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Autoradiographic analysis on agar plates of antigens from subcellular fractions of rat liver slices. 大鼠肝切片亚细胞抗原琼脂板放射自显影分析。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.411
W S MORGAN, P PERLMANN, T HULTIN

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of (14)C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.

用14C氨基酸孵育大鼠肝脏切片,匀浆,差速离心。用非离子洗涤剂Lubrol W和EDTA进一步提取微粒体。这些提取物和游离微粒体的“细胞液”,从pH 5可沉淀的部分中释放出来,随后使用琼脂扩散技术与抗血清反应。所采用的抗血清是用不同的大鼠肝脏亚细胞部分或大鼠血清蛋白注射兔获得的。当琼脂扩散板进行自放射线照相时,发现一些沉淀物具有放射性,而另一些则没有。对照实验表明,这种标记是由于(14)C氨基酸在切片孵育期间特异性结合到各种大鼠肝脏抗原中,而不是由于放射性物质对免疫沉淀的非特异性吸附。当切片与同位素孵育长达30分钟时,可以用洗涤剂从微粒体中提取的血清蛋白被强烈标记,许多其他未知意义的微粒体抗原也被标记。相比之下,存在于细胞液中的血清蛋白仅被弱标记。大多数典型的细胞液蛋白,包括可沉淀的和可在pH 5下溶解的,似乎都没有被标记。用洗涤剂提取微粒体后剩余的核糖体残留物的EDTA提取物没有获得一致的可重复性结果。肝切片孵育较长时间(长达120分钟)导致细胞液中血清蛋白的强标记,以及在其他细胞液蛋白中出现标记。结果讨论了亚细胞合成位点和不同抗原的代谢。
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引用次数: 33
Ultrastructural cytochemistry. Enzyme and acid hydrolysis of nucleic acids and protein. 超微结构的细胞化学。核酸和蛋白质的酶和酸水解。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.437
E H LEDUC, W BERNHARD

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 micro sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3 degrees C are more rapidly digested by proteinases than those fixed for the same length of time at 20 degrees C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 micro sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.

通过酶或酸水解,可以从固定在福尔马林中并包埋在水溶性聚氧化物中的组织的超薄切片(用于电子显微镜)和平行微切片(用于光学显微镜)中选择性地提取特定的细胞成分,产品X133/2097。正常的大鼠组织在3℃的福尔马林中固定15分钟,蛋白酶的消化速度比在20℃固定相同时间的大鼠组织更快。胰蛋白酶选择性地攻击核染色质和星浆的核糖核蛋白颗粒,而线粒体和酶原颗粒则抵抗胰蛋白酶的消化。胃蛋白酶迅速攻击线粒体和酶原颗粒。乳糜质和细胞核最初抵抗消化,但随着时间的推移,整个细胞质和细胞核的染色质间部分受到攻击。核糖核酸酶在2个显微切片上消除细胞质嗜碱性,但在平行的超薄切片上,用醋酸铀酰染色并在电镜下检查,显示细胞质的核糖核蛋白颗粒没有变化。单独的去氧核糖核酸酶没有作用,但在用胃蛋白酶或盐酸预处理切片后,去氧核糖核酸酶特异性攻击核染色质。切片中含有核酸的结构被高氯酸或盐酸逐渐分解。
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引用次数: 70
Morphological and chemical studies of collagen formation. II. Metabolic activity of collagen associated with subcellular fractions of guinea pig granulomata. 胶原形成的形态学和化学研究。2与豚鼠肉芽肿亚细胞部分相关的胶原代谢活性。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.373
D A LOWTHER, N M GREEN, J A CHAPMAN

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-(14)C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-(14)C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-(14)C-valine into TCA-insoluble material by various combinations of subcellular fractions.

从角叉菜胶诱导的肉芽肿中获得的核、微粒体和线粒体薄片的电子显微照片显示,较重的部分被较轻的部分污染了。在细胞核+碎片中可见条状胶原原纤维。线粒体部分仅出现少量条纹原纤维;在这部分中可以看到非常细的细丝(直径50 A),但不能肯定地与来自破碎核的纤维状物质区分开来。用0.2 M NaCl可提取35%的线粒体和80%的微粒体胶原蛋白,并可通过标准的溶液和再沉淀方法纯化。离子交换色谱法测定的这些胶原蛋白组分的氨基酸组成在其他哺乳动物物种的胶原蛋白和明胶通常发现的范围内,允许微粒体胶原蛋白的一些非胶原污染物的10%至20%。用L-(14) c -脯氨酸孵育组织切片后,用纸层析法从细胞核、线粒体和微粒体胶原蛋白的水解产物中分离出羟基脯氨酸和脯氨酸。胶原中羟基脯氨酸的比活性约为1:2:6,而上清中结合的羟基脯氨酸的比活性仅为1,说明胶原在微粒体中进行了初级合成。试图证明L-(14) c -脯氨酸与胶原蛋白或游离羟脯氨酸在无细胞系统中的结合是不成功的,也不可能通过各种亚细胞组分的组合证明L-(14) c -缬氨酸与tca不溶性物质的非特异性结合。
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引用次数: 51
Fine structure of an unusual intracellular supporting network in the Leydig cells of Amblystoma epidermis. 浅裂瘤表皮间质细胞中不寻常的细胞内支撑网络的精细结构。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.457
E D HAY
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引用次数: 31
Serum fractionation and the effects of bovine serum fractions on human cells grown in a chemically defined medium. 血清分离和牛血清对在化学确定的培养基中生长的人细胞的影响。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.389
R HOLMES, S W WOLFE

