bluuebunny/crossref_metadata_2025_split huggingface dataset:
A critical evaluation of Bernhard’s EDTA regressive staining technique for RNA
Authors: Pearson E. C. , Davies H. G. 
Published: April 2021

ABSTRACT
The EDTA regressive staining procedure to detect RNA (Bernhard’s technique) is based on the proposition that after staining ultrathin sections with uranyl the stain is preferentially removed from DNA rather than RNA by the action of the chelating agent EDTA. Whilst attempting to use the EDTA regressive staining procedure to detect the presence of RNA in the large granule complexes of chicken erythrocyte nuclei, certain anomalous staining patterns were observed in the chromatin of these nuclei. Essentially, these were that the edges of condensed chromatin bodies stained positively for RNA even though this molecule is known not to be present there in significant quantities. The staining patterns suggested that chromatin was retaining its stain in a manner expected of RNA but not DNA as a consequence of EDTA-containing species failing to pass freely through the section. This hypothesis was tested by carrying out the EDTA procedure on embedded specimens of a DNA-containing virus, simian virus 40 (SV40), small enough not to be exposed at the surface of the section. In this way it has been shown that virus particles completely surrounded by resin destain so much more slowly than chromatin, which is accessible at the surface of the section, that without any other information it would be concluded that the viruses contained RNA, not DNA. This apparently anomalous result arises because the difficulties encountered by stain or EDTA molecules in passing through a plastic section were not appreciated at the time of the initial pubheation of the technique. The observations are discussed in the light of recent knowledge that has been gained on the kinetics of staining by measuring the electron-scattering densities of stained sections, similar measurements having been made on sections stained by Bernhard’s technique. A model for the mechanism of the EDTA regressive staining technique consistent with the experimental observations is proposed and the conditions under which Bernhard’s staining procedure retains its specificity are defined. Briefly, these conditions are that: the sections be stained for only a short period with uranyl before treating them with EDTA even though such brief staining is undesirable for quantitative measurements of stain uptake into biological material; andthat sections stained in lead only be compared as controls with sections stained by Bernhard’s technique so that any specificity of lead for sub-cellular components is not confused with a positive indication of the presence of RNA.
Unless these conditions are fulfilled, results obtained by the use of the regressive staining technique may be highly misleading.
Qualitative and quantitative differences in spreading of human fibroblasts on various protein coats. modulation by treatment of the cells with amines
Authors: Cassiman J. J. , Van Der Schueren B. , Van Leuven F. , Van Den Berghe H. 
Published: April 2021

ABSTRACT
In contrast to established cell lines, normal human skin fibroblasts spread on their own fibronectin. The present investigation has examined whether human fibroblasts, like established cell lines, would be capable of spreading on substrata coated with proteins with different reactivity towards the cell surface. Coverslips were coated with human serum, fibronectin, α2macroglobulin–trypsin, a polyspecific anti-fibroblast antibody and a polyspecific anti-calfserum antibody. The attachment of the cells to these substrata was of the same extent. Spreading was examined qualitatively using phase, interference contrast and reflection contrast optics on live cells, as well as scanning electron microscopy on fixed cells. To quantitate the maximum degree of cell spreading a semi-automated system was used, which measured the cell perimeter on a large number of cells. The distributions of the degree of cell spreading on the five substrata were compared statistically. The qualitative and quantitative differences observed on the various substrata could be further differentiated by adding various amines to the cells during 60 min spreading or during a 30 min preincubation before spreading. No strict correlation could be found between the effect of the amines on attachment or on spreading and their presumed effects on cellular transglutaminases. The results clearly indicate that the spreading of human fibroblasts can be modulated by the nature of the substratum and that, by using quantitative methods, these differences in behaviour can be measured accurately.
Alveolar sacs of Tetrahymena: ultrastructural characteristics and similarities to subsurface cisterns of muscle and nerve
Authors: Satir Birgit H. , Wissig S. L. 
Published: April 2021

