Expression of paraoxonase isoform did not confer protection from acute sarin poisoning in the Tokyo subway terrorist attack.

We previously reported the polymorphism of the high density lipoprotein-associated enzyme paraoxonase (PON1), in the 10 sarin poisoning victims in the Tokyo subway terrorist attack. Arg192 PON1, which has low sarin hydrolysing activity, was found to be more common in the Japanese population than in people of other races. However, from our analyses seven of the victims expressed the PON1 phenotype with high sarin hydrolysing activity and three with low sarin hydrolysing activity. These results indicate that the main factor contributing to the tragedy of the Tokyo subway terrorist attack was the high toxicity of sarin rather than the race-dependent genetic difference in the Arg192 PON1 polymorphism.

Possibility of paternity testing using RFLP analysis on a very small amount of material.

Paternity testing by DNA analysis was carried out using dental pulpal and chorionic villous tissue from two children respectively, and fresh blood samples obtained from the alleged parents. DNA was extracted spectroscopically from the pulp of an upper wisdom tooth (16 micrograms) and the chorionic villi (53 micrograms). The RFLP method was used for DNA analysis of the parent-child relationships because both of the DNAs extracted had a high molecular weight. Distinct bands were detected with 32P-labelled multi-locus (Myo) and single locus (pYNH24) DNA probes. In the case of the dental specimen all of the bands of the child's DNA were found to be derived from either of the alleged parents, demonstrating a consistent parent-child relationship (the probability of established paternity was 99.86%) whilst in the case of the villous specimen the father-child relationship was denied. This procedure can provide much information using very little material for analysis but where the samples are in a good condition.

Preclinical evaluation of an immunotherapeutic peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens.

BACKGROUND: Peptide immunotherapy is a new approach to treating allergic diseases, but a therapeutic peptide for Japanese cedar pollinosis has not yet been developed. OBJECTIVE: The aim of this study is to prepare and preclinically evaluate a hybrid peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens. METHODS: The recombinant hybrid peptide was prepared after immunodominance of 7 T-cell determinants was confirmed by means of PBMC proliferation assay in 113 volunteers with pollinosis. The hybrid peptide was compared with a mixture of the 7 T-cell determinants in a dose-dependent PBMC proliferation assay in 6 volunteers with pollinosis. PBMC proliferation and binding activity of serum IgE antibody against the hybrid peptide, Cry j 1, and Cry j 2 were investigated in 48 volunteers with pollinosis. RESULTS: The hybrid peptide induced T-cell proliferation with an average 100-fold lower concentration than a mixture of the 7 peptides. PBMCs from 44 (92%) of 48 volunteers proliferated against the hybrid peptide, with significant correlation (r = 0.87) in T-cell proliferation against Cry j 1 and Cry j 2. No serum IgE antibodies specific to Cry j 1 or Cry j 2 bound to the hybrid peptide. CONCLUSION: A hybrid peptide comprising 7 T-cell determinants has the potential for inducing T-cell proliferative responses that is superior to the potential of a mixture of the T-cell determinants and comparable with that of Cry j 1 and Cry j 2. The hybrid peptide will be of use in specific immunotherapy against Japanese cedar pollinosis.

Bunina body in frontal lobe dementia without clinical manifestations of motor neuron disease.

We report here an early autopsy case of a 60-year-old woman clinically diagnosed as having frontal lobe dementia without other neurological deficits. Postmortem examination revealed mild spongiosis in layers II and III of the frontal cortex, together with depletion of melanin-containing neurons in the substantia nigra. In addition to ubiquitin-positive neurites, ubiquitin-positive, tau-negative inclusions, which were previously considered to be a hallmark for motor neuron disease with or without dementia, were identified in neurons of the hippocampal dentate gyrus and of the temporal cortex. Although the patient lacked lower motor symptoms, the presence of Bunina bodies identified in the hypoglossal nuclei further supported the relationship of this case to motor neuron disease. Bunina bodies might be present in some cases of frontal lobe dementia. The presence or absence of Bunina bodies should be scrutinized even in cases without motor symptoms. In this case, creatine kinase of skeletal muscle origin was elevated, which might also be a potential indicator that suggests subclinical involvement of lower motor neurons.

Diagnosis of cyanide intoxication by measurement of cytochrome c oxidase activity.