Serum has been fractionated by curtain electrophoresis using carboxymethyl cellulose dissolved in sodium bicarbonate electrolyte. Various fractions were produced from bovine serum and added to replicate cultures of Chang's endoepithelial cells and HeLa cells grown in a chemically defined medium. The effects of each of the various fractions on the appearance of the cultures and on cell multiplication were studied. Three different fractions were obtained and two were subjected to further purification. One fraction associated with albumin promoted survival, attachment, and flattening as well as cell multiplication. A second fraction associated with the alpha globulins promoted survival and multiplication of some cells. A third fraction caused cells to aggregate and form free floating clumps. An adequate chemically defined medium for continuous growth of human cells was used throughout the study. The response of cells to alterations in their environment which simulated some of the effects produced by serum fractions is described.

用溶解在碳酸氢钠电解质中的羧甲基纤维素幕电泳分离血清。从牛血清中提取不同的组分,并将其添加到在化学定义的培养基中生长的张氏内皮细胞和HeLa细胞的重复培养中。研究了不同组分对培养物外观和细胞增殖的影响。得到三种不同的馏分,其中两种进行进一步纯化。一个与白蛋白相关的部分促进了存活、附着、扁平以及细胞增殖。与α球蛋白相关的第二部分促进了某些细胞的存活和增殖。第三个部分导致细胞聚集并形成自由漂浮的团块。在整个研究过程中,使用了一种适当的化学定义的培养基,用于人类细胞的连续生长。描述了细胞对环境变化的反应,模拟了血清组分产生的一些影响。
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引用次数: 27
Electron stains. I. Chemical studies on the interaction of DNA with uranyl salts. 电子污渍。1 . DNA与铀酰盐相互作用的化学研究。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.335
C R ZOBEL, M BEER

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO(2) (++)/P mole ratio of about (1/2) and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about (1/3). Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.

人们对DNA与铀酰盐的相互作用进行了化学研究。研究了pH值、盐浓度和DNA结构完整性对盐-底物复合物化学计量学的影响。在pH为3.5时,DNA与低浓度的铀酰离子相互作用,生成UO(2) (++)/P摩尔比约为(1/2)的底物金属离子配合物,并具有较大的结合常数。在低pH(约2.3)时,摩尔比降至约(1/3)。在HCHO溶液中加热破坏DNA的结构完整性会导致金属离子结合量的类似下降。将pH值提高到3.5以上,就像增加盐溶液的浓度一样,结合力明显增加。这种附加绑定具有较低的关联常数。在类似条件下,DNA结合铀酰离子的能力是牛血清白蛋白的7倍,这表明在电子显微镜染色中具有有用的选择性。
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引用次数: 108
Spermiogenesis in the crayfish (Procambarus clarkii) II. Description of stages. 克氏原螯虾(Procambarus clarkii)的精子发生2。阶段描述。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.301
M J MOSES

The sperm of the crayfish, Procambarus clarkii, is relatively simple among decapod sperm and was described in the first paper of this series (28). The present paper details the development of this sperm as followed with the light and electron microscopes. The process is divided into six stages for purposes of description. The main points of interest discussed are the absence of mitochondria or mitochondrial derivatives in the mature sperm, the development of a complex acrosome in the absence of highly organized characteristic Golgi apparatus but in the presence of small stacks of annulate lamellae, and the changes in the nucleus. Of the latter, the elaborate convoluted sheets of membrane that are extensions of the nuclear envelope are unique. The nucleus undergoes unusual changes in size and shape that are accompanied by several phases of organization of the chromatin. In the mature sperm the nucleus is empty-appearing and notably lacking in any apparent high degree of order. The entire development of the sperm is consonant with the idea that the fate of the mitochondria and centrioles, structures that figure prominently in the elaborate architecture of flagellate sperm, is associated with the lack of a flagellum.

