家兔晶状体纤维的超微结构:纤维细胞的间隙连接

本文报道晶状体纤维细胞间间隙连接的形态结构。我们利用冰冻断裂技术,在不同部位的球-和-凹连结的头部以及在纤维细胞和纤维细胞之间都观察到间隙连接的存在。通过极其丰富的上述连接,可实现细胞间代谢物和离子的传递。作者认为:对正常晶状体纤维细胞之间的间隙连接的深入了解,将会为晶状体发病机制的研究提供新的线索。     

人类白内障晶状体的超微结构和元素分析

本文报道人成熟型白内障晶状体的超徽结构和元素分析。作者使用附有能谱仪的JSM-35C扫描电镜对晶状体纤维细胞的巯、磷、钙进行微区分析。结果表明,硫的谱峰很高,磷和钙的谱峰则很低。此外,线扫描分析证明,沿扫描线硫在晶状体纤维细胞内的分布比较均一。通过扫描电镜观察,发现其纤维细胞外形不规则,球-和-凹的连接不易看到。在不同区域的晶状体纤维细胞出现大量破坏及球体的形成。而最明显的改变是在品状体局部纤维细胞的扁平面形成无数刺状突起,这些突起具有不同的大小和形态,分布也不均匀。     

冰冻断裂电子显微镜技术研究鸡败血支原体细胞膜

近年来冰冻断裂电子显微镜技术是研究生物膜的超微结构最有效的方法之一。有人采用此种技术对莱氏衣原体、丝状支原体山羊变种、鸡败血支原体A5969、以及螺旋支原体等进行研究中。发现有些为家畜、家禽病原体,其膜断裂面复型上存在直径约为50—100A的球形蛋白质颗粒。本文报道用鸡败血支原体S_6菌株为实验材料,用仿制的Bullivant-AmesⅡ型简易冰冻断裂装置,对完整的支原体细胞或分离的支原体膜进行的实验结果。     

适合于植物花器官的冰冻切片技术

通过对4种植物主要花器官冰冻切片技术的各个环节及参数的研究,建立了一种适合于植物花器官的冰冻切片技术,即蔗糖保护-液氮速冻-冰冻切片法。其具体程序是：材料经固定和冷冻保护(蔗糖为冷冻保护剂)后进行速冻包埋(液氮为包埋剂);尔后进行冰冻切片;切片经干燥和染色(或者不染色)后,在显微镜下观察并摄影。此法为植物花器官的细胞生物学和分子生物学研究提供了简便、快速和高效的切片技术。     

自噬在晶状体发育及白内障的研究进展

自噬,或细胞自我消化,是参与蛋白质和细胞器降解细胞途径。晶状体是由一层晶状体上皮细胞及一系列正在分化的纤维细胞组成,纤维细胞发育通过线粒体、细胞核等细胞器退化完成的。在晶状体中,自噬在晶状体纤维细胞成熟,构成无细胞器区(organell-free zone,OFZ)的过程中,起着重要在作用。很多的研究证明对Atg5,Vsp34,FYCO1等基因在维持晶状体透明性中的作用进行了讨论,并且在晶状体上皮细胞,未成熟纤维细胞中证实自噬小泡的存在。本文旨在概括目前自噬的研究进展,重点介绍自噬与人晶状体发育及其与白内障形成机制的研究进展。     

一种冰冻刻蚀装置的设计及应用

冰冻刻蚀(或断裂)技术与其他常规电镜技术相比具有许多优点。样品的快速冰冻可起到物理固定的作用这不仅减少了化学固定引起的人为改变,而且也省去了固定和脱水等步骤;对生物标本进行不同时期的冰冻可以得到不同阶段的细胞复型;在膜的研究方面;它可与生物化学方法相辅,同样起到生物化学方法所起的弱化膜疏水键的作用,从而使两层疏水性面的任何侧面的三维结构暴露出来,远比超薄切片所得到的两维片层结构要丰富。     

