The Tokyo subway sarin attack has long-term effects on survivors: A 10-year study started 5 years after the terrorist incident.

OBJECTIVES: The Tokyo subway sarin attack in 1995 was an unprecedented act of terrorism that killed 13 people and sickened more than 6,000. The long-term somatic and psychological effects on its victims remain unknown. METHODS: We conducted analyses on the self-rating questionnaire collected annually by the Recovery Support Center (RSC) during the period from 2000 to 2009. The RSC is the only organization that has large-scale follow-up data about sarin attack victims. The prevalence of self-reported symptoms was calculated over 10 years. We also evaluated the prevalence of posttraumatic stress response (PTSR), defined as a score ≥ 25 on the Japanese-language version of the Impact of Event Scale-Revised. The multivariate Poisson regression model was applied to estimate the risk ratios of age, gender, and year factor on the prevalence of PTSR. RESULTS: Subjects were 747 survivors (12% of the total) who responded to the annual questionnaire once or more during the study period. The prevalence of somatic symptoms, especially eye symptoms, was 60-80% and has not decreased. PTSR prevalence was 35.1%, and again there was no change with time. The multivariate Poisson regression model results revealed "old age" and "female" as independent risk factors, but the passage of time did not decrease the risk of PTSR. CONCLUSIONS: Although symptoms in most victims of the Tokyo subway sarin were transient, this large-scale follow-up data analysis revealed that survivors have been suffering from somatic and psychological long-term effects.

The sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate induces ER stress in human astrocytoma cells.

Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents.

The Sarin-like Organophosphorus Agent bis (isopropyl methyl)phosphonate Induces Apoptotic Cell Death and COX-2 Expression in SK-N-SH Cells.

Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression.

Acute effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate, on cardiovascular parameters in anaesthetized, artificially ventilated rats.

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective ß-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.

[Comparison of four immunoassay screening devices for detection of benzodiazepine and its metabolites in urine: mainly detection of etizolam, thienodiazepine and its metabolites].

The immunoassay screening of benzodiazepines in urine is one of the most important methods of drug analysis in clinical and forensic laboratories. We experienced an unusual case of poisoning wherein the result of Triage DOA immunoassay screening was negative, although Depas (etizolam) was detected in the blood of the victim who had been suspected to prescribe Depas by gas chromatography-mass spectrometry. Depas has been widely used for the treatment of anxiety in Japan. Three immunoassay screening devices (AccuSign BZO, Monitect-3, and Fastect II) were evaluated for their specificity for etizolam, its 2 major metabolites M-III and M-VI, and other metabolites of benzodiazepines in urine. With AccuSign BZO, etizolam, M-III, and M-VI could be detected at concentrations of 1,000 ng/mL in urine; however, they could not be detected even at concentrations of 25,000 ng/mL with the other kits. In the case of etizolam poisoning, the result of AccuSign BZO was positive; however, Triage DOA, which is mainly used for the detection of drugs in urine at intensive care units (ICUs) or forensic laboratories, showed negative result for benzodiazepines. The concentrations of etizolam and its metabolites in urine were measured by the established high-performance liquid chromatographic method. The concentrations of M-III and M-V were 700 and 1,600 ng/mL, respectively. AccuSign BZO demonstrated higher specificity-than the other screening kits for the detection of etizolam and its metabolites in urine. Therefore, the types of drugs detected would be increased by combining Triage DOA with AccuSign BZO in ICUs or forensic laboratories.

Transcriptional coactivator with PDZ-binding motif is essential for normal alveolarization in mice.

