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Abstract RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model. Key messages • Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified. • A splicing reporter assay demonstrated that the variant affected the TTN splicing. • Rbm20I538T mice showed neither DCM nor cardiac dysfunction. • Rbm20I538T mice showed different splicing patterns and the gene expressions.

A Japanese man in his 30s died suddenly. Postmortem computed tomography and autopsy revealed a pulmonary embolism from an organizing thrombus in the inferior vena cava as the cause of death. Genomic analysis of congenital thrombophilia-related genes (i.e., SERPINC1, PROC, PROS1, F2, F5, PLG, and MTHFR) revealed a heterozygous variant of PROS1 (p.A139V), which has been reported in patients with congenital protein S deficiency. After a genetic conference that included forensic pathologists, molecular scientists, genetic researchers, genetic clinicians, and clinical physicians, the results of the genetic analysis were explained to the family. Biochemical analyses of protein S (PS) activity and total PS antigen levels were performed with samples from the deceased's family and genetic analysis was not performed until clinical symptoms appear. Herein we demonstrate the importance of genetic background in cases of a sudden death due to pulmonary embolism.

A Japanese girl with polycystic kidney disease (PKD) developed normally, but at 8 months of age, she was hospitalized for acute onset dyspnea. On the day after admission to hospital, her general condition suddenly became worse. An echocardiogram showed left ventricular dilatation with thin walls, severe mitral valve regurgitation, and a reduced ejection fraction. She died of acute cardiac failure 3 hours after the sudden change. Postmortem analysis with light microscopy showed disarray of cardiomyocytes without obvious infiltration of lymphocytes, and we diagnosed her heart failure as idiopathic dilated cardiomyopathy (DCM). Clinical exome sequencing showed compound heterozygous variants in JPH2 (p.T237A/p.I414L) and a heterozygous nonsense mutation in PKD1 (p.Q4193*). To date, several variants in the JPH2 gene have been reported to be pathogenic for adult-onset hypertrophic cardiomyopathy or DCM in an autosomal dominant manner and infantile-onset DCM in an autosomal recessive manner. Additionally, autosomal dominant polycystic kidney disease is a systemic disease associated with several extrarenal manifestations, such as cardiomyopathy. Here we report a sudden infant death case of DCM and discuss the genetic variants of DCM and PKD.

The aim of this study is to determine the molecular mechanism of sudden death in a previously healthy patient. Clinical exome sequencing revealed I536T-RBM20 variant, which alters RNA splicing of TTN and is causative for dilated cardiomyopathy. Comprehensive RNA sequencing (RNA-seq) was also performed in the patient samples and the control samples. Splicing abnormality was compared in cardiac muscle and skeletal muscle. RNA-seq analysis of the cardiac and skeletal muscle showed abnormal splicing of LDB3, not of TTN. Exon 11 of LDB3 was abnormally included in the patient samples compared with the control samples. This abnormal LDB3 splicing pattern in skeletal muscle has been reported in myotonic dystrophy type 1 (DM1) patients. We, thus, confirmed that the patient had expanded CTG repeat in DMPK and the diagnosis was genetically DM1. This finding suggest that one of the molecular mechanisms of sudden cardiac death in this asymptomatic subclinical DM1 patient might be LDB3 abnormal splicing due to the CTG repeat in DMPK, rather than RBM20 variant. RNA-seq analysis is useful to determine the exact molecular diagnosis for sudden cardiac death.

Methamphetamine, a representative stimulant, is one of the most commonly abused drugs globally. Although originally used in healthcare settings as a psychostimulant among the psychotropic drugs, it has been illegally bootlegged and abused because of its strong pharmacological action. Methamphetamine is a central nervous system stimulant that induces hypernoia and reduces fatigue and drowsiness. However, its abuse can lead to dependence and the development of psychosis, with the chief complaints of hallucinations and delusions. Its use is therefore restricted by law. In terms of psychotropic drugs, whether old or new type of drugs are prescribed depends on symptoms even now. Psychotropic drugs that have high lethality associated with overdose include typical antipsychotics, tricyclic antidepressants, and barbiturates. Even drugs that have few side effects and are considered safe, such as atypical antipsychotics, selective serotonin reuptake inhibitors, benzodiazepines, and non-benzodiazepines, are associated with a risk for overdose, increased suicide rates, and onset of discontinuation syndrome where they are prescribed more than necessary. Individuals can form physical and psychological dependence to benzodiazepines and barbiturates, and therefore attention must be paid to their long-term use.

