Collaborative Research: Novel Thermal Hysteresis Glycolipid Antifreeze in Insects and Plants

项目名称：合作研究：昆虫和植物中的新型热滞性糖脂防冻剂项目负责人：Duman， John g.项目名称编号：IOS 1025929暴露在零度以下温度下的生物通过变得耐冻（它们在冷冻中存活）或必须变得防冻以防止冻结来适应。结构多样的抗冻蛋白（AFPs）已经在许多不同的生物中进化：动物、植物、细菌、真菌等，但昆虫的AFPs可以说是最活跃的。afp通过结合冰晶表面和/或冰成核表面来抑制冻结，从而阻止水分子加入晶体表面。因此，afp降低了水溶液的凝固点，但不改变熔点，产生了它们存在的热滞后（TH，凝固点和熔点之间的差异）特征。在此之前，人们只知道抗冻蛋白具有这种活性。知识价值。最近，我们发现了一种新的糖脂，其TH与昆虫afp的TH相等。这些新的抗冻糖脂（AFGLs）在几种抗冻昆虫（包括抗冻和抗冻）、青蛙、鱼和抗冻植物中被发现。由于热滞后以前只在蛋白质中被发现，这一新发现有可能改变我们对生物如何适应零度以下温度的看法。AFP的功能在避冻物种中得到了最好的研究，在避冻物种中，AFP的功能是通过阻断来自外部冰的表面接种冻结和抑制体液中的冰核来防止冻结。我们研究的一种阿拉斯加甲虫，Cucujus clavipes，产生典型的甲虫型afp，帮助它们进入超低温，所以即使放在零下150摄氏度，它们也不会冻结。在~-70℃时，体内的水玻璃化，变成玻璃，但不冻结。C. clavipes是产生AFGLs的物种之一。我们建议继续对这种有趣的昆虫进行研究，以确定这些抗冻液在生理功能方面的潜在协同作用。我们还将研究两种抗冻昆虫的AFGLs结构和生理功能，分别是来自阿拉斯加（耐~-60℃）的Upis ceramboides和来自印第安纳州（耐~-28℃）的Tipula trivittata，以及来自印第安纳州的苦甜茄属植物Solanum dulcamara。抗冻th蛋白（AFPs或AFGL）在抗冻物种中的作用尚不清楚。回想一下，这些物种已经进化到能够冷冻和生存，那么为什么要防冻液呢？一种可能性是，由于这些生物通常只能在细胞外的水被冻结后存活，AFGLs可能起到防止冰从细胞外水扩散到细胞内水的致命作用。事实上，这可能是事实，因为这些物种中的大多数AFGL都与细胞膜有关，而细胞膜正好处于这种功能的位置。本研究的主要科学目标是：(1)确定AFGLs的结构；(2)确定其在抗冻和避冻生物中的生理功能。这项研究的广泛影响有三个方面：(1)生物医学材料的潜在低温保存，(2)作物和园艺植物的潜在耐寒性提高，(3)对生物教育的积极影响。AFPs和现在的新型AFGLs在细胞、组织和器官的低温保存方面有可能应用。AFGLs可以为细胞提供冷冻保护，使它们更容易被冷冻保存。afgl还可以在不冻结的状态下将材料储存在零度以下，模仿它们在阿拉斯加的深层过冷C. clavipes中的功能。这也可以应用于农业，培育出更耐寒的植物。高中、本科、博士和博士后阶段的学生将直接参与这项研究，从而接受从野外生物学和生理生态学到生物化学和分子生物学的跨学科培训。此外，阐明这些适应有可能吸引来自不同背景的新学生和实践科学家。在这个日益专业化的时代，生化学家和生态学家、生物学家和物理学家或化学家似乎没有什么共同兴趣，这样的研究可以促进相当多的跨学科理解和合作。我们最近首次发表的AFGLs得到了广泛的关注，从纽约时报到自然杂志。此外，我们还将为耐寒性研究人员提供服务，筛选他们的生物体进行afgl。

PROJECT TITLE: Collaborative Research: Novel Thermal Hysteresis Glycolipid Antifreeze in Insects and PlantsPRINCIPAL INVESTIGATOR: Duman, John G.PROJECT TITLE NUMBER: IOS 1025929Organisms that are exposed to subzero temperature adapt by becoming either freeze tolerant (they survive being frozen) or they must become freeze avoiding to prevent freezing. Structurally diverse antifreeze proteins (AFPs) have evolved in many different organisms: animals, plants, bacteria, fungi, etc., but insect AFPs are arguably the most active. AFPs inhibit freezing by binding to the surface of ice crystals and/or ice nucleating surfaces, thereby reventing water molecules from joining the crystal surface. Consequently, AFPs lower the freezing point of an aqueous solution, but do not change the melting point, producing the thermal hysteresis (TH, difference between the freezing and melting points) characteristic of their presence. Previously, only antifreeze proteins were known to have this activity. Intellectual Merit. Recently, we identified novel glycolipids with TH equal to that of insect AFPs. These new antifreeze glycolipids (AFGLs) were found in several cold tolerant insects (both freeze tolerant and freeze avoiding), a frog, a fish, and a freeze tolerant plant. Since thermal hysteresis has previously been identified only in proteins, this novel discovery has the potential to transform our ideas on how organisms adapt to subzero temperatures. AFP function has been best studied in freeze avoiding species where they function to prevent freezing by blocking inoculative freezing across