ULTRAVIOLET LIGHT INDUCED PHENOMENA IN BIOMEMBRANES

生物膜中的紫外线诱导现象

• 批准号: 2391909
• 负责人: IRENE KOCHEVAR
• 金额: $ 24.79万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391909
• 关键词: DNA damage; apoptosis; calcium flux; cell membrane; dyes; enzyme activity; fluorescent dye /probe; free radical oxygen; gene induction /repression; light adverse effect; mathematical model; membrane activity; membrane model; membrane permeability; membrane potentials; membrane structure; nonvisual photosensitivity; phospholipase A2; phospholipase C; photochemistry; photosensitizing agents; regulatory gene; singlet oxygen; ultraviolet radiation

Photochemistry in cellular membranes is responsible for initiating many responses of cells to ultraviolet and visible radiation with and without added photosensitizing molecules. The plasma membrane and mitochondrial membranes are frequently the sites of primary photosensitization in the use of dyes plus visible radiation to treat tumors (photodynamic therapy, PDT). The long range objective of this application is to provide a rational basis for design of membrane-photosensitizing dyes for applications in medicine and biology. The specific aims of this proposal are: Specific Aim 1: To produce, separately, singlet oxygen (1O2*) and free radicals from the same photosensitizer molecules in the plasma membrane. Singlet oxygen will be produced using low intensity light; radicals will be produced using pulsed lasers. The ability of mathematical modeling to predict optimal laser intensity and wavelength for photosensitization of cells will be evaluated. Specific Aim 2: To establish the influence of the location and identity (1O2* or free radicals) of the primary reactive species formed during photosensitization on biochemical alterations produced in the plasma membrane. Photosensitizers will be used that localize at different sites in the plasma membrane or generate different reactive species. Changes in membrane functions will be quantitatively compared between photosensitizers. Membrane functions to be measured include proline uptake, activity of Na+,K+-ATPase, membrane permeability, intracellular Ca2+, and membrane potential. The kinetics for alteration of plasma membrane potential and intracellular Ca2+ will be monitored on a microsecond to minute time scale after laser excitation using fluorescent reporter dyes. Specific Aim 3: To determine whether cellular responses to membrane photosensitization reflect the identity or location of formation of the reactive species. The activation of the membrane-associated enzymes, phospholipases A2 and C, will be assessed after photosensitization with dyes producing either 1O2* or free radicals in the plasma membrane. Induction of early response genes will be used as used to measure the effects of reactive species on an early nuclear response to plasma membrane photosensitization. Measurements will include induction of c- jun, c-fos and c-myc mRNA. Induction of apoptosis by photosensitizers that cause plasma membrane damage will be assessed. After photosensitization, DNA will be isolated and cleavage into nucleosome- size fragments will be quantitated.

细胞膜中的光化学反应是引发许多 细胞对紫外线和可见光辐射的反应， 添加了光敏分子。质膜和线粒体 膜通常是主要光敏化的部位， 使用染料加可见辐射治疗肿瘤（光动力疗法， PDT）。本申请的长期目标是提供一种 膜用光敏染料设计的合理依据 在医学和生物学中的应用。本提案的具体目标 为： 具体目标1：分别生产单线态氧（1 O2 *）和游离氧 来自质膜中相同光敏剂分子的自由基。 单线态氧将使用低强度光产生;自由基将 使用脉冲激光器生产。数学建模的能力， 预测光敏化的最佳激光强度和波长 细胞将被评估。 具体目标2：确定地点和身份的影响 (1O2* 或自由基）的主要反应性物种形成的过程中， 对血浆中产生的生化变化的光敏化 膜的将使用定位于不同部位的光敏剂 或产生不同的活性物质。变化 将在膜功能之间进行定量比较 光敏剂待测量的膜功能包括脯氨酸 摄取，Na+，K+-ATP酶活性，膜透性，细胞内 Ca ~（2+）和膜电位。等离子体变化动力学 膜电位和细胞内Ca 2+将在 在使用荧光激发的激光激发后的微秒到分钟的时间尺度 报告染料。 具体目标3：确定细胞对膜的反应是否 光敏化反应的身份或形成的位置， 活性物种膜相关酶的激活， 磷脂酶A2和C，将在光敏化后用 在质膜中产生1 O2 * 或自由基的染料。 早期应答基因的诱导将用于测量 反应性核素对等离子体早期核反应的影响 膜光敏化测量将包括诱导c- jun、c-fos和c-myc mRNA。光敏剂诱导细胞凋亡 将评估导致质膜损伤的因素。后 光敏化，DNA将被分离并裂解成核小体- 大小片段将被定量。