Cytochrome c oxidase (CCO), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. We measured CCO activity, in the major organs of the rat at various times after death caused by cyanide intoxication. Tissue samples were homogenized, and the CCO activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. The CCO activity inhibition was highest in the brain, although the cyanide concentration was lowest level. As a result of this and the clinical symptoms displayed, we consider the brain to be the primary organ of cyanide intoxication. As cyanide is highly toxic to humans, in small amounts and many patients and victims have already had some medical care, it is difficult to detect cyanide in criminal investigations. The CCO activities in various organs remained significantly low for 2 days after the cyanide intoxication, suggesting that the diagnosis may be possible by measuring not only the cyanide concentration but also the CCO activity.

Two cases of right ventricular ischemic injury due to massive pulmonary embolism.

In general, massive pulmonary embolism induces severe right ventricular overload, but pathological changes in the right ventricle due to pulmonary embolism is rarely seen. In this report, we describe two autopsy cases of massive pulmonary embolism without pre-existing cardiopulmonary disease. Both cases were accompanied by myocarditis-like changes in the right ventricle and infiltration of a number of polymorphonuclear neutrophils and mononuclear cells into the dilated right ventricular wall. Transmural or subendocardial coagulation necrosis was not apparent. Almost all of the mononuclear cells were immunohistochemically revealed to be CD68-positive macrophages. We speculated that these findings resulted from ischemia due to massive pulmonary embolism.

The ratio of insulin to C-peptide can be used to make a forensic diagnosis of exogenous insulin overdosage.

A 25-year-old man was killed by his lover by an intravenous injection of insulin and then air. We had difficulty in determining whether insulin had really been injected, so we have 18 control cases and proved that the ratio of insulin to C-peptide in a corpse can be used as positive evidence for the insulin overdosage.

The mechanism of human adipocere formation.

In adipocere, some specific fatty acids possessing higher melting points, together with soap, play an important role in the formation and stabilization of adipocere. These fatty acids were shown to be mainly 10-hydroxy stearic and 10-hydroxy palmitic acids. Slight amounts of 10-oxo stearic and 10-oxo palmitic acids, which have higher melting points than those of hydroxy fatty acids (OHFAs), exist in the adipocere as well. The substantial adipocere is formed and stabilized mainly by these specific fatty acids. The OHFA and oxo fatty acid (OXOFA) are biosynthesized by some bacterial enzymes. Various aerobic and anaerobic bacteria are involved in the formation of adipocere. For instance, microbial conversion of various unsaturated fatty acids to 10-OHFA by Micrococcus luteus was investigated. It turned out that 10-OHFA was synthesized only from fatty acids possessing cis-9-unsaturatin. It was also shown that 10-OHFAs were converted to the corresponding 10-OXOFAs but 10-OXO compounds were inactive as substrates. It was further found that the enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxy stearic acid, but also dehydrogenation of this product in the presence of deuterium. On the other hand, we found out that there was 10-hydroxy-12-octadecenoic acid (10-OHODA) in the linoleic acid in human adipocere and that there were 9-chloro-10-methoxy (9-methoxy-10-chloro) palmitic acid and 9-chloro-10-methoxy (9-methoxy-10-chloro) stearic acid in human neonate adipocere.

Purification and characterization of diquat (1,1'-ethylene-2, 2'-dipyridylium)- metabolizing enzyme from paraquat-resistant rat liver cytosol.

To establish a paraquat-resistant Wistar rat strain, we carried out continuous sister-brother mating among rats that survived high-dose intraperitoneal administration of paraquat dichloride (360 mg/kg). The percentages of paraquat-resistant rats among wild rats and among the fifth-generations were 7.1% and 20.6%, respectively. After high-dose paraquat administration, the serum paraquat concentration in sensitive rats was much higher than that in paraquat-resistant rats. The cytosol fraction of liver from paraquat-resistant rats had higher paraquat- and diquat-metabolizing activities than that of liver from paraquat-sensitive rats. By contrast, microsomal fractions from livers of paraquat-resistant and paraquat-sensitive rats had no paraquat- or diquat-metabolizing activity. This paraquat/diquat-metabolizing enzyme was partially purified from paraquat-resistant rat liver cytosol using affinity chromatography for diquat. At the end of the purification procedure, rat liver diquat-metabolizing enzyme was purified 1154-fold to a final specific activity of 32.32 mol/h/mg protein, and an overall recovery of about 0.46% was obtained. This enzyme oxidized diquat to diquat-dipyridone during overnight incubation at 37 degrees C, but only metabolized traces of paraquat. The molecular mass of the enzyme was estimated as 190 kDa, and its isoelectric point of it was 4.6-4.7. Kinetic study revealed the values of K(m) and V(max) to be 35.0 micromol/l and 0.81 micromol/h/ml, respectively.

