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

项目标题：合作研究：昆虫和植物中的新型热滞糖脂防冻剂PRINCIPAL调查员：Duman，John G.ProjECT标题编号：iOS 1025929暴露在零下温度下的组织通过变得耐寒(它们在被冻结中存活)或必须变得避免冻结以防止结冰来适应。结构多样的抗冻蛋白(AFP)在许多不同的生物中进化，如动物、植物、细菌、真菌等，但昆虫AFP被认为是最活跃的。AFPS通过结合到冰晶表面和/或冰核表面来防止冻结，从而阻止水分子加入晶体表面。因此，AFP降低了水溶液的冰点，但不改变熔点，产生了它们存在的热滞(TH，冰点和熔点之间的差异)。此前，只有抗冻蛋白具有这种活性。智力上的功绩。最近，我们发现了一种新的糖脂，其TH与昆虫的AFP相当。这些新的抗冻剂糖脂(AFGL)在几种耐寒昆虫(包括耐冻和避冻)、一只青蛙、一条鱼和一种耐寒植物中发现。由于以前只在蛋白质中发现了热滞现象，这一新发现有可能改变我们对生物体如何适应零下温度的看法。AFP功能在避免结冰的物种中得到了最好的研究，在这些物种中，AFP通过阻止从外部冰穿过表面的接种性结冰和通过抑制体液中的冰核来防止结冰。我们研究的一个物种是阿拉斯加甲虫，Cucujus clavipe会产生典型的甲壳虫型AFP，帮助它们进行深度过冷，这样即使温度达到？150摄氏度，它们也不会冻结。在摄氏零下70度时，体内的水会玻璃化，变成玻璃，但不会结冰。棒状梭菌是产生AFGL的物种之一。我们建议继续对这种有趣的昆虫进行研究，以确定这些防冻剂在生理功能上的潜在协同作用。我们还将研究两种耐冻昆虫的AFGL的结构和生理功能，它们来自阿拉斯加(耐冻至~60oC)和来自印第安纳州的(Tipula Trivittata)(耐冻至~28oC)，以及耐寒植物印第安纳州苦甜茄子。TH-抗冻剂(AFP或AFGL)在耐寒物种中的作用尚不清楚。回想一下，这些物种已经进化到可以冻结和生存，那么为什么要防冻剂呢？一种可能性是，由于这些生物通常只在细胞外水冻结时存活，AFGL可能起到防止冰从细胞外水向细胞内水的致命扩散的作用。事实上，情况可能是这样，因为这些物种中的大多数AFGL都与细胞膜有关，非常适合这一功能。本研究的主要科学目标是：(1)确定AFGL的结构；(2)鉴定AFGL在耐冻和避冻生物中的生理功能。这项研究的更广泛的影响有三个方面：(1)生物医学材料的潜在超低温保存；(2)潜在地提高作物和园艺植物的耐寒性；(3)对生物教育的积极影响。AFPS和现在的新型AFGL在细胞、组织和器官的冷冻保存中有可能应用。AFGL可以为细胞提供冷冻保护，使它们更容易被冷冻保存。AFGL还可能允许在未冻结状态下将材料储存在零度以下，模拟它们在阿拉斯加深过冷的棒状梭菌中的功能。还可能在农业上应用，从而培育出更耐寒的植物。高中、本科、博士和博士后水平的学生将直接参与这项研究，从而接受从田间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，对这些适应的阐明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，当生物化学家和生态学家、生物学家和物理学家或化学家似乎几乎没有共同兴趣时，这样的研究可以促进相当多的跨学科理解和合作。我们最近首次出版的AFGL受到了从《纽约时报》到《自然》杂志的广泛关注。此外，我们还将为耐寒研究人员提供服务，筛选他们的生物体是否存在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.