ABSTRACT
The ultrastructure of the layer of alveolar sacs of Tetrahymena thermophila and its relationship with the plasma membrane were examined in thin sections and freeze-fracture replicas.
In thin sections, the plasma membrane and outer alveolar membrane are seen to parallel each other closely and are linked by struts 9 nm wide spaced at 9 nm intervals. The plasma and alveolar membranes have different ultrastructural characteristics. The unit-membrane structure of the plasma membrane is asymmetric and thicker than that of the alveolar membrane. The unit-membrane structure of the alveolar membrane is symmetric. The lateral borders of adjacent alveolar sacs are closely apposed and flattened against one another, forming alveolar sutures. The pellicle at the cell surface, consisting of the plasma membrane, the alveolar sacs and the underlying epiplasm, overlies a single layer of mitochondria and a single layer of flattened cisterns of rough-surfaced endoplasmic reticulum (RER).
Examination of freeze-fracture replicas reveals that the plasma membrane and the inner and outer alveolar membranes each have a unique population of unit-membrane particles. This suggests that they are separate membrane domains. The appearance of alveolar sutures, i.e. the apposed lateral borders of adjacent alveolar sacs, suggests that the alveolar membranes in this region are more rigid than elsewhere and may be sealed together by an adhesive substance. Interruptions in the sutures are interpreted as indicating that adjacent alveolar sacs are connected by small isthmuses or channels, confirming earlier reports that alveolar sacs are large chambers within a continuous, interconnected system.
Alveolar sacs, in their relationship with the plasma membrane, closely resemble the subsurface cisterns of nerve and muscle cells. In muscle cells, the cisterns participate in the regulation of the cytoplasmic calcium ion concentration that controls contraction of myofilaments. Several functions of the surface of Tetrahymena, e.g. secretion of mucocysts and ciliary motility, are believed to be calcium-dependent, and the alveolar sacs may play a role in mechanisms that control the concentration of this divalent cation in localized regions along the cell surface.
The alveolar sacs contain laminated inclusion bodies that appear to be rich in phospholipid. The inclusion bodies may represent reserves of phospholipid material stored in preparation for membrane formation.
Effect of tubulin-binding agents on the infiltration of tumour cells into primary hepatocyte cultures
Authors: Roos Ed , Van de Pavert Ilja V. 
Published: April 2021

ABSTRACT
The influence of tubulin-binding agents on the infiltration of murine MB6A lymphosarcoma and TA3 mammary carcinoma cells into primary rat hepatocyte cultures was studied. Colchicin, nocodazole and vinblastin reduced the number of infiltrating lymphosarcoma cells, probably by interfering with the adhesion of these cells to the exposed hepatocyte surface. However, the subsequent infiltration of cells that did adhere was not affected or even slightly stimulated. The reduced adhesion appears to be due to an effect on both the MB6A cells and the hepatocytes.
In contrast, adhesion of TA3 cells was not reduced and infiltration was markedly enhanced by these agents, due to an effect on the TA3 cells but probably not on the hepatocytes.
These observations support previously described morphological evidence for differences between the infiltration mechanisms of the two tumour cell types. It is concluded that the system within the hepatocytes involved in adhesiveness of the exposed surface to MB6A cells is distinct from that mediating other types of adhesion. The tendency of TA3 cells to invaginate hepatocytes may be due to disturbances in tubulin-dependent processes.
A re-evaluation of the relationships between the fibrillar centres and the nucleolus-organizing regions in reticulated nucleoli: ultrastructural organization, number and distribution of the fibrillar centres in the nucleolus of the mouse sertoli cell
Authors: Mirre C. , Knibiehler B. 
Published: April 2021