Metabolic diseases are one of the main causes of sudden infant death, but definitive postmortem diagnosis is difficult, because metabolic diseases are functional and have fewer morphological abnormalities. Metabolic autopsy is a procedure that focuses on metabolic diseases, which includes the analysis of metabolic products or genetic analysis. However, it is not routinely performed, and only a small number of cases of sudden death with metabolic diseases have been reported in Japan. Furthermore conventional genetic analysis is “one gene, one exon at a time” and is therefore time-consuming and effort-intensive. Recently, next-generation sequencing methods have become available and can comprehensively screen numerous genes, making the technique practical for sudden death in the field of forensic science. In this chapter, we describe the basis of metabolic disease and discuss several case reports. Furthermore, we introduce a recent study into sudden unexpected death in infancy from the perspective of “functional autopsy.” Some causes of sudden infant death are inherited diseases, and accurate diagnosis, even postmortem, can prevent further tragedies among the remaining family.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) results in an increased risk of sudden death. We sought mutations of desmoglein-2 (DSG2), desmoplakin (DSP), and plakophilin-2 (PKP2) in 15 cases of sudden death whose causes of death could not be determined at autopsy. In three victims, mutations were identified in DSP. Two of these mutations were novel; one had previously been reported in a patient with ARVC that had been diagnosed clinically. Histological findings were not typical of ARVC; however, it was notable that these mutations were present in three of 15 cases, a relatively high proportion. The causal relationship between the mutations and ARVC is unclear, but the mutations might have been associated with faulty desmosomal proteins resulting in fatal arrhythmia. Combining information gathered by the traditional means of gross and histological examination with postmortem genetic analysis of young victims would assist in identifying their cause of death.

Background: Effects of drug abuse on LINE-1 retrotransposition (L1-RTP) in somatic cells are unknown. Results: Methamphetamine and cocaine-induced L1-RTP depends on CREB. Conclusion: Methamphetamine and cocaine activate CREB and L1-RTP in neuronal cell lines. Significance: To our knowledge this is the first study to demonstrate the induction of L1-RTP by drugs of abuse., Long interspersed element 1 (L1) is a retroelement constituting ∼17% of the human genome. A single human cell has 80–100 copies of L1 capable of retrotransposition (L1-RTP), ∼10% of which are “hot L1” copies, meaning they are primed for “jumping” within the genome. Recent studies demonstrated induction of L1 activity by drugs of abuse or low molecular weight compounds, but little is known about the underlying mechanism. The aim of this study was to identify the mechanism and effects of methamphetamine (METH) and cocaine on L1-RTP. Our results revealed that METH and cocaine induced L1-RTP in neuronal cell lines. This effect was found to be reverse transcriptase-dependent. However, METH and cocaine did not induce double-strand breaks. RNA interference experiments combined with add-back of siRNA-resistant cDNAs revealed that the induction of L1-RTP by METH or cocaine depends on the activation of cAMP response element-binding protein (CREB). METH or cocaine recruited the L1-encoded open reading frame 1 (ORF1) to chromatin in a CREB-dependent manner. These data suggest that the cellular cascades underlying METH- and cocaine-induced L1-RTP are different from those behind L1-RTP triggered by DNA damage; CREB is involved in drug-induced L1-RTP. L1-RTP caused by drugs of abuse is a novel type of genomic instability, and analysis of this phenomenon might be a novel approach to studying substance-use disorders.

Cardiac ryanodine receptor gene (RyR2) mutations cause fatal arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. The N-terminal region of RyR2 is one of the hot spots for mutations. In this study, we investigated cardiac phenotypes of a knock-in mouse model carrying R420W mutation of RyR2. The N-terminal R420W mutation has already been found in juvenile sudden death cadavers of unrelated families. The depolarization-induced Ca(2+) transient amplitude was significantly lower in cardiomyocytes from RyR2(R420W/R420W) mice compared with wild-type mice. The time to peak of the Ca(2+) transient was significantly increased in RyR2(R420W/R420W) mice. Furthermore, the prolonged decay time from the peak of the Ca(2+) transient was detected in RyR2(R420W/R420W) mice. ECG telemetry revealed that various types of arrhythmias were induced in RyR2(R420W/R420W) mice in response to administration of caffeine and adrenaline. The mutant mice showed high occurrences of arrhythmias in response to heart stimulants compared with wild-type mice. These findings suggest that R420W mutation impairs depolarization-induced Ca(2+) oscillation in cardiomyocytes, which possibly results in sudden death due to stress-induced arrhythmias.

