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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Energy dispersive X-ray fluorescence spectrometry (EDX) enables rapid, non-destructive, multi-elemental analysis. Using EDX, lead and some other metallic elements were detected in paraffin-blocks in which skin samples from a gunshot wound were embedded. Lead was not identified in control samples from non-injured cases. These findings indicate that EDX is a useful method for rapid non-destructive analysis of paraffin-embedded blocks. This technique can provide scientific evidence for identification of a firearm even after storing the sample for a long time.

Congenital long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) are known to be involved in some sudden unexplained death (SUD) cases. We examined possible mutations of the genes responsible for LQTS and CPVT in 17 SUD cases. Three cases showed RYR2 mutations, which are responsible for CPVT. We also found a novel KCNQ1 mutation. The KCNQ1 mutation is most frequently found in patients with congenital LQTS. Since morphological abnormalities have not been reported in patients with LQTS and CPVT, postmortem molecular screening for LQTS and CPVT-causative genes may be necessary for diagnosis of a SUD case. It may also useful for preventing living family members with the disease-causing mutation from cardiac events.

We have performed haplotyping of 16 Y-STRs (giving 17 PCR products): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y GATA H4 in 47 biological paternity cases (father and son) in Japan. We have detected two unusual alleles at the DYS439 and DYS448 locus in two paternity trio cases. In one case, DYS439 allele 15 of the son was found not to derive from the father (allele 14). In the other case, the father (allele 18) and the son (allele 17) shared no bands at DYS448. Sequencing showed that the son gained a single unit (GATA) at DYS439 in one case and the son losed 1 unit (AGAGAT) at DYS448 in the other case.