不同固定方法对植物细胞扫描电镜观察用冷冻断裂样品的影响

用冷冻断裂法在扫描电镜下研究了洋葱(Allium cepa)根端分生组织细胞内部的三维结构。采用了两种固定方法。冷冻断裂前只用1％锇酸固定的材料容易在细胞质和核之间断开,而用卡诺固定液(无水乙醇:冰醋酸3:1)前固定,然后再用1％锇酸固定的材料容易使细胞核断裂。前一固定方法适于研究细胞质的内部结构(细胞骨架的纤维、线粒体、内质网等及其三维分布关系):后一固定方法适于研究核内结构(染色质、核仁、核基质纤维)的三维形象,特别是核仁纤维中心染色质的三维结构。     

丙线照射所致人血淋巴细胞与中国白鼠卵巢成纤维细胞DNA单链断裂及其修复的观察

用微孔滤膜碱洗脱法观察了丙线照射引起人血淋巴细胞(D_o为400拉德)及中国仓鼠卵巢成纤维细胞(D_o为200拉德)的DNA单链断裂及其修复。在0～3000拉德范围内,两种细胞DNA单链断裂的程度基本一致,照射剂量与单链断裂的对数之间呈直线相关。800、1500及3000拉德照射后,经过0.5～7小时的孵育,中国仓鼠卵巢成纤维细胞DNA单链断裂的修复优于人血淋巴细胞,说明这两种细胞辐射敏感性与DNA单链断裂修复能力无关。     

丙线照射所致人血淋巴细胞与中国仓鼠卵巢成纤维细胞DNA单链断裂及其修复的观察

用微孔滤膜碱洗脱法观察了丙线照射引起人血淋巴细胞(Do为400拉德)及中国仓鼠卵巢成纤维细胞(Do为200拉德)的DNA单链断裂及其修复。在0—3000拉德范围内,两种细胞DNA单链断裂的程度基本一致,照射剂量与单链断裂的对数之间呈直线相关。800、1500及3000拉德照射后,经过0.5—7小时的孵育,中国仓鼠卵巢成纤维细胞DNA单链断裂的修复优于人血淋巴细胞,说明这两种细胞辐射敏感性DNA单链断裂修复能力无关。     

部分感觉器官的超微结构

图1.角膜的扫描电镜图像该图为家兔角膜,上半部为复层扁平上皮的游离面,下部为角膜断面。可见复层扁平上皮由5—6层细胞组成(EP),基底面平滑无乳头,固有层(LP)中胶原纤维层的走行方向与上皮表面相平行。×600 图2.晶状体的扫描电镜图像图为大鼠晶状体纤维的表面形象,不少部位可见其断面,完整的晶状体纤维呈长扁平带状,定向平行排列,结构非常规律。×1,800 图3.视网膜的扫描电镜图像图为人视网膜,左     

Lens fiber organization in four avian species: a scanning electron microscopic study

The three-dimensional organization of the eye lenses of the chicken, the canary, the song-thrush and the kestrel was studied using light and scanning electron microscopy. The lenses of birds are characterized by the presence of two distinct compartments: the annular pad and the main lens body, separated by a cavum lenticuli. The annular pad fibers had a hexagonal circumference all contained a round nucleus and except for the canary were smooth-surfaced and lacking anchoring devices. In the canary, however, the annular pad fibers were studded with edge protrusions and ball-and-socket junctions. The semicircular main lens body fibers of all four species were studded with ball-and-socket junctions and edge protrusions. In contrast with mammals these anchoring devices were present throughout the lens up to the embryonal nucleus. Superficially the main lens body fibers were extremely flat. Additionally membrane elevations and depressions and globular elements were found on these central fibers in three species, the kestrel being the exception. At the transition between annular pad and main lens body the fibers turned their course and the nuclei became oval and disappeared in the deeper aspect of the main lens body. The cavum lenticuli was filled with globules tied off from the annular pad fibers. It seems attractive to assume that the presence of a separated annular pad, a cavum lenticuli filled with globular elements, the extreme flatness of the superficial central fibers and the studding of these central fibers with anchoring devices up to the embryonal nucleus are morphological expressions of the mouldability of the bird's eye lenses and consequently would explain their efficient accommodative mechanism including formation of a lenticonus. The presence of nuclei in the annular pad fibers and their typical change at the transitional zone between annular pad and main lens body are suggestive for a two-phased differentiation in bird's lens fibers: differentiation of the germinative epithelial cells to annular pad fibers which migrate to the main lens body after which they differentiate further to main lens body fibers.     

