Formation of leukotoxin (9,10-epoxy-12-octadecenoic acid) during the autoxidation of phospholipids promoted by hemoproteins.

Myoglobin (Mb) and cytochrome c (Cyt c) are known to promote lipid peroxidation when mixed with certain types of phospholipids. In the presence of phospholipids such as cardiolipin (CL), ferrous Mb and Cyt c were converted to ferric hemoproteins, and autoxidation of the phospholipids and the oxidation of free linoleic acid (LA) added to the reaction mixture were observed. When the reaction mixture comprising 0.01 mM Cyt c, 0.2 mM CL and 0.1 mM LA was incubated, 92.7% of LA was consumed, and the LA products included 2.49 microM 9,10-epoxy-12-octadecenoic acid (leukotoxin) and its isomer which are potent inhibitors of mitochondrial respiration and have toxic effects on cardiac function. Hemoglobin (Hb) could promote almost no lipid peroxidation in the presence of any kinds of phospholipids. The experiments using some scavengers of active oxygen species revealed that tocopherol and ascorbic acid could strongly reduced LA oxidation caused by Cyt c or Mb. As LTx production was also observed when LA was mixed with Fe2+, LTx may be a common product where non-enzymatic lipid peroxidation occurs.

Formation of keto and hydroxy compounds of linoleic acid in submitochondrial particles of bovine heart.

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A2, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and alpha-tocopherol. The cardiolipin-cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid-hemoprotein lipid peroxidation systems such as the cardiolipin-cytochrome c system.

A method for monitoring ADP-induced thromboembolism in mice.

A method for monitoring ADP-induced thromboembolism in mice by means of the measurement of respiratory rate and depth was studied. The measurement were accurately carried out with a new method devised by us. In mice intravenously injected with 5-40 mg ADP/kg, it was found that the respiratory rate decreased transiently and dose-responsively, and that the platelet count also decreased transiently. On the other hand, no decrease in respiratory rate after administration of ADP was observed in neuraminidase-pretreated mice, in which the platelet count was significantly reduced. In the histological examination, platelet thrombi formed in the pulmonary vessels were observed 10 sec after administration of 10 and 40 mg/kg of ADP in mice which had not been treated with neuraminidase. The extent of thromboembolism at the dose of 40 mg/kg showed a tendency to be more marked than that at 10 mg/kg. Pretreatment with 300 mg dipyridamole/kg p.o. prevented the respiratory depression at 40 mg ADP/kg. However, with 10 mg dimorpholamine/kg i.v. or 30 mg warfarin/kg p.o., no preventive effect was observed. Therefore, it was concluded that it is possible to monitor ADP-induced thromboembolism in mice by means of the accurate measurement of the respiratory rate and depth.

Screening of milk aspiration in 105 infant death cases by immunostaining with anti-human alpha-lactalbumin antibody.

To determine the frequency and degree of milk aspiration in infant death cases, immunohistochemical examinations were performed on lung sections from 41 sudden death cases and 64 in-hospital death cases using anti-human alpha-lactalbumin antibody. Milk aspiration to some degree was detected in more than half of the sudden death cases and in about one-third of the in-hospital death cases. A semi-quantitative examination of the amount of aspirated milk was subsequently performed in the positive cases. The amount of aspirated milk in the sudden death cases was significantly higher than that in the in-hospital death cases. The frequency distribution of the amount of aspirated milk was similar in shape in both groups. In most cases, a very small amount of aspirated milk was detected. The aspirated milk was assumed to be a result of occasional gastroesophageal reflux or cardiopulmonary resuscitation. However, in five cases, much larger amounts of aspirated milk were found. In these cases, milk aspiration may have been an important part of the cause of death. We concluded that slight milk aspiration is not rare in infant death cases, and that in a few cases, the aspiration is lethal. An immunohistochemical screening test is available to perform a postmortem diagnosis in these cases.

Reconsideration of the sequence of rigor mortis through postmortem changes in adenosine nucleotides and lactic acid in different rat muscles.

We examined the changes in adenosine triphosphate (ATP), lactic acid, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in five different rat muscles after death. Rigor mortis has been thought to occur simultaneously in dead muscles and hence to start in small muscles sooner than in large muscles. In this study we found that the rate of decrease in ATP was significantly different in each muscle. The greatest drop in ATP was observed in the masseter muscle. These findings contradict the conventional theory of rigor mortis. Similarly, the rates of change in ADP and lactic acid, which are thought to be related to the consumption or production of ATP, were different in each muscle. However, the rate of change of AMP was the same in each muscle.

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.

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.

Production of monoclonal antibody reactive with leukotoxin and its immunohistochemical application.

A sensitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ALM-1) was developed for the determination of leukotoxin (9,10-epoxy-12- octadecenoic acid), which was reported to exist in human burned skin and neutrophils, and was regarded as a toxic and/or defensive substance in living beings. The leukotoxin was conjugated with bovine serum albumin (BSA) by means of the mixed anhydride technique as immunogen, and BALB/c mice were immunized over 6 months. The detection limit of leukotoxin was at least as low as 5 ng in this ELISA. This antibody had a strong specificity to leukotoxin and no cross-reactivity with the other analogs tested, and was usable for an immunohistochemical application. By using the antibody, leukotoxin was immunohistochemically observed not only in neutrophils, but also in alveolar macrophages in an oxygen-exposured rat lung.

Immunohistochemical detection of human milk components aspirated in lungs of an infant.

We examined an autopsy case of an infant whose cause of death was suspected to be asphyxia due to human breast milk aspiration. In order to demonstrate aspirated milk in the lungs, we conducted immunohistochemical staining using eight antibodies against the components of human milk. Seven of the eight antibodies reacted positively with the substances suspected to be aspirated milk. We concluded that immunohistochemical staining with these antibodies is useful to demonstrate human milk aspiration and provide some keys for detecting the causes of unexpected infant deaths.

Evidence for an antemortem injury of a burned head dissected from a burned body.

A 41-year-old woman was killed following blows to the head with a bat. Her body was burned and her head was dissected with a saw and hidden in the soil. Although the body was discovered within a few days of her death, the putrescent head was not located until four months later. A fracture of the left temporal bone was found in the head, which was partially charred. The confession of the murder suspect was unreliable, and the police suggested that the cause of death may not have been a head injury but asphyxia, and that the bone fracture to the head may have been caused after its dissection. It was therefore necessary to determine when the fracture was formed. The fracture line continued to the base of the skull, indicating that it was not due to heat. Magnetic resonance computed tomography (MR-CT) indicated the existence of a blood clot in the left mastoid cells, across which the fracture line passed. Upon sectioning of the skull with a saw, a dark red clot was located in the left mastoid cells, indicating that bleeding had occurred before the body was set alight. It seemed most logical to assume that the bone fracture was formed when the victim was alive, or before her body was burned.

Analysis of pedestrian injuries in traffic accidents (2)--Especially on leg injury.

A deep case study on 118 pedestrians struck to fronts of vehicles or vehicle derivatives was carried out. In this paper we investigated a leg injury which occurs the most frequently in a vehicle-pedestrian collision. The data obtained from the case study were compared with those of autopsied cases. Influence of impact speed and point, and of an accident pattern were considered. The leg injury showed higher AIS (Abbreviated Injury Scale) than that of the arm injury caused by direct impact against the front exterior of vehicles. The impact speed and ISS (Injury Severity Score) showed strong correlation, but the former and AIS did not show significant correlation. The higher impact speed and the run-over are likely to cause a bilateral leg fracture.

[An autopsy case of Smith-Lemli-Opitz syndrome].

A 17 month-old girl was found dead in her bed. The findings of a juditial autopsy revealed that she had a cleft palate, low-set and accessory ears, and syndactyly of right and left second and third toes and right third and fourth toes. Her birth size was small and subsequently she failed to thrive. Organ anomaly, such as atriaol septal defect, accessory spleens, and uterus bicornis bicollis were found. From these findings, this case was diagnosed as a Smith-Lemli-Opitz syndrome.

Experience with cloned mitochondrial DNA analysis to forensic practice.

The sequencing method of the D-loop region of cloned mitochondrial DNA (mtDNA) together with multilocus and single locus DNA probe patterns was applied to identification of the dismembered and putrefied remains of a pregnant woman. The multilocus DNA fingerprinting was useful to identify whether or not 4 dismembered remains were from the body of the same person, whereas the single locus DNA analysis was useful to decide that the suspect was the father of the fetus. On the other hand, the sequencing method of the D-loop region of cloned mtDNA after amplification by polymerase chain reaction (PCR) was particularly advantageous to highly degraded and contaminated DNA specimens.

Intercellular localization of leukotoxin (9:10-epoxy-12-octadecenoic acid) in lungs in rats by an immunoelectron microscopy.

In this paper intercellular localization of leukotoxin (9:10-epoxy-12-octadecenoic acid, LTx) in neutrophils and alveolar macrophages of rats exposed to the 100% oxygen was demonstrated by a pre-embedding method of an immunoelectron microscopy. The Sprague-Dawley rats were treated with the 100% oxygen for 72 hrs, and the lungs were stained with anti-LTx IgG (Fab) by an immunocytochemical method and examined by a transmission electron microscope. The positive reactions with anti-LTx IgG (Fab) was shown strongly in the rough-surfaced endoplasmic reticulum (rER) and cell membranes of the neutrophils and alveolar macrophages. This result indicates that LTx is produced in biomembrane-rich organelle of alveolar macrophages as well as neutrophils dependently on the treatment period under hyperoxia conditions, suggesting that LTx is closely related to biological reactions and is an important chemical mediator on pulmonary diseases.