ABSTRACT
In mouse testis, the diploid Sertoli cell displays one large nucleolus flanked symmetrically by two heterochromatic masses. The hybridization in situ with [3H]rRNA confirmed that the ribosomal cistrons are localized within the central nucleolar mass. At the ultrastructural level this nucleolar mass appears to be reticulated and contains numerous fibrillar centres. These fibrillar centres are surrounded and interconnected by an electron-opaque fibrillar network, which constitutes the reticulated nucleolonema of the nucleolus. Ag-NOR staining reveals the presence of the argyrophilic proteins associated with active nucleolus-organizing regions (NORs) within both the fibrillar centres and the electron-opaque fibrillar component. Autoradiographic studies after [‘H]uridine incorporation show that ribosomal DNA transcription only takes place in this dense fibrillar component. Three-dimensional reconstruction of four Sertoli cell nucleoli after serial sectioning reveals that the size and number of the fibrillar centres are very variable from one cell to another (26, 35, 38 and 41 fibrillar centres). The analysis of the volume occupied by the fibrillar centres as compared to the whole nucleolar volume demonstrates that the larger the nucleolus, the more fibrillar centres it contains, but also the more numerous the fibrillar centres, the larger their total volume. While in each case the number of the NORs is virtually the same, i.e. ten.
In the light of these results we concluded that, at least in reticulated nucleoli, there is no numerical relationship between the number of fibrillar centres and the number of NORs, and that the fibrillar centres cannot be considered only as the nucleolar counterparts of the NORs. Moreover, the increasing number of fibrillar centres from the smallest nucleolus to the largest one is difficult to explain by the previously postulated hypothesis of a reserve of inactive rDNA packaged in the fibrillar centres. These data led us to reconsider the role of the fibrillar centres in the transcriptional activity of reticulated nucleoli.
Structural changes in dividing sea-urchin eggs induced by the volatile anaesthetic halothane
Authors: Hinkley Robert E. , Chambers Edward L. 
Published: April 2021

ABSTRACT
Fertilized Lytechinus eggs exposed to the volatile anaesthetic halothane before metaphase do not undergo cytoplasmic cleavage. This effect has been correlated with the failure of the contractile ring to assemble. Comparative studies on mitotic apparatuses isolated from control and halothane-treated cells show that halothane significantly impairs both spindle and aster growth as early as metaphase. When transferred to control solutions, halothane-treated cells initiate furrowing activity in association with either the first or second mitotic division, depending on the duration of the exposure to anaesthetic and the concentration employed. In contrast to these effects, halothane has no effect on any aspect of the cleavage process if applied later than metaphase. In this case, furrows develop even in the presence of halothane, deepen progressively, and complete cell division. These observations confirm and extend previous studies on echinoderm eggs exposed to volatile anaesthetics, and support the view that anaesthetic agents indirectly prevent cell cleavage by inhibiting the growth of the mitotic apparatus.
Electron microscopy and x-ray microanalysis of calcium-binding sites on the plasma membrane of Beroe giant smooth muscle fibre
Authors: Nicaise G. , Hernandez-Nicaise M. L. , Malaval L. 
Published: April 2021

ABSTRACT
When they are fixed with glutaraldehyde in the presence of calcium ions, the plasma membranes of Beroe giant smooth muscle fibres display micropapillae filled with an electron-dense deposit. After freeze-fracturing of fixed tissue, the micropapillae are still present, therefore their shape and size are determined during or before glutaraldehyde fixation, and are not due to rearrangement during subsequent steps of tissue processing; intramembranous particles are seen at the periphery rather than at the top of micropapillae. In conventional stained sections, the surface of most micropapillae is surrounded by fine fibrils; when the fuzzy coat is separated from the muscle cell by a clear space, this fibrillar material becomes conspicuous and links the micropapillae to the coat. After calcium-free (EGTA) fixation, the plasma membrane is completely free of electron-dense sites but ‘empty’ micropapillae can be seen. X-ray microanalysis of single electron-dense deposits by wavelength-dispersive spectrometry reveals a high calcium content. A weak osmiophily is suspected, but does not seem to interfere with this analysis of calcium. The highest peak-to-background ratios for calcium were obtained using the smallest aperture of the Wehnelt of the analytical microscope.
In the Discussion, the micropapillae are compared to similar structures described by other authors in a variety of cell types.
The accessibility of certain proteins on embryonic chick neural retina cells to iodination and tryptic removal is altered by calcium
Authors: Cook James H. , Lilien Jack 
Published: April 2021