RATIONALE: Transcriptional coactivator with PDZ-binding motif (TAZ) is assumed to act as a coactivator of several transcription factors including smad2/3. In the lung, surfactant protein C (Sftpc) is known to be a downstream target of thyroid transcription factor-1 (TTF-1)-TAZ transcriptional coactivation. OBJECTIVES: The lung phenotype of Taz-deficient mice was explored. METHODS: Taz-deficient mice were analyzed pathologically and physiologically. Next, we performed microarray analysis to determine the genes closely related to abnormal lung development. Finally, Taz-heterozygous mice were injected with bleomycin. MEASUREMENTS AND MAIN RESULTS: Taz-deficient homozygotes showed abnormal alveolarization during lung development, which caused in adult mice airspace enlargement mimicking emphysema. There was no significant difference in the expression of Sftpc between wild-type and Taz-deficient lungs. Instead, microarray analysis identified some candidate downstream genes related to the pathogenesis, including the connective tissue growth factor (Ctgf) gene, which is required for normal lung development. In vitro studies showed that TAZ up-regulated Ctgf expression not only by reinforcing transforming growth factor-beta/smad signals, but also by interfering in the more proximal Ctgf promoter region (from bp -123 to -76), defined as the TAZ response element. Furthermore, Taz-heterozygous mice were resistant to bleomycin-induced lung fibrosis. CONCLUSIONS: The results indicate the importance of TAZ in lung alveolarization and its involvement in the pathogenesis of lung fibrosis.

The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ.

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

Transcriptional activity of Pax3 is co-activated by TAZ.

Pax3 is a transcription factor which functions in embryonic development and human diseases. In a yeast two-hybrid screen with full-length Pax3 as bait, we isolated a clone encoding transcriptional co-activator with PDZ-binding motif (TAZ) from an E10.5 mouse embryo cDNA library. Co-immunoprecipitation and nuclear co-localization of TAZ with Pax3 suggest that their association is functionally relevant. In situ hybridization revealed TAZ and Pax3 expression to partially overlap in the paraxial mesoderm, limb buds, and the neural tube. In C2C12 myoblast cells and NIH3T3 cells, TAZ enhanced the transcriptional activity of Pax3 on artificial and microphthalmia-associated transcription factor promoter-luciferase constructs, suggesting that TAZ can function as a co-activator of Pax3. Functional interaction between Pax3 and TAZ may provide a clue to clarifying the mechanism by which Pax3 serves as a transcriptional activator during embryogenesis.

Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice.

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human beta-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2beta1, termed mouse beta-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the beta-defensin family. Our findings indicated that multiple beta-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.

Transgenesis of newt with exogenous gene expression facilitated by satellite 2 repeats.

Genetiç transduction of newt was undertaken in search of conditions allowing high exogenous gene expression in stages from late embryo to larva when regeneration experiments, which are advantageous for this group of animals, are possible. DNA of a ß-galactosidase-encoding gene miwZ was injected in the cytoplasm of fertilized eggs in different molecular forms, and its fate and gene expression were studied in individual embryos. When DNA was injected in a linear form, DNA concatemerized quickly and the majority of the exogenous sequences were localized in the nuclear fraction from early gastrula onwards. By contrast, when circular DNA was injected, the exogenous DNA sequence largely remained in the cytoplasmic fraction, and the entry of the sequence into the nuclear fraction was delayed to mid-gastrula stage. Reflecting these differences, ß-galactosidase expression in an embryo occurred earlier and more efficiently by linear DNA injection. Whole mount staining for ß-galactosidase activity in tail bud embryos showed only spotty staining with circular DNA injection while spreading staining was also observed with linear DNA, suggesting difference in clone sizes of expressing cells depending on the DNA form. Expression of ß-galactosidase, however, became low in the hatching stage. To augment and stabilize ß-galactosidase expression to this stage, repeats of satellite 2 sequence, which is a transcriptionally active middle repetitive sequence highly conserved among urodeles, were added to the DNA in a way to bracket the miwZ gene. Injection of this DNA in linear form resulted in one order of magnitude higher ß-galactosidase expression compared to without satellite 2 in the neurula embryos, and in high and persistent expression in the hatching larvae, without significant increase in the copy number of the exogenous DNA. Satellite 2 sequence probably provided a genetic environment favorable for transcription of a gene.