Kazuhito Hatanaka

Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart

Abstract — Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 &mgr;mol/L) only during IP blunted the infarct size limitation by IP (37.3±6.3% versus 7.4±2.1% in the IP group, P <0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3±9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8±3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.

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.

Immobilization stress-induced thymocyte apoptosis in rats.

Immobilization of rats is a well known model of emotional stress. We studied apoptosis in rat thymocytes during immobilization for 2-8 hours (h). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells increased with time of immobilization. The positive cells increased significantly after 2 h and comprised about 33% of total cells after 8 h. The apoptotic cells aggregated as scattered foci predominantly in the cortex of the thymus and diffusely distributed with time of the stress. The DNA laddering also increased with time of immobilization, which was inhibited by a glucocorticoid receptor antagonist, RU-486. These results suggest that the emotional stress evoked by immobilization induces thymocyte apoptosis through glucocorticoid overflow.

Development of rigor mortis is not affected by muscle volume

There is a hypothesis suggesting that rigor mortis progresses more rapidly in small muscles than in large muscles. We measured rigor mortis as tension determined isometrically in rat musculus erector spinae that had been cut into muscle bundles of various volumes. The muscle volume did not influence either the progress or the resolution of rigor mortis, which contradicts the hypothesis. Differences in pre-rigor load on the muscles influenced the onset and resolution of rigor mortis in a few pairs of samples, but did not influence the time taken for rigor mortis to reach its full extent after death. Moreover, the progress of rigor mortis in this muscle was biphasic; this may reflect the early rigor of red muscle fibres and the late rigor of white muscle fibres.

Emotional stress activates MAP kinase in the rat heart

Emotional stress evoked by immobilization of the rat induces c-fos mRNA or other immediate early genes. This response is mediated by activation of alpha- and beta-adrenoceptors, through mechanisms that have not yet been elucidated. Here we show that immobilization stress activates p44/p42 Mitogen-Activated Protein kinase (p44/p42 MAP kinase, Erk1/Erk2). Pretreatment with the beta1-blocker, metoprolol, did not inhibit the activation of stress-induced MAP kinase, while blockage of the alpha1-adrenoceptor by pretreatment with alpha1-blocker, prazosin or the alpha/beta-blocker, amosulalol, attenuated the activation. Application of the alpha1-agonist, phenylephrine, but not the beta-agonist, isoproterenol, to the perfused rat heart elicited MAP activation. Thus, emotional stress activates the alpha1-adrenoceptor-mediated MAP kinase pathway, whereas the pathway of the response mediated by the beta-adrenoceptor remains unknown.

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

Abstract 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 °C or 25 °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 °C and at 25 °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 °C than at 37 °C in each type of muscle.

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.

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.

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.

Thiol-Oxidizing Agent Diamide and Acidic pH Enhance Lipid Peroxidation of Rat Heart Mitochondria and Cardiolipin? Cytochrome c Complex

In this study, we investigated lipid peroxidation in rat heart mitochondria hydrolyzed by phospholipase A2 (PLA2) and lipid peroxidation in a mitochondrial‐mimetic lipid peroxidation system, where phospholipids such as cardiolipin (CL) and cytochrome c (Cyt c) were first mixed together and then PLA2 and calcium chloride were added to the mixture (CL‐Cyt c‐PLA2 system). Production of hydroperoxy and hydroxy compounds of linoleic acid (LA)1