The retroelement long interspersed element-1 (LINE-1 or L1) comprises about 17% of the human genome. A single human cell has 80 to 100 copies of retrotransposition-competent L1, approximately 10% of which are 'hot' and actively 'jump' around the genome. Recent observations demonstrated that low-molecular weight compounds may induce L1 retrotransposition through unknown mechanisms. Herein, we demonstrated that the painkillers morphine and fentanyl citrate trigger L1 retrotransposition in neuronal cells without inducing DNA damage or up-regulating L1 mRNA expression. This effect was blocked by an antagonist of Toll-like receptor 4 (TLR4). Taken together, the data suggest that L1 retrotransposition due to morphine and fentanyl citrate is distinct from that triggered by DNA damage, requires TLR4, and is a novel type of genomic instability. Thus, we propose that L1 retrotransposition should be characterized as a component of the pharmacological activity of these analgesic agents.

A simple and reliable method to determine acephate and methamidophos in mammalian tissues is presented. The method includes solid-phase extraction of tissue extracts with active carbon cartridges followed by gas chromatography–mass spectrometry analysis. During the study, a matrix effect was observed especially at low concentrations of acephate and methamidophos in serum and in brain. To minimize the effect, we prepared calibration curves with relatively short ranges. The validation data, such as the linearity of calibration curves, limits of detection, and coefficients of variation for recovery rates, were generally satisfactory. The present method is useful for determination of acephate and methamidophos in mammalian tissues because of its simplicity and speed, keeping in mind the presence of the matrix effect.

Nonalcoholic steatohepatitis (NASH) can develop into end-stage disease such as cryptogenic cirrhosis and hepatocellular carcinoma. Hence, it is important to understand the pathogenesis of NASH. In general, the "two-hit theory" has prevailed as a pathogenic mechanism of NASH. According to this theory, lipopolysaccharides (LPS) contained in normal portal blood are the "second hit," but their role is not completely understood. Based on this theory, we evaluated the role of LPS in NASH pathogenesis. For the first hit to develop metabolic abnormalities, a synthetic diet rich in disaccharide (synthetic diet: 12.1 cal% disaccharide) was fed to Zucker (fa/fa) rats for 12 weeks. For the second hit, 100 microg/kg LPS was injected intraperitoneally once daily for 2 weeks. Synthetic diet-fed rats treated with LPS showed an increase in the triglyceride content and higher expression of profibrogenic mRNAs in the liver. Plasma alanine aminotransferase levels were significantly elevated using this protocol. Furthermore, histological examination demonstrated that this protocol induced mild hepatic fibrosis and focal necrosis in the livers of all rats. Synthetic diet-fed Zucker (fa/fa) rats treated with LPS could be useful for understanding the development of hepatic fibrosis in the two-hit theory.

The KCNQ1 gene encodes a voltage-dependent potassium ion channel, and mutations in this gene are the most common cause of congenital long QT syndrome (LQTS). In the present study, we investigated the various phenotypic characteristics of vertigo 2 Jackson (C3H/HeJCrl-Kcnq1(vtg-2J)/J) mice with a Kcnq1 mutation. Both heterozygotes (vtg-2J/+) and homozygotes (vtg-2J/vtg-2J) showed prolonged QT intervals in electrocardiograms (ECGs) compared to C3H/HeJ control (+/+) mice. Furthermore, vtg-2J/vtg-2J mice showed gastric achlorhydria associated with elevation of their serum gastrin levels. The serum corticosterone levels were also significantly increased in vtg-2J/vtg-2J mice. In addition, vtg-2J/vtg-2J mice exhibited significantly higher blood pressure. These findings indicate that the Kcnq1 mutation in vtg-2J mice alters various physiological functions in the cardiac, gastric and adrenocortical systems, and suggest that vtg-2J mice may represent a useful model for studying Kcnq1 functions.

Background Mutations of the cardiac ryanodine receptor type 2 (RyR2) gene are known to cause effort-induced polymorphic ventricular arrhythmia, syncope and sudden death. Methods and Results The possible mutations in the RyR2 gene were examined in 18 autopsy cases of sudden unexplained death (SUD). Two cases were found to have the heterozygous missense mutation in exon 14 (nucleotide change C1258T, coding effect R420W). Both cases showed mild fatty infiltration of the right ventricular apex. Interestingly, 1 case showed an enlarged thymus with accompanying hypertrophy of the tonsils and mesenteric lymph nodes. In addition, a narrowing of the aorta was observed in this case. These phenotypic characteristics are consistent with status thymico-lymphaticus, which combines sudden death with an enlargement of lymphoid organs and hypoplasia of the cardiovascular system. The second case also displayed some characteristics of status thymico-lymphaticus. Conclusion The R420W mutation has already been reported in families with juvenile sudden death and may be causative of sudden death in our cases. Postmortem molecular screening of the RyR2 gene could be useful for investigation for cause of death in SUD. The possible association of the RyR2 mutation with status thymico-lymphaticus is discussed. (Circ J 2006; 70: 1402 - 1406)

