FINE STRUCTURE OF CEREBROSIDE OCCURRING IN GAUCHERS DISEASE
FINE STRUCTURE OF CEREBROSIDE OCCURRING IN GAUCHERS DISEASE
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DOI：
10.1073/pnas.61.2.484
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发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
LEE, RE
中科院分区：
文献类型：
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作者：
LEE, RE
材料与方法脾和/或肝组织从6例戈谢病患者中获得，无论是在脾切除术时还是在尸检时。对这些组织进行光镜和电子显微镜检查，并冷冻储存。通过在1%乙酸铵溶液中切碎小片段，可以容易地制备解冻的组织用于负染色。将该溶液的小滴加入到1%磷钨酸的液滴中，并使用300目涂层和无涂层网格支撑染色材料。当应用于甲醛固定的组织时，该过程同样令人满意。采用阴影技术以确定负染色制备物中观察到的螺旋方向。遮蔽在真空蒸发器中进行，使用以大约100 °的角度遮蔽的碳铂。在整个研究过程中使用了菲利普斯200电子显微镜。在阴影形式的印刷阶段给予了照顾，以保持正确的螺旋线的螺旋感。4密度梯度离心法分离肾小管与其他细胞膜。对于该程序，用2.5倍体积的含有0.35 M蔗糖、0.025 M KCl和0.01 Al MgCl 2的Tris缓冲液（pH 7.6）研磨组织。在Sorvall离心机中以27，000 X g初始离心10分钟后，将10 ml上清液小心地加到含有10 ml 1.5 Mf蔗糖的30 ml离心管中，所述蔗糖在10 ml 2.0 M蔗糖上分层。在75，000 X g下离心17小时后，将在0.3和1.5 M蔗糖之间收集的白色层单独收集，沉淀，并重悬于不添加蔗糖的Tris缓冲液中，并将2 ml加入到15 ml范围为0.3至1.5 Al蔗糖的连续梯度溶液中。然后将材料再次以78，000 X g离心17小时。用极性脂质溶剂系统（组成：100 ml氯仿，40 ml甲醇，6 ml蒸馏水）对连续梯度密度级分进行薄层色谱，通过电子显微镜测定，该级分主要含有管状形式。还分别根据Lowry等人5和Bartlett和Shin，6 '7的方法对该级分进行蛋白质和磷测定。在试图产生侧小管形态的变化中，该部分也暴露于2.0 A尿素以及1% Triton、0.1 N NaOH和0.1 N HCl过夜。结果。- (1)形态学：在切碎的组织的负染色制备物中或在梯度密度级分中，管状形式很容易被识别为长形式。通常，几个小管缠绕在一起。单个小管的宽度从250到500埃不等，有的长度可达5埃。每个小管似乎是由10-12个纤维轻轻地扭曲在小管的长轴。阴影技术揭示了一个螺旋与右手螺旋的意义（图。第2和第3段）。相邻的小管常常相互缠绕（图4）。
Materials and Methods.-Splenic and/or liver tissues were obtained from six patients with Gaucher's disease, either at the time of splenectomy or at autopsy. These tissues were processed for light and electron microscopy, and were stored frozen. The thawed tissues could easily be prepared for negative staining by mincing small fragments in a solution of 1% ammonium acetate. Small drops of this solution were added to drops of 1% phosphotungstic acid, and 300-mesh coated and carbonized grids were used to sup-port the stained material. This process was equally satisfactory when applied to form-aldehyde-fixed tissues. Shadowing techniques were employed in order to determine the direction of the helix noted in negatively stained preparations. Shadowing was carried out in a vacuum evaporator, using carbon platinum shadowed at an angle of approximately 100. A Phillips 200 electron microscope was used throughout this study. Care was given during the printing phase of the shadowed forms in order to preserve the correct screw sense of the helix. 4Density gradient centrifugation was carried out in order to separate the tubules from other cellular membranes. For this procedure, tissue was ground with 2.5 times the volume of Tris buffer (at pH 7.6) containing 0.35 M sucrose, 0.025 M KCl, and 0.01 Al MgCl2. After initial centrifugation for 10 min at 27,000 X g in a Sorvall centrifuge, 10 ml of the supernatant was carefully applied to a 30-ml centrifuge tube containing 10 ml of 1.5 Mf sucrose layered on 10 ml of 2.0 M sucrose. After centrifugation for 17 hr at 75,000 X g, the white layer that had collected between the 0.3 and 1.5 M sucrose was separately collected, pelleted, and resuspended in Tris buffer without added sucrose, and 2 ml was applied to a 15-ml continuous gradient solution ranging from 0.3 to 1.5 Al sucrose. The material was then centrifuged again for 17 hr at 78,000 X g. Thin-layer chromatography with a polar lipid solvent system (composition: 100 ml chloroform, 40 ml methanol, 6 ml distilled water) was carried out on the continuous gradient density fraction which contained predominantly tubular forms as determined by electron microscopy. Protein and phosphorus determinations were also performed on this fraction according to the methods of Lowry et al. 5 and Bartlett and Shin, 6'7 respectively. In an attempt to produce changes in the morphology of the cerebroside tubule, this fraction was also exposed overnight to 2.0 A urea, as well as to 1% triton, 0.1 N NaOH, and 0.1 N HCl. Results.-(1) Morphology: Tubular forms are easily identified as long forms in the negatively stained preparations of the mincedtissues or inthe gradient density fractions. Frequently, several tubules are twisted together. The individual tubules have a width varying from 250 to 500 A, and someattain a length of 5, i. Each tubule appears to be made up of 10-12 fibrils gently twisted about the long axis of the tubule. Shadowing techniques reveal a helix with a right-handed screw sense (Figs. 2 and 3). Adjacent tubules are often noted to be twisted about each other (Fig. 4).