Onset of rigor mortis is earlier in red muscle than in white muscle.

Rigor mortis is thought to be related to falling ATP levels in muscles postmortem. We measured rigor mortis as tension determined isometrically in three rat leg muscles in liquid paraffin kept at 37 degrees C or 25 degrees C--two red muscles, red gastrocnemius (RG) and soleus (SO) and one white muscle, white gastrocnemius (WG). Onset, half and full rigor mortis occurred earlier in RG and SO than in WG both at 37 degrees C and at 25 degrees C even though RG and WG were portions of the same muscle. This suggests that rigor mortis directly reflects the postmortem intramuscular ATP level, which decreases more rapidly in red muscle than in white muscle after death. Rigor mortis was more retarded at 25 degrees C than at 37 degrees C in each type of muscle.

The effects of sarin-like and soman-like organophosphorus agents on MAPK and JNK in rat brains.

One sarin-like and one soman-like organophosphorus agent [bis(isopropyl methyl)phosphonate, BIMP and bis(pinacolyl methyl)phosphonate, BPMP] were injected intravenously (iv) in rats. An increase in the tyrosine phosphorylation of several proteins in the cytosol fraction of the brain was observed. Activation of c-Jun N-terminal kinase (JNK) and slight activation of mitogen-activated protein kinase (MAPK) in the cytosol were also observed. The activation of these enzymes may be related to the high toxicity of these nerve agents.

Immunohistochemical examination of the lungs in infant death cases using antibodies against milk components.

To examine the use of immunohistochemical staining with antibodies against milk components for detection of aspirated milk on lung sections, eighteen infant death cases were investigated. Immunostaining was performed with anti-human alpha lactalbumin, anti-human IgA, anti-human milk fat globulin 1, and anti-cow whey antibody. Reactivity with each antibody was examined, and semi-quantitative examinations were performed to compare the amount of aspirated milk using anti-human alpha lactalbumin antibody. Materials in the alveoli or bronchioli on lung sections suspected to be aspirated milk showed the most sensitive and clearest reaction with anti-human alpha lactalbumin antibody. Of the eighteen cases, ten cases showed positive reaction with this antibody. The amount of aspirated milk varied widely in each case. In conclusion, immunohistochemical staining with antibodies against human milk components, especially anti-human alpha lactalbumin antibody, can detect small amounts of milk. Using this method, we were able to compare the relative amount of aspirated milk among cases.

Effect of cytochrome c on the linoleic acid-degrading activity of porcine leukocyte 12-lipoxygenase.

Hemoproteins are known to have quasilipoxygenase activity that converts linoleic acid (LA) to its hydroperoxides. However, it is not still clear whether, like lipoxygenases, hemoproteins can produce LA hydroperoxides when the LA is part of a mixture containing many different saturated and unsaturated fatty acids. In this study, we found that such hemoprotein as cytochrome c (Cyt c) did not produce LA hydroperoxides from the phospholipase A(2) (PL-A(2)) hydrolysis products of egg yolk phosphatidylcholine (PC). We also found that traces of hydroperoxides and a high concentration of the target unsaturated fatty acid (LA) needs to be present in a fatty acid mixture before the quasi-lipoxygenase activity of Cyt c becomes apparent. We also attempted to elucidate how Cyt c interact with porcine leukocyte 12-lipoxygenase (12-LOX). Hemoproteins are known to possess pseudo-lipohydroperoxidase activity, and can remove the hydroperoxides of unsaturated fatty acids from a reaction mixture. However, we found that Cyt c catalyzed the reaction by which hydroperoxides degrade LA, and thus enhanced the LA-degrading activity of 12-LOX. This hemoprotein-induced promotion of the ability of 12-LOX to degrade LA was observed even when the reaction mixture contained many different saturated and unsaturated fatty acids.

Postmortem changes in cytochrome c oxidase activity in various organs of the rat and in human heart.

Cytochrome c oxidase (COX), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. To test whether cytochrome c has potential as an indicator of these toxins in cadavers, we measured COX activity in the main organs of the rat, and in the human heart, at various times after death. Each tissue sample or organ was homogenized and the COX activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. COX activity was significantly higher in rat brain, heart and kidney than in lung and liver from 0 to 4 days after death. The loss of COX activity was significantly slower in the brain and heart than in the lung, liver and kidney. Most importantly, COX activity correlated with the time-since-death for each of the rat organs we tested (r2=0.70-0.95), but for the human heart (r2=0.47). It may be possible that COX activity is likely to be a useful indicator of the time-since-death, and is worth pursuing as an indicator of the tissue cyanide and CO content.