Tryptase is a neutral protease of human mast cells, and an important indicator of mast cell activation and degranulation in anaphylactic events. The elevation of serum mast cell tryptase (SMCT) is used for postmortem diagnosis of anaphylaxis. We have quantified the SMCT levels of 122 forensic autopsy cases with various causes of death and found only three where the SMCT levels were remarkably elevated, with values of 179, 68.9 and 69.4 ng/ml (normal level

We report a case of a 65-year-old worker who suffered a third-degree skin burn to 5% of his total body surface area as a result of being splashed in the face with hydrofluoric acid (HF). He died shortly thereafter without having received adequate first aid. His serum fluoride concentration was markedly increased at 6.38 mg/dl with hypocalcemia and hyperkalemia. We concluded that he died of HF poisoning. In this case he might have managed to avoid death if he had not been working alone and if he had received adequate first aid on the scene as soon as possible. We re-emphasize the need for the immediate initiation of first aid on the scene and the distribution of information on the risks of HF to workers.

We have detected two unusual alleles at the DXS10011 locus in two paternity trio cases. In one case, one allele of the daughter was found not to have been derived from the mother but the other allele was shared with the father. In the other case, the mother and the son shared no bands. Paternity in both cases was established using conventional polymorphic markers in addition to DNA markers (probabilities: >0.999999). Sequencing showed that the two de novo alleles of the children acquired a single unit (GAAA).

Tyrosine phosphorylation signaling has been reported to be involved in regulatory mechanisms of ethanol-induced modulation of the central nervous system.We investigated the effect of ethanol administration on tyrosine phosphorylation signaling in rat brain and also examined the possible involvement of Fyn kinase in the process.Immunoprecipitation experiments showed that Crk-associated substrate (Cas) was tyrosine-phosphorylated in response to ethanol administration. Fyn kinase was shown to be activated by ethanol administration and to phosphorylate Cas on tyrosine residue in vitro. Furthermore, Fyn kinase was co-localized with Cas in rat cerebellum and cerebral cortex.Cas was tyrosine-phosphorylated in rat brain by ethanol administration, and Fyn kinase was most likely involved in the process.

Mitogen-activated protein kinases (MAPK)-signaling pathways play key roles in cytoplasmic-nuclear signal transmission in response to various extracellular stimuli. In this study, we investigated the effect of repeated fasting stress on activation of the 3 members of the MAPK family, the extracellular signal-regulated kinase (ERK), the c-Jun NH(2)-terminal kinase (JNK), and the p38 mitogen-activated protein kinase (p38 kinase), in rat liver. Immunecomplex kinase assays showed that ERK and JNK were significantly activated in the liver extract from fasted rats whereas p38 kinase showed no activation. In an immunohistochemical study, the phosphorylated and activated form of ERK (p-ERK) was abundantly expressed in pericentral hepatocytes of fasted liver compared with those of the control. On the other hand, the phosphorylated and activated form of JNK (p-JNK) was highly expressed in irregular-shaped cells along the sinusoidal lining of fasted liver. A double immunofluorescent study to identify p-JNK immunoreactive cells revealed them to be Kupffer cells, which are the resident hepatic macrophages. In conclusion, ERK and JNK are selectively activated in distinct cell types of rat liver by repeated fasting stress.

A case of a 40-year-old female who died of amniotic fluid embolism is presented. This case showed typical histological findings of this syndrome. Postmortem serum of this case showed an elevated tryptase level (67.2ng/ml, normal levels

Summary. A novel cell line, designated OHK, was established from ascites of a 59-year-old Japanese woman with diffuse large B-cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed‐Sternberg-like giant cells. On ultrastructural examination, rich intermediate filaments, and well-developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell-associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposi’s sarcomaassociated herpesvirus and Epstein‐Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage-colony-stimulating factor (M-CSF), granulocyte-CSF, interleukin 6 and transforming growth factor b1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.