Identification and dynamics of paraquat in the bone marrow, thymus and spleen in rats using immunohistochemical techniques.

We reported the immunohistochemical identification of paraquat in the immune and hematopoietic systems such as bone marrow, spleen and thymus. Twelve paraquat-poisoned rats were sacrificed in 3 h, 12 h, 3 days, 7 days and 10 days after intravenous administration of paraquat. In the bone marrow, paraquat was localized in several types of blood cells (granulocyte, erythrocyte and megakaryocyte) and their precursors between 24 h and 7 days after the injection. In the thymus, paraquat was mainly localized in the medulla between 12 h and 7 days after the injection, whereas in the spleen, it was mainly localized in the red pulp between 12 h and 10 days after administration of paraquat.

Personal identification from human remains using morphological characteristics and DNA analysis.

This report describes personal identification of four parts of a dismembered human body on the basis of physical characteristics and DNA analysis. A 69-year-old woman suffering from depression went missing from her home in May 1992. Five days after her disappearance, a female torso was found in a river. In a two-week period thereafter, a left forearm, a female head and neck and a right forearm were found consecutively in the same river. Although the end of the cervical vertebra was anatomically identical to that of the torso, the two forearms did not match. However, DNA analyses with a single-locus DNA probe pYNH24, DO alpha typing and a multi-locus DNA probe "Myo", indicated that these four parts came from the same person. The missing woman had undergone lobectomy of the right pulmonary upper lobe, and had suffered from slight scoliosis of the thoracic vertebrae and a second lumbar vertebral compression fracture. These characteristics were also observed in the torso. In order to obtain more conclusive evidence, DNA fingerprinting using DNA extracted from the torso, the missing woman's brother and her daughter was performed using the DNA probes described above. The data obtained indicated that the remains were those of the missing woman.

[Analysis of pedestrian injuries in traffic accidents: impact point, injury portion, and injury severity].

We conducted in-depth case studies of traffic accidents involving 118 pedestrians, who were struck by the fronts of bonnet-type cars. The data were reviewed in order to correlate injury severity with pedestrian age, and area of body contact, contact surface of vehicle or the local environment, and impact speed. It was found that head injuries caused by impacts against the top surface of car bonnets and leg injuries caused by front bumpers both showed high incidence rates. Both head and leg injuries caused by striking the vehicle were more severe than those caused by striking the road surface. In cases of impact between the head and the solid portion of the vehicle, such as the A-pillar, or in cases of secondary impact between the head and underhood solid structures such as shock tower, injuries tended to be more severe. The location of head impact varied according to the stature of the pedestrians.

Wolbachia and nuclear-nuclear interactions contribute to reproductive incompatibility in the spider mite Panonychus mori (Acari: Tetranychidae).

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are responsible for cytoplasmic incompatibility in a wide range of arthropods such as insects and mites. Spider mites often show uni- and bidirectional incompatibilities among populations with and without Wolbachia. Therefore, we surveyed the presence of Wolbachia by PCR and then conducted crossing experiments among 25 populations of Panonychus mori to determine how Wolbachia are related to the incompatibility in this species. Five out of the 25 populations were infected with Wolbachia. These five populations were treated with an antibiotic (rifampicin) to eliminate Wolbachia. We carried out round-robin crosses among 20 Wolbachia-uninfected populations, five infected populations and five rifampicin-treated populations (30 x 30=900 crosses in total). Incompatibility among P. mori populations was caused by Wolbachia infection, nuclear-cytoplasmic interactions or nuclear-nuclear interactions. Wolbachia-mediated incompatibility was observed in crosses between uninfected females and infected males or between females and males harboring different Wolbachia strains. Nuclear-cytoplasmic interactions may be responsible for the unidirectional incompatibility in crosses between the two northern populations and one of the southern populations. Bidirectional incompatibility caused by nuclear-nuclear interactions was observed in 99 combinations of interpopulation crosses (99/300=0.33). Although no geographical trends were detected in the distribution of bidirectionally compatible populations, the results reveal a genetic divergence among P. mori populations.

Death due to a methane gas explosion in a tunnel on urban reclaimed land.

Studies of four male victims who were killed in an accidental tunnel gas explosion on urban reclaimed land are described. The studies were judicial autopsy examinations to determine the precise causes of death. Two men died of carbon monoxide intoxication, one died of massive brain damage, and the fourth died of drowning. The concentrations of methane in several organs were much lower than the lethal level, whereas those in adipose tissue were relatively high. These findings indicated that a low concentration of methane was almost always present in the atmosphere at the construction site. Recently, coal mine accidents have been decreasing in Japan. However, there is still a possibility of underground explosions or gas leaks in confined spaces other than coal mines. To determine the precise cause of death in such cases, careful autopsies and other examinations should be performed using methods similar to those used in coal mine accidents.