克氏原螯虾(Procambarus clarkii)的精子在十足动物的精子中是相对简单的,在本系列的第一篇论文中有描述(28)。本文用光学显微镜和电子显微镜对该精子的发育进行了详细的描述。为了便于描述,这个过程分为六个阶段。讨论的主要兴趣点是成熟精子中线粒体或线粒体衍生物的缺失,在没有高度组织的高尔基体的情况下复杂顶体的发育,但存在小堆环状片,以及细胞核的变化。在后者中,作为核膜延伸的复杂卷曲的膜片是独一无二的。细胞核在大小和形状上经历了不同寻常的变化,并伴随着染色质组织的几个阶段。在成熟精子中,细胞核是空的,明显缺乏任何明显的高度有序。精子的整个发育过程都符合这样一种观点,即线粒体和中心粒的命运与鞭毛的缺失有关。线粒体和中心粒是鞭毛精子精细结构的重要组成部分。
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引用次数: 90
Fine structure of the larval anuran epidermis, with special reference to the figures of Eberth. 无尾蝇幼虫表皮的精细结构,特别参考了埃伯的外形。
Pub Date : 1961-07-01 DOI: 10.1083/jcb.10.3.425
G B CHAPMAN, A B DAWSON

Small pieces of skin from 8 cm long Rana clamitans larvae were fixed in OsO(4), washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on a Porter-Blum ultramicrotome and were examined in an RCA electron microscope, type EMU 2D. The sections showed that aggregates of fibrous material in the cells of the inner layer of epidermal cells are identical in disposition and size with the classical figures of Eberth. It is conclusively shown that these figures do not arise from an aggregation of mitochondrial filaments. The tendency of the fibrils to concentrate on attachment points, or thickenings of the basal plasma membrane, is noted. It is also observed that numerous mitochondria are located in the distal region of the cells of the outer layer of epidermis in association with the secretory vacuoles. Microvilli are seen occasionally on the free surface of the skin. Cisternae are found only in the cells of the outer epidermal layer, while vesicular endoplasmic reticulum is found in the cells of both epidermal layers.

将8厘米长的蛤蛙幼虫的小片皮肤固定在OsO(4)中,洗涤,脱水,并包埋在甲基丙烯酸酯混合物中。超薄切片在Porter-Blum超微切片机上切片,在EMU 2D型RCA电子显微镜下检查。切片显示,表皮细胞内层细胞内的纤维物质聚集体在分布和大小上与经典的埃伯斯图相同。结论表明,这些数字不是来自线粒体细丝的聚集。注意到原纤维集中于附着点或基底质膜增厚的趋势。我们还观察到许多线粒体位于与分泌液泡相关的表皮外层细胞的远端区域。皮肤游离表面偶尔可见微绒毛。池池只存在于外表皮层的细胞中,而泡状内质网存在于两个表皮层的细胞中。
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引用次数: 43
Centriole replication. A study of spermatogenesis in the snail Viviparus. 中心体复制。蜗牛胎生精子发生的研究。
Pub Date : 1961-06-01 DOI: 10.1083/jcb.10.2.163
J G GALL

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO(4) and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mmicro in diameter and 330 mmicro long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.

本文描述了长尾螺(Viviparus malleatus Reeve)精子发生过程中中心粒的复制。用OsO(4)固定并包埋在聚酯树脂Vestopal w中的睾丸切片切片,电镜观察可见两种精子细胞。这就产生了典型的携带9条染色体单倍体数量的单鞭毛精子和只有一条染色体的非典型多鞭毛精子。两个中心粒存在于最年轻的典型精母细胞中。每个都是直径约160微米,长330微米的空心圆柱体。壁由9组以特征图案排列的三联体纤维组成。在粗粒形成之前的某个时候,一个未成熟的中心粒,或称为前中心粒,在每个成熟的中心粒旁边出现。前中心孔在大部分方面类似于成熟的中心孔,除了长度:它是环状的而不是管状的。子核的轴线垂直于母核的轴线。虽然在电子显微镜下还没有观察到成熟阶段,但它可能在前期后期发育到完全大小。认为中心粒具有恒定的极化。远端形成鞭毛或其他中心粒产物,而近端代表前中心粒并与复制有关。前期的四个中心粒(两个亲本和两个子代)通过两次减数分裂分布到四个典型的精子中,它们在其中起着鞭毛基体的作用。非典型精母细胞最初含有两个正常的中心粒。它们中的每一个都被一簇前核所包围,在前期后期逐渐拉长。在两次异常减数分裂后,中心粒簇产生多鞭毛精子的基体。这些事实是根据Pollister首先提出的理论来讨论的,即非典型细胞中多余的中心粒来自退化染色体的着丝粒。
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引用次数: 194
THE PLASMA MEMBRANE OF STAPHYLOCOCCUS AUREUS. 金黄色葡萄球菌的质膜
Pub Date : 1961-06-01 DOI: 10.1083/jcb.10.2.292
A Suganuma
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引用次数: 49
期刊
The Journal of Biophysical and Biochemical Cytology
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