In the present study, we have investigated the effect of intraperitoneal injection of ethanol (3.5 g/kg) on tyrosine phosphorylation in rat brain. Immunoblot analysis using an antiphosphotyrosine antibody revealed that a 130-kDa protein band was detected in the brain extract in response to ethanol administration. This ethanol-stimulated tyrosine phosphorylation of the 130-kDa protein was found in the brain but not in the heart, liver or thymus. The 130-kDa phosphotyrosine-containing protein was identified by immunoprecipitation to be Cas, a crk-associated src substrate. This ethanol-stimulated tyrosine phosphorylation of Cas was observed most prominently in the cerebellum and the cerebral cortex. We further examined the possible involvement of Fyn kinase in ethanol-stimulated Cas tyrosine phosphorylation. Immunecomplex kinase assay showed that Fyn was activated in the cerebellum and cerebral cortex of ethanol-administered rats. Immunoprecipitation experiments also showed that Fyn was co-immunoprecipitated with an anti-Cas antibody in these regions from ethanol-administered rats. Furthermore, exogenous Fyn was shown to phosphorylate Cas from cerebellum and cerebral cortex in vitro. These findings indicate that ethanol stimulates tyrosine phosphorylation of Cas in rat cerebellum and cerebral cortex, and that Fyn may be involved in the process.

c-Jun NH2-terminal kinase is a key enzyme mediating the cellular response to a variety of extracellular stimuli. In the present study, we performed immunohistochemical studies of the expression of the phosphorylated form of the kinase in 51 human aortas of various ages. The phosphorylated kinase immunoreactivity was strongly detected in vascular smooth muscle cells of the medial vessel layer of atherosclerotic lesions from adults. Immunoreactivity was also strongly detected in similar cells of the intima. On the other hand, immunoreactive phosphorylated kinase was only weakly detected in the medial vascular smooth muscle cells of non-atherosclerotic lesions from adults. We also investigated the expression of the phosphorylated kinase in infant aortas. In contrast to its weak immunoreactivity in adult non-atherosclerotic lesions, the kinase immunoreactivity was detected in high amounts in vascular smooth muscle cells of non-atherosclerotic lesions from infants. Thus, the abundant expression of the phosphorylated kinase in these cells in atherosclerotic lesions of adults and non-atherosclerotic lesions of infants suggests that the activation of c-Jun NH2-terminal kinase may be an important element initiating the proliferation of vascular smooth muscle cells during atherogenesis and aortic development.

We report here a sudden death case of a patient previously diagnosed as Marfan syndrome (MFS). The victim was dead on the wheel and the cause of death was diagnosed to be a rupture of the thoracic aorta by autopsy findings. MFS is an autosomal dominant disorder of the connective tissue and can be a cause of sudden death. Postmortem genetic analysis revealed a heterozygous p.C1307Y of the FBN1 gene, which is responsible for pathogenesis of MFS, was evident. This substitution was not found in 400 alleles from control individuals. In addition, the position 1307 is highly conserved among species. Because the position 1307 serves as part of the Cys1307-Cys1320 disulfide bond of the fibrillin-1, the p.C1307Y substitution results in loss of the intramolecular disulfide bond. The p.C1307Y substitution may be associated with the pathology of the present case, and show a higher risk for aortic rupture and subsequent sudden death.

Postmortem diagnosis of neuroleptic malignant syndrome (NMS) is difficult to perform, because the clinical symptoms just before death are not usually available. Malignant hyperthermia (MH) is a catastrophic, life-threatening hypermetabolic syndrome triggered by certain anesthetics. Ryanodine receptor type 1 (RYR1) gene mutations are known to be involved in susceptibility to MH. Similarities in clinical features, such as elevated body temperature, between NMS and MH have led to the suggestion that NMS is a neurogenic form of MH. In this study, we analyzed possible mutations of the RYR1 gene in 11 psychiatric patients suspected at autopsy to have died of NMS. All cases were suspected of having elevated body temperature at death, and their causes of death could not be determined by autopsy examinations. Two mutations (R4645Q and A612T) in the RYR1 gene were identified. The R4645Q mutation has previously been reported in MH patients, but five heterozygous mutations were also found in 400 Japanese control alleles. The other mutation was novel, and was not found in the same control alleles. The results of this study provide the first successful identification of RYR1 mutations in psychiatric patients suspected at autopsy of having died of NMS. However, the association between RYR1 gene mutations and cause of death in psychiatric patients suspected of dying of NMS remains unclear.