Scanning electron-microscopic study of lens fibers of the pig

Summary Whole pig lenses were fixed, critical-point dried and fractured, and the internal surfaces examined with a scanning electron microscope. At various locations from the equator to the center of the lens four types of fibers can be distinguished. The superficial fibers have small interdigitations. Cortical lens fibers, which are hexagonal in shape exhibit well developed ball-and-socket junctions. Other cortical fibers appear slightly undulated and show fine granulations. The core lens fibers are characterized by microplicae on the cell surfaces and by a more rounded or rectangular form. Results are discussed in relation to previous electron-microscopic studies of other species.     

Membrane contacts and lens transparency

Two kinds of membrane contacts in the vertebrate lens are described. Fiber gap junctions are domains where small molecules can pass between lens cells. Membrane structures of ball-and-socket type interlock adjacent lens fibers and thus contribute to the structural integrity of the lens. Both of these membrane contacts appear crucial for the maintenance of lens transparency.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic delta-crystallin.

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.     

Ultrastructure of traumatic cataractogenesis in the frog: a comparison with mouse and human lens.

Normal and needle-punctured lenses of Rana pipiens were examined with the electron microscope in order to characterize the sequence of ultrastructural changes that follow the injury over a 5-month period. Results were compared with those obtained previously in experimentally injured mouse and accidentally injured human lenses. The normal adult frog lens was found to have a morphology similar to that of mammalian lenses. As in the human, frog lens epithelial cells contained scattered microfilaments and were connected by desmosomes and gap junctions. They differed from mouse cells, which had been shown to lack desmosomes and to have microfilaments organized into dense bundles. These differences are postulated to be related to the degree of accommodative deformation of the lens displayed by these species. After injury, cellular debris and fibrin, accumulated in the wound, were phagocytized by extrinsic cells derived from the blood and ocular tissues. Leucocytes, pigmented cells and fibroblasts remained in the wound for eight weeks, along with epithelial cells which proliferated and migrated from the wound margins.Epithelial cells showed an increase in those organelles associated with protein synthesis and transport, and in microfilaments. In cataractous lenses, epithelial cells showed changes in matrix, and lens fibers became organized into smaller, denser compressed units. At five months, considerable healing had taken place, but localized opacities persisted in many frog lenses.     

A New Method Using Hexamethyldisilazane for Preparation of Soft Insect Tissues for Scanning Electron Microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Lipid composition of lens plasma membrane fractions enriched in fiber junctions

Little is known about the lipid environment of lens fiber junctions, the plasma membrane structure proposed to be responsible for passage of low molecular weight metabolites between adjacent lens fiber cells. Plasma membranes of the ocular lens are especially rich in fiber junctions. The resistance of junctional domains to disruption by detergent or alkali treatment provides the opportunity to isolate a lens plasma membrane fraction enriched in fiber junctions. When examined by electron microscopy, the fiber junction fraction prepared from bovine lenses was enriched with junctional structures by about twofold when compared to total plasma membrane. We compared the protein, phospholipid, and cholesterol concentration of total plasma membrane with fiber junctional membrane from rat and cow lens and from aged normal cataractous human lenses. The principal finding was that junctional membrane contained 20-40% more total lipid than that of the total plasma membrane. This was due to a proportionate increase in the relative content (mg/mg protein) of both phospholipid and cholesterol. Exclusive of one exception (nucleus of bovine lens), the cholesterol/phospholipid molar ratios of the two fractions were similar. In the bovine nucleus, the cholesterol/phospholipid molar ratio was substantially higher in the fiber junctional-enriched membrane fraction than in the total plasma membrane, suggesting a special association of cholesterol with bovine nuclear fiber junctions. The relative lipid compositions of the plasma membrane and fiber junction-enriched fractions from human normal and cataractous lenses were similar, suggesting that human senile cataractogenesis involves changes in the lens plasma membrane more subtle than would be reflected by gross changes in the membrane lipid composition.