ABSTRACT
We have used cell-surface-specific labelling techniques and two-dimensional gel electrophoresis to identify proteins on embryonic chick neural retina cells and to determine the effects of Ca2+ on their accessibility to labelling and tryptic removal. A number of proteins on these cells are, in the presence of Ca2+, relatively inaccessible to iodination and/or tryptic removal. Of these, a glycoprotein of M, approx. 130 × 103, with a pl of approx. 4·8, is the major cellsurface-iodinatable species that is retained during trypsinization in the presence of Ca2+. The removal of Ca2+ renders this glycoprotein much more accessible to both procedures. Its accessibility to these probes decreases on re-addition of Ca2+. The accessibility of its oligosaccharide moiety to galactose oxidase is, however, unaltered by the removal of Ca2+. These characteristics, together with immunological data presented elsewhere suggest that this glycoprotein may be a component of the Ca2+-dependent adhesive system that can be demonstrated on these cells.
Proplastid dna synthesis at pachytene in pollen mother cells of Lilium henryi
Authors: Smyth D. R. 
Published: April 2021

ABSTRACT
About three-quarters of the DNA synthesis occurring in pachytene pollen mother cells of Lilium henryi takes place in proplastids. Only around 15 % can be attributed to mitochondrial labelling and 10% to nuclear DNA synthesis. Label was identified in the proplastid genome by its location in electron microscopic autoradiographs, by its buoyant density (1·698 g/ml), and by its specific hybridization to chloroplast DNA sequences from spinach. Proplastids, while apparently not dividing at pachytene, may be replicating their DNA in readiness for subsequent proliferation in developing microspores. The annealing properties of plastid DNA closely parallel those of labelled pachytene DNA sequences implicated in meiotic exchange events.
A survey of dna repair incision activities after ultraviolet irradiation of a range of human, hamster, and hamster-human hybrid cell lines
Authors: Collins Andrew , Jones Carol , Waldren Charles 
Published: April 2021

ABSTRACT
We describe a rapid and simple assay for DNA damage and repair in mammalian cells, based on the partial unwinding of nicked DNA in alkali, and involving transfer of this DNA to nitrocellulose and the digestion of single-stranded DNA with S, nuclease, all steps taking place on tissue-culture chamber slides. Direct breakage of DNA by ionizing radiation has been examined, and we have developed a standard procedure for measuring enzymic breakage of DNA as an index of excision-repair capacity following ultraviolet irradiation.
We report a wide range of repair capacities among various hamster and human cell lines, with considerable overlap between the two species. Hybrids between hamster and human cells tend to display repair activity characteristic of the hamster parent.
Nuclear transplant studies on the reduction in numbers of presumptive germ nuclei in excon jugants of Paramecium caudatum
Authors: Mikami Kazuyuki 
Published: April 2021

ABSTRACT
Nuclear differentiation in exconjugants of Paramecium caudatum is closely associated with a brief localization of the postzygotic nuclei near the opposite ends of the cell, with the germinal nucleus (micronucleus) in the anterior region and the somatic nuclei (macronuclei) in the posterior region. The posterior nuclei cannot regenerate to produce micronuclei when all four anterior nuclei are removed. There is no difference among the anterior four presumptive micronuclei, because, when any three of them were removed, the remaining nucleus was able to divide at each postconjugational fission and to persist as a micronucleus during the vegetative phase. This conclusion agrees with the results of transplanting a presumptive micronucleus into a vegetative cell. Cells during the vegetative phase, however, normally have only one micronucleus. Micronuclear number must be reduced to arrive at the uni-micronucleate condition after the stage of macro- and micronuclear differentiation. Elimination of supernumerary presumptive micronuclei, which had been indicated by morphological observations, was confirmed by the results of nuclear transplantation studies.
Infiltration of lymphosarcoma cells into hepatocyte cultures: inhibition by univalent antibodies against liver plasma membranes and lymphosarcoma cells
Authors: Middelkoop Otto P. , Roos Ed , Pavert Ilja V. Van De 
Published: April 2021