Paternity determination of a fetus whose mother was admitted to an institution for the welfare and health of handicapped persons was requested of us by a doctor and lawyer of the institution. The fetus was recovered by a legal artificial abortion based on the Act on Maternity Health and Welfare (Japan) with the permission of the custodian. Commercially available MCT118, HLADQA, PM, and 9 STRs were tested for DNA samples from the fetus, the mother, her younger brother, her father, her grandfather, and 4 staff members of the institution. Only the brother was not excluded and the paternity probability was estimated at 99.857% on the basis of newly formulated expressions for multiallelic loci on the assumption of sibling incest. We concluded then that the fetus was fathered by the brother. DNA fingerprinting with multilocus and single locus minisatellite probes which were performed to confirm the paternity also support the conclusion. Bandsharing frequencies between the family members, however, did not necessarily reflect their actual kinship, which findings suggest that multilocus DNA fingerprinting requires further accumulation of data for consanguineous cases such as incest. Universal formulation for calculating paternity probability for a sibling incest case on the basis of multiallelic monolocus polymorphisms is also presented.

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays an important role in various physiological functions including T cell activation. This enzyme is a target molecule for the immunosuppressants, cyclosporin A and FK506. In the present study, we investigated immunohistochemical localization of calcineurin and FKBP12, an FK506-binding protein, in human thymus and epidermis. The catalytic subunit (calcineurin A) of calcineurin was abundantly expressed in Hassall's corpuscles which were localized in the thymic medulla and represented the terminal stages of thymic medullary epithelium. The regulatory subunit (calcineurin B) of calcineurin was also expressed in high amounts in Hassall's corpuscles. In the epidermis, which shows similarities to Hassall's corpuscles, both subunits were also abundantly expressed, and their expression increased with the differentiation of keratinocytes. FKBP12 was observed to be expressed abundantly, both in Hassall's corpuscles and the entire epidermis. These findings suggest that the differentiated forms of the two cell types, which are the thymic medullary epithelial cell and the keratinocyte, are the target for pharmacological actions of FK506.

In the present study, we investigated whether tyrosine phosphorylation was involved in thymic involution, which has been reported to correlate well with the effects of various kinds of stresses. Immunohistochemistry using the anti-phosphotyrosine antibody showed that the immunoreactivity decreased remarkably in the involuted thymus of stressed rats as compared with the control thymus. Immunoblot analysis using the anti-phosphotyrosine antibody revealed the tyrosine-phosphorylated proteins with apparent molecular masses of 120, 90, and 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were detected in the control thymus. The immunoreactive band corresponding to the three proteins decreased remarkably in the involuted thymus. Further, we found by immunoprecipitation experiments that the 120 kDa protein was p130cas, a crk-associated src substrate. These findings suggest that tyrosine phosphorylation signaling may be involved in thymic involution.

We investigated a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWF) gene (nucleotides [nt] 1639-2404; i.e., F8VWF). We identified 13 alleles and 33 genotypes in 49 unrelated Japanese individuals. The heterozygosity of the region was 0.897. Direct sequence analyses revealed five single-base substitutions, one tetranucleotide (TTAT) insertion, and seven short tandem repeats (STRs) in the intron; four of the STRs and one single-base substitution had been reported previously. The four new base substitutions we identified were 1849T>A, 2122C>T, 2180C>T, and 2192C>T. The novel TTAT tetranucleotide was inserted between nt 2057 and 2058. The three newly identified STRs were 1978(TATC)(1-2), 2193(ATCT)(5-13), and 2234(TGTA)(5-7). The five single-base substitutions and the TTAT insertion were identified only with 3' downstream of vWA allele 14.

We herein present an autopsy case of fatal methemoglobinemia due to suicidal ingestion of sodium nitrite. A man in his 30s who had exhibited mild depression was found dead in his house. A glass containing a white turbid solution and a suicide note were found on the table near the decedent. At autopsy, the lividity of the face and back was greenish gray, and the blood was tinted a chocolate brown color. Toxicological analysis of nitrite and nitrate using a colorimetric method showed that the nitrite concentrations were

Traumatic cerebellar hemorrhagic contusions are infrequent, and the pathogenic mechanism involves a coup injury that is associated with motor vehicle accidents in most cases. Traumatic basal subarachnoid hemorrhage (TBSAH) is commonly reported after blunt trauma to the neck or unrestricted movement of the head, and the source of the hemorrhage is most frequently identified in the vertebrobasilar arteries. A 55-year-old woman who was addicted to alcohol was found dead in her bed. She had a bruise on the left side of her posterior parietal region, and autopsy revealed massive subarachnoid hemorrhage at the base of the brain; the hematoma was strongly attached to the right lower surface of the cerebellar hemisphere. No ruptured cerebral aneurysms, arteriovenous malformations or vertebrobasilar artery leakage were detected. Hemorrhagic cerebellar contusions were regarded as the source of the TBSAH. This is the first report of TBSAH suspected to have been caused by contrecoup cerebellar contusions.