the surface from external ice and by inhibiting ice nucleators in body fluids. One species that we study, an Alaskan beetle, Cucujus clavipes, produces typical beetle type AFPs that assist them to deep supercool, so that they do not freeze even if taken to ?150oC. At ~-70oC the body water vitrifies, turns to glass, but does not freeze. C. clavipes is one of the species that produces AFGLs. We propose to continue studies of this interesting insect to determine the potential synergy in physiological function of these antifreezes. We will also investigate the structure and physiological function of AFGLs in two freeze tolerant insects, (Upis ceramboides) from Alaska (freeze tolerant to ~-60oC) and (Tipula trivittata) from Indiana (freeze tolerant to ~-28oC), and in a freeze tolerant plant, the bittersweet nightshade Solanum dulcamara from Indiana. The function of TH-antifreezes (AFPs or the AFGL) in freeze tolerant species is not well understood. Recall that these species have evolved to freeze and survive, so why have antifreeze? One possibility is that, since these organisms generally only survive freezing of their extracellular water, the AFGLs may function to prevent the lethal spread of ice from the extra- to the intra-cellular water. In fact, this may be the case since most of the AFGL in these species is associated with cell membranes, perfectly situated for this function. The primary scientific goal of this study is to (1) determine the structure of the AFGLs, and (2) to identify their physiological functions in both freeze tolerant and freeze avoiding organisms. The broader impacts of this study are three-fold: (1) potential cryopreservation of biomedical materials, (2) potential improved crop and horticultural plant cold tolerance and (3) a positive effect on biological education. AFPs, and now the novel AFGLs, have possible applications in the cryopreservation of cells, tissues and organs. AFGLs may provide freeze protection to cells, permitting them to be more easily freeze-preserved. AFGLs may also permit subzero storage of materials in the unfrozen state, mimicking their function in the deep supercooling C. clavipes from Alaska. There could also be applications in agriculture resulting in more cold tolerant plants. Students at the high school, undergraduate, PhD and post-doctoral levels will be directly involved in this study, thereby receiving interdisciplinary training ranging from field biology and physiological ecology to biochemistry and molecular biology. In addition, elucidation of these adaptations has the potential to attract new students and practicing scientists from diverse backgrounds. In this era of increased specialization when biochemists and ecologists, biologists and physicists or chemists seem to have few common interests, such studies can foster considerable interdisciplinary understanding and cooperation. Our recent initial publication of AFGLs has received widespread general attention, ranging from the NY Times to Nature magazine. Also, we will provide a service to cold tolerance researchers by screening their organisms for AFGLs.