Masataka Nagao

Novel detection of plankton from lung tissue by enzymatic digestion method

Studies are reported on the enzymatic digestion method for detection of plankton from lung tissue by using proteinase K with sodium dodecyl sulphate. This method is simple, safe and effective for detection of not only phytoplankton including diatoms but zooplankton which are destroyed by the acid digestion method. The present method is, therefore, much more advantageous for diagnosis of drowning than the disorganization method using strong acids.

Estimating stature from the length of the lumbar part of the spine in Japanese.

In order to estimate the stature from the length of the lumbar part of spine (LLPS), studies were made on Japanese males (n=42) and females (n=29) autopsied in our laboratory during 1984–1987. Somatometry was performed on the stature and LLPS in centimetres, the latter being measured from the upper edge of the first lumbar vertebral body, to the promontorium, along the anterior surface of the spine. LLPS were 19.9 ± 1.19 cm in males and 18.6 ± 0.84 cm in females (mean ± S.D.). The regression equations calculated were as follows: stature in males = LLPS × 3.23 + 101.7; stature in females = LLPS × 2.31 + 110.8. The standard errors of estimate were 6.16 cm in males and 4.05 cm in females. This method is useful for estimating the stature of severely burned or mutilated bodies which have no limbs.

The mechanism of experimental adipocere formation: Hydration and dehydrogenation in microbial synthesis of hydroxy and oxo fatty acids

The enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid but dehydrogenation of this product. The mechanism of the hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxostearic acids produced in the presence of D2O or H2(18)O. The activity of these enzymes was increased by preincubating Flavobacterium meningosepticum with oleic acid.

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.

Detection of sarin hydrolysis products from sarin-like organophosphorus agent-exposed human erythrocytes

A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID50 and LD50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarins hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.

The mechanism of experimental adipocere formation: Substrate specificity on microbial production of hydroxy and oxo fatty acids

Studies are reported on microbial conversion of various unsaturated fatty acids to 10-hydroxy and/or 10-oxo fatty acids by Micrococcus luteus. Four fatty acids possessing cis-9-unsaturation produced 10-hydroxy and 10-oxo fatty acid products, but three enoic acids possessing trans-9-unsaturation or double bond(s) in other than the 9-carbon position were inactive as substrates. 10-Hydroxy palmitic and stearic acids were converted to the corresponding 10-oxo fatty acids, but the 10-oxo compounds were inactive as substrates. This indicates that the metabolic sequence of cis-9-enoic fatty acid by the microbial enzyme(s) is first converted to 10-hydroxy fatty acid and then to its 10-oxo compound.

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.

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

Abstract 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.

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.

Transport characteristics of paraquat across rat intestinal brush-border membrane

The mechanism of absorption of paraquat, which is a type of quaternary ammonium compound (QAC), was studied using rat intestinal loops and brushborder membrane vesicles. Approximately 47% and 37% of radioactively labeled paraquat injected into jejunal and ileal loops disappeared, respectively, after 60 min. Since only a small amount of radioactivity was detected in the mucosal fraction, most of the paraquat that disappeared from the intestinal lumen was considered to have been carried away by the bloodstream, indicating that paraquat absorption was greater than expected. In spite of its low lipid solubility, the uptake of paraquat by brush-border membrane vesicles reflected smooth penetration into the intravesicular space rather than binding to the membrane. According to the increase in extravesicular paraquat concentration, paraquat uptake in the early stage was saturable. Moreover, early paraquat uptake was significantly inhibited by structurally-related QACs such as tetramethylammonium and choline, but not by an endogenous dicationic amine (putrescine). On the other hand, inside-negative membrane potential had no significant effect on the time course of paraquat uptake. From these results, it is suggested that paraquat is absorbed through a specialized mechanism associated with the carrier-mediated transport system for choline on the brush-border membrane.