A population study of the short tandem repeat locus DXS10011 was carried out in a sample of 104 unrelated Japanese individuals (56 males, 48 females). Thirty-six different alleles were distinguished. The power of discrimination was 0.933 (male) and 0.997 (female). Sequence analysis of alleles was examined for 34 samples. The sequence structures of the alleles were classified into three types: A, B and C. Type A had a regular repeat structure of the repeat region: (GAAA)(n). Types A and B differed in the base sequence of the repeat region. As an A (adenine) insertion in 5' upstream of (GAAA)(n) repeat unit was type C, this type was differentiated from type A.

We report an autopsy case of a man whose death was caused by a combination of aspirin (acetylsalicylic acid) poisoning and chlorine gas inhalation. Toxicological analysis of aspirin and salicylic acid using high performance liquid chromatography with photodiode array detection showed that the concentration of salicylic acid was 444.7 μg/ mL in the heart blood and 68.3 μg/g in the brain. Existence of chlorine gas was proven, in the form of chloride ions, in a plastic bag near the body using energy dispersive X-ray fluorescence spectrometry. It was thought that damage to the mucosa, respiratory tract and skin had been caused by hydrochloric and hypochlorous acid associated with chlorine gas inhalation that rapidly aggravated the respiratory distress and pulmonary edema caused by aspirin and salicylic acid. This case report shows that the risk of death is increased when two substances, aspirin and chlorine gas, are taken through different routes, even if the concentration of one of them does not reach the lethal dose.

Summary. We investigated whether hyaluronan (HA) was produced by a malignant lymphoma cell line with a primary effusion lymphoma immunophenotype, OHK. OHK secreted high levels of HA into the culture supernatant, and reverse transcription polymerase chain reaction showed mRNA for the HA synthases 2 and 3. HA binding protein-reactive HA was present in the plasma membrane and in the cytoplasm. The inoculation of OHK into severe combined immunodeficiency disease mice produced marked viscid ascites with concentrated HA and a vascular-rich mesenteric solid tumour. These results show that HA is produced and secreted by the malignant lymphoma cell line OHK.

Intron 40 of the vWF gene is known to harbor three different STRs, which were separately reported by Kimpton et al. (vWA), by Peake et al. (vWF-P), and by Ploos van Amstel et al. (vWF-A). En bloc detection of the three STRs (F8VWF) and sequencing of the F8VWF alleles revealed novel base substitutions, tetranucleotide insertions, and tetranucleotide repeats. It is noted that the nucleotide substitutions and insertions were observed exclusively in those F8VWF alleles having the vWA allele 14.

A fatal poisoning case involving etizolam, phenobarbital, promethazine and chlorpromazine is presented. Quantitative toxicological analysis showed that the concentrations of etizolam, phenobarbital, promethazine and chlorpromazine in the femoral blood were 86 ng/ml, 5082 microg/ml, 0.107 microg/ml and 0.144 microg/ml, respectively, and large amounts of drugs were also detected in the stomach contents. We conclude that the cause of death was due to the interaction of multiple psychotropic drugs.

One type of arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder resulting in fatty replacement of the right ventricular wall and consequent fatal arrhythmias. It is a major cause of sudden death in young people. Mutations in the genes encoding desmosomal proteins have been identified as a cause of ARVC. We report a case of sudden death during exercise in a juvenile. This case showed fatty replacement of the right ventricular wall, which suggests that ARVC may have been associated with the cause of death. Further genetic analysis showed a novel homozygous R292C substitution of the desmoglein-2 gene (DSG2), which encodes a desmosomal protein. In addition to morphological examination, genetic analysis may be useful for diagnosis of ARVC-suspected autopsy cases.

We present a case of fatal carbamazepine poisoning. Quantitative analysis of carbamazepine using high performance liquid chromatography, revealed that the concentrations of carbamazepine were 50.2 microg/ml in the femoral venous blood and 60.3 microg/ml in the heart blood, respectively, and many unabsorbed tablets were also observed in the stomach contents. We concluded that the cause of death was due to carbamazepine overdose.

