Variation in membrane structure as revealed by negative staining technique

A study has been made of the structure of membrane material from various fractions of cellular homogenates. By the use of the negative staining technique several types of membrane structure have been observed. The variation occurs in both internal structure and in the surface features of the different membrane types. The structures observed are characteristic of different membrane fractions and are reproducible under the conditions described. We propose that the structures observed in response to the negative staining technique represent characteristic structural or chemical properties of each type of membrane and are therefore a clue to significant differences in the natural membrane structures, although the structures observed may not exist in the same configuration within the cell.     

Z-line structure of the rabbit psoas muscle studied by negative contrast staining

The ultrastructure of the Z-disc of the rabbit psoas muscle was elucidated by electron microscopy using negative staining technique. Conclusions summarized from this work are as follow: (a) Z-disc involves two layers of Z-filaments, i.e. connecting filaments, which bind thin filaments of adjacent I-discs in the Z-line region. These layers are spaced about 380 A apart. (b) Z-filaments measure 380 A X 30 A. (c) The angle between the connecting filaments and the thin filaments depends on ionic conditions and varies from 20 degrees to 90 degrees. (d) We conclude that alpha-actinin is a structural component of Z-filaments, since dimensions of Z-filaments and their interaction with thin filaments are similar to those of alpha-actinin.     

Electron microscope studies on the structure of collagen fibrils by negative staining

Summary Electron microscope studies on collagen from rat-tail tendon using a negative staining technique have indicated the presence of filaments 15–20 Å in diameter within the fibres. These filaments are thought to correspond to the collagen macromolecule.We would like to thank Prof. R. A. McCance for supplying the specimens of fowl tendon used in this investigation, and Dr. F. H. C. Crick, Dr. S. Fitton-Jackson and Dr. T. Gillman for a number of valuable discussions. One of us (W. J. T.) wishes to thank the Medical Research Council for financial support during this work.     

Studies by negative staining on the structure of collagen fibrils in normal and lathyritic rats

Summary Electron microscope studies on collagen from rat-tail tendon using the negative staining technique indicate the presence of fine filaments 15–30 Å in diameter within the fibres. The banding of the fibrils was only slightly affected by aminoacetylnitrile bisulphate, but an increased amount of fine fibrous material was present in preparations from experimental animals. It is suggested that this material represents a soluble form of collagen which is known to predominate after administration of the drug. Despite the fact that this would be a chemically abnormal collagen, its structure by electron microscopy corresponds remarkably well with the form suggested from X-ray diffraction and physico-chemical studies on normal soluble collagen.The author would like to thank Mr. J. Clements for technical assistance and Mr. R. Thomas (Dental School, Bristol) for discussion on the effects of aminoacetylnitriles in rats.     

Structure of the lac repressor studied by negative staining

Extensively purified lac repressor from Escherichia coli was observed under an electron microscope after negative staining with sodium phosphotungstate. It is shown that a negatively stained repressor molecule has at least an axis of 2-fold rotational symmetry and an inter-subunit space (hole or cleft) filled with the stain, in which double helical DNA may fit in a native state of the repressor.     

Filament-carrying tubules demonstrated by negative staining in various mammalian cell types

Summary Among other tubular elements obviously representing the endoplasmic reticulum, tubules carrying filaments with a diameter of about 4 m were found in negatively stained specimens of a variety of mammalian cell strains. They have been found in strains of epithelial and fibroblastic, normal and malignant, human and animal origin. So far, it is not possible to identify the filament-carrying tubules with equivalent structures in thin sections.     

Chromosome core structure revealed by silver staining

Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichrormatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichrormatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).     

The estimation of sugar concentration in individual sieve-tube elements by negative staining

The presence of water-soluble compounds in sectioned plant tissue can be visualized in negative contrast by freeze-substitution in acetone, followed by embedment in Epon containing 6% Sudan B. The contents of mature sieve tubes and companion cells of bean (Phaseolus vulgaris L.) showed strong, and mostly uniform, negative staining. The degree of negative staining was measured by microspectrophotometry. Since, in sieve tubes, virtually all of the solute is the translocated sugar, the sugar concentration can be estimated by comparison with similar measurements made on sections from pith blocks which were infiltrated with sugar solutions and processed by the same procedures. Sieve tubes contained a solution of about 11.2% (w/v) sucrose; companion cells contained a similar concentration of sucrose. Negative staining, and therefore the sucrose concentration, in immature sieve-tube elements and companion cells was much less than in their mature counterparts.This work was presented in part at a joint U.S.-Australia Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

Direct correlation of structure changes and thermal events in hydrated lipid established by simultaneous calorimetry and time-resolved x-ray diffraction.

In many lipid systems, polymorphic and mesomorphic behavior depends on sample thermal history. To establish unequivocally the structural origin of endothermic and exothermic events in such systems, we have performed simultaneous calorimetry and time-resolved x-ray diffraction (SCALTRD). To this end, aluminum calorimetry crucibles were used to contain the hydrated lipid sample, and the calorimeter was mounted with the base of the crucible oriented perpendicular to a synchrotron-derived focused monochromatic x-ray beam for SCALTRD data collection. Measurements were made with hydrated monoelaidin and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) contained in hermetically sealed crucibles. Time-resolved x-ray diffraction (TRXRD) data were collected using an x-ray image intensifier/video system and a streak camera containing an x-ray sensitive image plate and/or film. SCALTRD analysis of the lamellar gel to lamellar liquid crystalline phase transition in hydrated monoelaidin gives identical progress curves by calorimetry and TRXRD at a scan rate of 1 degree C/min. At faster rates, calorimetry shows a broader phase transition that starts at a lower and ends at a higher temperature than is observed by TRXRD. The disparity arises in part because the x-ray beam used in TRXRD interrogates only a small portion of the sample, whereas the calorimeter responds to the entire sample volume. Because data collection times are relatively long, radiation damage is an important potential problem for SCALTRD measurements. Such an effect was observed with DEPE/water in that TRXRD shows the lamellar gel to lamellar liquid crystalline phase transition occurring at a lower temperature than observed by calorimetry. We speculate that the sample accumulates impurities locally as a result of radiation damage that has the effect of lowering the phase transition temperature at the site of interrogation by the x-ray beam. This "methods-in-combination" SCALTRD approach facilitates the direct correlation of structure rearrangements and thermal events in the same sample under identical conditions of thermal history. The information content of the data so derived far surpasses that available from either method used in isolation.