 2.33, 95%CI 1.21–4.48, p = 0.011). The ORs for the MUTYH Gln324His and the APEX1 Asp148Glu were statistically significant for colon cancer (adjusted OR 3.95, 95%CI 1.28–12.20, p = 0.017 for MUTYH Gln324His ; adjusted OR 3.04, 95%CI 1.38–6.71, p = 0.006 for APEX1 Asp148Glu). The joint effect of tobacco exposure and the MUTYH Gln324His showed a significant association with colorectal cancer risk in non-smokers (adjusted OR 4.08, 95%CI 1.22–13.58, p = 0.022) and the APEX1 Asp148Glu was significantly increased in smokers (adjusted OR 5.02, 95%CI 1.80–13.99, p = 0.002). However, the distributions of OGG1 Ser326Cys and XRCC1 Arg399Gln were not associated with a colorectal cancer risk.Our findings suggest that the MUTYH Gln324His and the APEX1 Asp148Glu constitutes an increased risk of colorectal cancer, especially colon cancer. The MUTYH Gln324His is strongly associated with colorectal cancer susceptibility in never smoking history, whereas the APEX1 Asp148Glu genotype constitutes an increased risk of colorectal cancer when accompanied by smoking exposure.Colorectal cancer is a major cause of death and is influenced by genetic characteristics and environmental factors. Humans are exposed daily to a large variety of toxic and carcinogenic compounds due to habits such as tobacco smoking. Tobacco smoking produces major classes o
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