Acquisition of Transmission Electron Microscopy Equipment for Invertebrate and Vertebrate Pigment Cell Studies, Undergraduate Laboratory Demonstrations, and Undergraduate Research

博士们已获得一笔赠款。肯塔基卫斯理学院的大卫·f·厄廷格和史蒂文·d·威尔特。这笔拨款将允许研究人员购买仪器，以装备透射电子显微镜设备。具体来说，该设备将包括一个用于研究细胞成分的透射电子显微镜——放大倍数在1000到30万之间；一种带有金刚石刀的超微切片机，用于切割厚度在60至80纳米范围内的组织切片；第二个微型小组，用于筹备工作和培训目的；玻璃刀匠，也用于准备工作和训练；还有合适的暗室设备。Oetinger博士将研究淡水虫（等足类）细胞中共色色素颗粒的结构和发育。随后，他将寻求更好地了解感染幼虫寄生虫（棘头虫）如何在结构上和生化上干扰正常色素颗粒的形成。威尔特博士对脊椎动物眼睛的视网膜色素上皮（RPE）感兴趣，特别是细胞之间形成“栅栏”的连接，限制脂质和蛋白质在相邻细胞之间的扩散。他将使用细胞培养和鸡胚胎来确定紧密连接分子在紧密连接的发育和维持中的作用。从水生等足类动物的年龄系列色素表皮将被固定，染色，包埋和切片。超微结构检查将评估细胞内色素颗粒的位置、大小、形状和数量。在某些昆虫中，共色色素被认为是色氨酸代谢的废物：这一假设将在淡水等足类动物（甲壳类动物）中进行测试，使用同步生化提取技术和色素颗粒的超微结构检查。因此，研究棘头虫幼虫发育与色素沉着变化的关系也将在超微结构水平上进行研究。威尔特博士将使用劳斯肉瘤逆转录病毒表达系统过度表达紧密连接分子（ZO-2）的亚域。然后，他将利用传统的电子显微镜和免疫金标记方法，从超微结构上确定ZO-2在RPE紧密连接的发育和调节中的作用。细胞色素和色素细胞在无脊椎动物和脊椎动物中发挥着多种生物学作用：隐色、生理颜色变化、紫外线屏蔽，以及作为视觉受体的重要组成部分。在等足动物-棘头动物系统中，Oetinger正在寻求一种寄生虫如何介导宿主形态变化的理解——这对寄生虫来说是有选择优势的。在威尔特的研究中，视网膜色素上皮的完整性对最佳视力至关重要。更好地理解维持这种完整性的超微结构-分子关系对于我们理解脊椎动物眼睛的进化和随后的结构和功能缺陷是很重要的。

A grant has been awarded to Drs. David F. Oetinger and Steven D. Wilt at Kentucky Wesleyan College. This grant will allow the investigators to purchase instrumentation to equip a transmission electron microscopy facility. Specifically, the equipment will include a transmission electron microscope for study of cellular components--in the 1,000 to 300,000 magnification range; an ultramicrotome with diamond knife for cutting tissue sections in the range of 60 to 80 nanometers in thickness; a second ultramicrotome for preparatory work and training purposes; a glass knife-maker, also for preparatory work and training; and appropriate darkroom equipment. Dr. Oetinger will study the structure and development of ommochrome pigment granules in the cells of freshwater sowbugs (isopods). Subsequently, he will seek a better understanding of how infection with larval worm parasites (acanthocephalans) interferes- structurally and biochemically with normal pigment granule formation. Dr. Wilt is interested in the retinal pigment epithelium (RPE) of vertebrate eyes-specifically the junctions between cells that form a "fence," restricting diffusion of lipids and proteins between adjacent cells. He will use cell cultures and chick embryos to determine the role of tight junction molecules in the development and maintenance of tight junctions.Pigmented epidermis from an age-series of aquatic isopods will be fixed, stained, embedded and sectioned. Ultrastructural examination will assess location, size, shape and number of pigment granules within cells. In certain insects, ommochrome pigments are known to be stored waste products from tryptophan metabolism: that hypothesis will be tested for freshwater isopods (crustaceans) using concurrent biochemical extraction techniques and ultrastructural examination of pigment granules. So too, study of the relationship between developing acanthocephalan larvae and altered pigmentation will be studied at the ultrastructural level. Dr. Wilt will use a Rous sarcoma retroviral expression system to overexpress subdomains of the tight junction molecule (ZO-2). Then, using traditional electron microscopy and immunogold labelling methods, he will determine the role of ZO-2 in the development and regulation of RPE tight junctions ultrastructurally.Cellular pigments and pigmented cells play of variety of biological roles in invertebrates and vertebrates: cryptic coloration, physiological color changes, ultraviolet light screening, and as important components of visual receptors. In the isopod-acanthocephalan system, Oetinger is seeking an understanding of how a parasite can mediate changes in a host's morphology--of selective advantage to the parasite. In Wilt's research, the integrity of the retinal pigment epithelium is critical for optimal vision. A better understanding of the ultrastructural-molecular relationships that maintain that integrity is important to our understanding of the evolution of the vertebrate eye and subsequent defects in structure and function.