[Creating language model of the forensic medicine domain for developing a autopsy recording system by automatic speech recognition].

For the purpose of practical use of speech recognition technology for recording of forensic autopsy, a language model of the speech recording system, specialized for the forensic autopsy, was developed. The language model for the forensic autopsy by applying 3-gram model was created, and an acoustic model for Japanese speech recognition by Hidden Markov Model in addition to the above were utilized to customize the speech recognition engine for forensic autopsy. A forensic vocabulary set of over 10,000 words was compiled and some 300,000 sentence patterns were made to create the forensic language model, then properly mixing with a general language model to attain high exactitude. When tried by dictating autopsy findings, this speech recognition system was proved to be about 95% of recognition rate that seems to have reached to the practical usability in view of speech recognition software, though there remains rooms for improving its hardware and application-layer software.

Does the sequence of onset of rigor mortis depend on the proportion of muscle fibre types and on intra-muscular glycogen content?

We examined the postmortem changes in the levels of ATP, glycogen and lactic acid in two masticatory muscles and three leg muscles of rats. The proportion of fibre types of the muscles was determined with NIH image software. The ATP levels in the white muscles did not decrease up to 1 h after death, and the ATP levels 1 and 2 h after death in the white muscles were higher than those in the red muscles with a single exception. The glycogen level at death and 1 h after death and the lactic acid level 1 h after death in masticatory muscles were lower than in the leg muscles. It is possible that the differences in the proportion of muscle fibre types and in glycogen level in muscles influences the postmortem change in ATP and lactic acid, which would accelerate or retard rigor mortis of the muscles.

Sarin-like and soman-like organophosphorous agents activate PLCgamma in rat brains.

We report that there is a time-related change in the phospholipase C (PLC) activities of rat brain cytosol and membrane fractions after iv injection of a soman-like or a sarin-like organophosphorous agent (bis(isopropyl methyl)phosphonate [BIMP] and bis(pinacolyl methyl)phosphonate [BPMP]). PLCgamma was activated in the brain cytosol fraction from BPMP-injected rats. The phosphorylating activity of rat brain membrane fractions were enhanced by BPMP treatment. The brain membrane fractions from BPMP-treated rats phosphorylated several proteins, including supposedly PLCgamma in the brain cytosol fraction from control rats in vitro. These results suggest that soman and sarin may stimulate a membrane tyrosine kinase, including growth factor receptors, directly or indirectly.

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart.

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.

A 'keyhole lesion' gunshot wound in an adipocere case.

A corpse completely converted into adipocere and showing two adjacent bone defects--a typical gunshot entrance wound and a keyhole lesion--is reported. Postmortem changes, a comminuted fracture of the cranial base, and destruction of the bullets made it impossible to determine the direction of fire through the keyhole lesion. Gunshot wounds may show various atypical forms, including keyhole lesions, and especially in old corpses, the distinction between an entrance wound and an exit wound can be very difficult, even if a careful complete autopsy is performed.

Calcium is required for quasi-lipoxygenase activity of hemoproteins.

Bovine and guinea pig heart homogenates, porcine leukocyte homogenate, and human hemolysate were found to vigorously oxidize linoleic acid, with lipoxygenase-like activity, to its hydroperoxy, epoxy, hydroxy-epoxy, and keto compounds in the presence of calcium chloride. In the absence of calcium, the reaction was significantly reduced. Attempts to characterize this quasi-lipoxygenase activity revealed that calcium potentiated the quasi-lipoxygenase activities of hemoproteins (hemoglobin, myoglobin, myeloperoxidase, catalase, cytochrome c) and hemin at the physiological pH of 7.5. Lipid peroxidation by hemoproteins was inhibited by albumin and erythrocyte membranes in blood, as well as by a low concentration of calcium in cells. However, it seems possible that in extracellular fluid, which contains a high concentration of calcium and a low concentration of albumin, hemoprotein released from damaged cells could oxidize unsaturated fatty acids derived by phospholipase-A2 from phospholipids of damaged cellular membranes. In a model of quasi-lipoxygenase activation under such conditions, lipids of erythrocyte membranes were oxidized by hemoglobin in the presence of phospholipase-A2 and calcium. The effect of nitrogen oxide, paraquat, and bleomycin on oxidation by hemoproteins and hemin was also discussed.