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease, and is commonly observed in patients with obesity or type 2 diabetes. The pathogenesis of hepatic fibrosis is clinically important to the outcome of NAFLD, however, is not well understood. Since dietary habits are often considered to be responsible for NAFLD, we used a synthetic diet rich in disaccharides (12.1% calorie sucrose and lactose), which can be considered for human consumption. We examined the long-term (24 weeks) effect of this diet on the liver of Zucker (fa/fa) rats. The synthetic diet-fed Zucker (fa/fa) rats showed hepatic fibrosis during the development of NAFLD with no apparent infiltration of inflammatory cells. They showed significantly elevated hepatic mRNA levels of proinflammatory and profibrogenic cytokines. These findings suggest that excess long-term feeding of a diet similar to the synthetic diet used in our study leads to hepatic fibrosis during the development of NAFLD in patients with obesity or type 2 diabetes. Our results were different from NAFLD accompanied by infiltration of inflammatory cells. However, they also suggest that long-term feeding of this synthetic diet to Zucker (fa/fa) rats is useful for studying the hepatic fibrogenesis during the pathogenesis of NAFLD.

The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.

Identification of a src-related tyrosine kinase and its regional and cellular distributions were studied in rat brain. The study was performed using a specific antibody raised against a synthetic peptide corresponding to the conserved autophosphorylation site of src-family tyrosine kinases. The antibody (α-src antibody) recognized a 43 kDa polypeptide in plasma membrane enriched fraction. The immunohistochemical data revealed that the polypeptide is localized in astrocytes of corpus callosum and fimbria hippocampus. The presence of this src-related protein in astrocytes suggests that it may have some control in their proliferation and differentiation.

Two isoforms of calcineurin beta subunit(beta 1 and beta 2) were identified in rat testis by a monoclonal antibody Va1. Both beta 1 and beta 2 were recovered in calmodulin binding protein fraction and showed calcium shift on SDS-polyacrylamide gel electrophoresis which is the specific character for EF-hand calcium binding protein. beta 2 showed same apparent molecular weight on SDS-PAGE as that of brain calcineurin beta and was found in wide variety of tissues. beta 1 was shown to have six amino acid polypepeptide sequence and it showed higher molecular weight than brain beta and was specific for testis.

Energy dispersive X-ray fluorescence spectrometry (EDX) enables rapid, non-destructive, multi-elemental analysis. Using EDX, bromide was detected in seawater but not in freshwater. We applied EDX to the detection of bromide in cardiac blood from medico-legal autopsy cases to obtain additional evidence supportive of seawater drowning. Bromide was detected in the blood of 4 out of 10 victims drowned in seawater. In contrast, bromide concentrations were below the quantification limit in both victims from freshwater drowning and non-drowning controls. No postmortem invasion of bromide was observed in animal experiments of postmortem immersion in seawater. These results indicate that the detection of bromide in blood by EDX could be a chemical marker supportive of drowning in seawater.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.

An elution technique for ABO blood grouping from hair samples is in wide use. However, difficulties may be encountered when identifying the ABO blood grouping using monoclonal antibody. In the present study, we have shown excellent results in ABO blood grouping from crushed hair samples using an improved immunohistochemical technique with a mesh pack.

We performed mutation analysis for genes implicated in long QT syndrome (KCNQ1, KCNH2, and SCN5A) in 17 sudden unexplained death autopsy cases. Single-strand conformation polymorphism and subsequent DNA sequencing analyses revealed that in one case, there was a variant, V207M of KCNQ1, a gene encoding a cardiac potassium channel. This case, a 40-year-old African male, was shown to have a heterozygous missense mutation (V207M), which has been previously reported to be ethnic-specific. The heterozygous V207M mutation was found in one case (0.23%) of 444 alleles from African individuals. We developed a knock-in mouse model carrier of the Kcnq1-V206M mutation, the mouse equivalent to the KCNQ1-V207M mutation identified in the victim. Significant prolongation of QT intervals was observed in the Kcnq1(V206M/V206M) mice. These findings suggest that the KCNQ1-V207M mutation might be pathogenic and might have been associated with the cause of death in the present case.

Malignant hyperthermia (MH) is a genetic disorder of skeletal muscle in susceptible individuals that is triggered by exposure to anesthetic agents, and can cause death. Mutations in the ryanodine receptor type 1 gene (RYR1) are associated with MH-susceptibility. MH is also triggered in susceptible individuals by severe exercise in hot conditions or by overheating in infants. Here, we report a case of a child, 2years, 9months of age, who was left in a car and exposed to a high environmental temperature. The child was suspected to have died of heat stroke by autopsy examinations. Postmortem mutation analysis revealed that the child possessed two distinct RYR1 mutations. Since each mutation had previously been identified in a separate MH-susceptible patient, MH-susceptibility with over-response to the environmental high temperature might have occurred in this child with RYR1 mutations. These findings suggest that a MH-susceptible case may have died with a presumed diagnosis of heat stroke at autopsy.