Research for biological and biochemical responses during nsh and shellfish cell death processes

对鱼类和贝类来说，每个细胞在身体死亡后仍然存在。因此，个体细胞在生存状态下的处理和处理会对死后代谢产生很大的影响。本研究的目的是揭示细胞死亡过程中生物生化变化的动态，并开发新的方法来评估鱼类和贝类的新鲜度。生物学方面，采用荧光探针如JC-1和罗丹明123测定急性切除的鱼类和甲壳类肌肉细胞的线粒体膜电位。结果表明，缺氧4小时后，鲤鱼肌肉细胞仍保持线粒体功能，缺氧4小时后，再氧化恢复了线粒体膜电位，表明鲤鱼肌肉线粒体能够在缺氧条件下存活并合成ATP维持细胞代谢。我们还观察到甲壳类动物肌肉细胞在尸体死亡后24小时仍保持线粒体膜电位。因此，线粒体膜电位是评价全体死亡后早期细胞活力和新鲜度的有力指标。红鲷在头部刺痛后5 - 30分钟剧烈抽搐。我们在这一现象中测量了脊髓中的单胺，发现脊髓中的去甲肾上腺素水平特异性降低。因此，我们认为红鲷的迟发性惊厥可能是由于脊髓去甲肾上腺素抑制脊髓运动神经元的急剧减少，导致脊髓运动神经元的急剧激活。因此，脊髓中的肾上腺素水平和迟发性惊厥是全身死亡后早期新鲜度的潜在指标。在数码显微镜下观察鱿鱼体死亡后染色质的反应。低温和缺氧处理诱导染色质囊缩回，并在体死后24小时内保持这种反应。因此，染色质反应也可用于评价鱿鱼的新鲜度。采用化学发光法检测脂肪酸过氧化氢早期脂质过氧化。黄尾马鲛鱼和马鲛鱼的深色肌肉由于过氧化脂肪酸的作用而表现出化学发光的增加，这表明过氧化脂肪酸也可能是评价鱼类肌肉新鲜度的一个潜在指标。我们还使用脂肪酸过氧化氢荧光探针DPPP来评价黄尾金枪鱼和金枪鱼肌肉的脂质过氧化。因此，DPPP也可用于评估鱼类肌肉脂质过氧化的早期阶段。总之，线粒体膜电位、脊髓去甲肾上腺素水平、延迟惊厥状态、染色质反应和脂肪酸过氧化氢水平是在死后变化的早期阶段有用和新颖的新鲜度指标。少

It is also true for fish and shellfish that each cell lives after body death. Therefore, metabolism postmortem would be strongly affected by handling and treatment during individual cell living state. The goals of this study is to disclose dynamics of biological and biochemical changes during cell death process and to develop novel methods for evaluating freshness of fish and shellfish. For biological approaches, mitochondrial membrane potentials in fish and crustacean muscle cells excised acutely were determined by the fluorescent probes such as JC-1 and rhodamine123. Then, carp muscle cells remained mitochondrial function during 4-hr anoxia treatment and that re-oxgenation revived mitochondrial membrane potential after 4-hr anoxia treatment, suggesting that carp muscle mitochondria could survive anoxia and synthesize ATP to sustain cell metabolisms. We also observed that crustacean muscle cells maintained the mitochondrial membrane potential 24 hours after body death. Thus, the mitoc … More hondrial membrane potential is a powerful index for evaluating the cell viability and freshness for early stages after whole body death. Red sea bream goes under drastic convulsion 5 - 30 min after cranial spiking. We measured monoamines in the spinal cord during this phenomenon and found that norepinephrine level in the spinal cord was reduced specifically. Then, we concluded that the delayed convulsion of red sea bream would be due to the drastic decrease in spinal cord norepinephrine suppressing spinal cord motoneuron, resulting in the drastic activation of spinal motoneurons. Thus, pinephrine levels in spinal cord and the delayed convulsion are potential indices for freshness for early stages after whole body death. Chromatophore responses were observed under digital microscope immediately after squid body death. Chilling and hypoxia treatment induced the retraction of chromatophore sacs and the responses were maintained over 24 hours after body death. Thus, the chromatophore responses afe also useful for evaluating freshness of squid. We adopted chemiluminescent detection of fatty acid hydroperoxide for the early stage of lipid peroxidation. Dark muscles of yellow tail and horse mackerel exhibited the increase in chemiluminescent due to fatty acid hydroperoxide, which suseested that fatty acid hydroperoxide would be also a potential index for evaluating fish muscle freshness. We also used DPPP, a fluorescent probe for fatty acid hydroperoxide, for evaluating lipid peroxidation of yellow tail and tuna muscle. Consequently, DPPP is also useful for evalulating very early stages of lipid peroxidation of fish muscle.In conclusion, mitochondrial membrane potential, norepinephrine level in spinal cord, delayed convulsion state, chromatophore responses, and fatty acid hydroperoxide levels afe useful and novel indices of freshness for very early stages during postmortem changes. Less