ABSTRACT
The number of murine MB6A lymphosarcoma cells that infiltrated rat hepatocyte cultures was found to be diminished after treatment of the lymphosarcoma cells with univalent antibodies raised against these tumour cells (anti-MB6A Fab), and also after treatment of the hepatocyte cultures with univalent antibodies directed against rat liver plasma membranes (anti-LPM Fab).
The inhibition of infiltration by anti-MB6A Fab and an anti-LPM Fab raised against sinusoidal face-enriched membranes could be entirely attributed to their interference with adhesion of MB6A cells to the exposed surface of the hepatocytes, because infiltration of the adherent cells was not inhibited. Anti-LPM Fab raised against contiguous face-containing LPM, on the other hand, inhibited the adhesion to the exposed surface and the subsequent infiltration of adherent cells. These observations suggest that specific membrane constituents of both MB6A cells and hepatocytes take part in liver infiltration, and that there may be two different hepatocyte components involved, one mediating adhesion to the exposed surface and the other taking part in the infiltration process proper.
Unrelated surface control of commitment to growth and attachment in isolated adult rat hepatocytes
Authors: Blanquet P. R. 
Published: April 2021

ABSTRACT
The relation between surface glycoconjugate molecules involved in the control of commitment to growth and attachment was investigated in adult Wistar rat hepatocytes in primary culture. On the one hand, the approach has been to analyse the binding of 3H-labelled con-canavalin A ([3H]Con A) to cell surfaces when these two cellular phemona were in turn modulated. The replacement of calf serum (CS) by foetal calf serum (FCS) in culture medium greatly enhanced cell attachment, its effect being about the same as that of FCS adsorbed to glass culture plates, but this change did not significantly affect the growth-stimulatory process. In contrast 2 or 3 μM-cytochalasin D (CD), which is known to act specifically on the inner face of the cell membrane, reversibly decreased the [*H]thymidine (dThd) incorporation in both CS-containing and FCS-containing media, but had only a very moderate effect on cell attachment. The reversible effect of the drug, 100, was followed by PH]leucine incorporation, as well as by measuring the [‘H]dThd incorporation in media with CD plus insulin and glucagon. Hepatocytes were assayed for binding either in buffered solution (at 4 or 37 °C) or in serum-containing media (at 37 °C). The Hill coefficients were calculated to be near 2·2 and 1·9, respectively for CS-stimulated and FCS-stimulated cells, and around 1·6 for CD-treated and glutaraldehyde-fixed cells. We interpret these results as showing that two different types of surface components might be involved in the control of commitment to growth and attachment. On the other hand, evidence was presented to indicate that these components really bind Con A. First, Con A treatment in buffered solution of cells previously attached in the presence of CS or FCS resulted in a progressive inhibition of EGTA-mediated cell detachment from substrata. Secondly, Con A reversibly decreased [3H]dThd incorporation in both CS-containing and FCS-containing media through a specific interaction with the cell surface. We conclude that entrance into S phase and attachment to serum protein substrata are unrelated phenomena in isolated adult rat hepatocytes and seem to be regulated by two species of surface glycoconjugateβ.
Cell-substrate contacts in cultured chick embryonic cells: an interference reflection study
Authors: Ireland Grenham W. , Stern Claudio D. 
Published: April 2021

ABSTRACT
Cell-substrate contacts in explants of different regions of early chick tissues were investigated using the technique of interference reflection microscopy. All the explants spread as epithelial sheets. During initial spreading a peripheral zone of 2–3 cells formed broad contacts with the substrate. In spread explants some cells in the centre made broad substrate contacts. A mat of extracellular material containing fibronectin was found under the explants. Focal contacts and focal adhesions increased in number during culture, and stress fibres were associated with them. These changes in cell contacts appeared more quickly in some tissues than in others. After 24 h, explants of hypoblast and definitive endoblast could easily be distinguished but by 7 days they were very similar. In the absence of serum, specialized cell contacts developed more quickly; in higher concentrations of serum, more slowly. Confrontations between explants were also examined. The most conspicuous feature was that cells in invading explants normally underlapped invaded cells. Invasion from above by an unspread explant could occur even if the invaded explant had formed many focal adhesions.