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

大卫F博士获得了一笔赠款。作者声明：Steven D.威尔特在肯塔基州卫斯理学院。 这笔赠款将允许研究人员购买仪器，以装备透射电子显微镜设施。 具体而言，这些设备将包括一台用于研究细胞成分的透射电子显微镜-放大倍数在1 000至300 000倍之间;一台带金刚石刀的超薄切片机，用于切割厚度在60至80纳米之间的组织切片;第二台用于准备工作和培训目的的超薄切片机;一台玻璃刀制造机，也用于准备工作和培训;和适当的暗室设备。 Oetinger博士将研究淡水播种虫（等足类动物）细胞中眼色素颗粒的结构和发育。 随后，他将寻求更好地了解如何感染幼虫寄生虫（棘头虫）干扰-结构和生物化学与正常的色素颗粒形成。 Wilt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，限制相邻细胞之间脂质和蛋白质的扩散。 他将使用细胞培养和鸡胚来确定紧密连接分子在紧密连接的发展和维持中的作用。来自一个年龄系列的水生等足类动物的色素表皮将被固定、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，眼色素被认为是色氨酸代谢产生的废物：这一假设将使用同步生化提取技术和色素颗粒的超微结构检查来测试淡水等足类动物（甲壳类动物）。 因此，也将在超微结构水平上研究发育中的棘头虫幼虫和改变的色素沉着之间的关系。 Wilt博士将使用Rous肉瘤逆转录病毒表达系统过表达紧密连接分子（ZO-2）的亚结构域。 然后，利用传统的电子显微镜和免疫金标记方法，他将从超微结构上确定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：隐蔽着色，生理颜色变化，紫外线屏蔽，以及作为视觉感受器的重要组成部分。 在等足棘头虫系统中，Oetinger正在寻求了解寄生虫如何介导宿主形态的变化-寄生虫的选择优势。 在Wilt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以及随后的结构和功能缺陷非常重要。

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.