bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of ehrlich ascites tumour cells with triton x-100: I. Biochemical analysis
Authors: Traub Peter , Nelson W. James 
Published: April 2021

ABSTRACT
Ehrlich ascites tumour cells, propagated in suspension culture, were extracted with Triton X-100 under different ionic conditions to study the sensitivity of intermediate-sized filaments formed in vivo to changes in the ionic environment in vitro. The following results were obtained : (1) in solution of low ionic strength and in the absence of di- and polyvalent cations, vimentin was quantitatively solubilized and recovered from the postnuclear supernatant by precipitation with (NH4),SO4 (23% saturation) or spermidine (2 BIM). (2) When the cells were extracted in low ionic strength buffer in the presence of 4 mM-Mg-acetate or 1 · 2 mM-spermidine, vimentin was quantitatively incorporated into Triton X-100-resistant residual cell structures. It could be easily extracted from Mg2+-stabilized cell residues with Mg2+- and polyamine-free buffer of low salt concentration; spermidine-stabilized cell residues were very resistant to dissociation. (3) In a solution of high salt concentration and in the absence of di- and polyvalent cations, little or no vimentin was retained in residual cell structures. Better, though not quantitative, binding was observed in solution at physiological NaCl or KC1 concentrations. (4) A synergistic effect of mono- and di- or polyvalent cations on the association of vimentin with the nuclei was detected when the extraction of cells was performed at concentrations of these ions, which individually were very inefficient in the formation of residual cell structures. (5) Triton X-100-resistant cell residues formed under any ionic conditions were rather resistant to shearing forces. If release of vimentin occurred, it was due to perturbance of the dissociation equilibrium between bound and free vimentin by dilution or washing. (6) Preincubation of cells in buffers of varying ionic composition and in the absence of Triton X-100, followed by titration of the amount of vimentin still incorporable into residual cell structures by extraction of cells with Triton X-100 in solution of 4 mM-Mg2+, showed that the incorporation of vimentin into cell residues was strictly dependent on the intracellular ionic conditions. (7) The intracellular dissociation of vimentin-containing intermediate-sized filaments, induced by preswelling of cells in low ionic strength buffer, could be reversed, to a considerable extent, by restoration of isotonicity. If the incorporation of vimentin into Triton X-100-resistant residual cell structures is taken as a measure of the intactness of the vimentin-type intermediate-sized filaments, the experimental results demonstrate that the stability of the filaments is largely dependent on the maintenance of physiological concentrations of mono-, di- and polyvalent cations.
Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication
Authors: Autuori F. , Brunk U. , Peterson E. , Dallner G. 
Published: April 2021

ABSTRACT
Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6·9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.
Control of histone gene expression in Physarum polycephalum : I protein synthesis during the cell cycle
Authors: Schofield P. N. , Walker I. O. 
Published: April 2021

ABSTRACT
Synchronous cultures of Physarum polycephalum were pulsed with [3H] lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to 5 phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.
Epidermal cell migration on collagen and collagen-derived peptides
Authors: Donaldson Donald J. , Smith Gerald N. , Kang Andrew H. 
Published: April 2021

ABSTRACT
Nucleopore filters coated with various genetic types of collagen and certain collagen-derived peptides were implanted under one margin of a skin wound on adult Notophthalmus viridescens (newt) hind limbs. In contrast to their behaviour on untreated filters, epidermal cells migrated readily and to equal degrees on human type I, newt type I, bovine type II, and bovine type IV collagen. Denaturation had no effect on the ability of collagen to support migration and all three cyanogen bromide peptides tested (α1 (I)CB3, 7 and 8) were able to support more migration than that seen on untreated filters. Glutaraldehyde-linked collagen gels supported migration but bovine serum albumin gels did not.
These results show that there is no species or collagen-type specificity shown by newt epidermal cells as they migrate over collagen-coated substrates. They also demonstrate that the tertiary structure of the collagen molecule is unimportant in its ability to bind to newt epidermal cells, and that the α1 (I) chain has at least three, and probably many epidermal binding sites. Finally, they indicate that the improved migration on collagen is not a non-specific response to protein on the substrate.
Chromatin from the unicellular red algaPorphyridiumhas a nucleosome structure
Authors: Barnes K. L. , Craigie R. A. , Cattini P. A. , Cavalier-Smith T. 
Published: April 2021

ABSTRACTWe have isolated a crude nuclear preparation from the unicellular red alga Porphyridium aerugineum and investigated the structure of Porphyridium chromatin.Electrophoresis of deproteinized DNA fragments produced by micrococcal nuclease digestion of Porphyridium nuclei gives a typical ladder pattern, indicative of a repeating structure. The DNA repeat-length, calculated from plots of multimer length against multimer number, varies somewhat between different digestions, ranging from 160 to 180 base-pairs (average 173). We interpret this as evidence of heterogeneity in repeat-length; the calculated repeat-length depends on the extent of digestion because chromatin sub-populations with longer repeatlengths are on average digested earlier.Polyacrylamide / sodium dodecyl sulphate gel electrophoresis of basic proteins purified from Porphyridium nuclear preparations gives a pattern characteristic of core histones. Although our interpretation is complicated by some degradation, the result strongly suggests that Porphyridium chromatin contains each of the four core histones and that they are similar to those of higher eukaryotes. This, together with the micrococcal nuclease digestion results, demonstrates that Porphyridium chromatin is not fundamentally different from that of higher eukaryotes.
Ultrastructural and immunocytochemical demonstration of gametophytic proteins in the pollen tube wall of the primitive gymnosperm Cycas
Authors: Pettitt J. M. 
Published: April 2021

ABSTRACT
Ripe pollen of the primitive gymnosperm Cycas armstrongii germinates 8–15 days after sowing on a suitable artificial medium. During the pre-germination interval, the tube cell of the tricellular male gametophyte is actively engaged in the synthesis and export of polysaccharide material. This polysaccharide is laid down as a continuous microfibrillar stratum between the tube cell plasmalemma and the pollen wall intine. While the polysaccharide layer is forming, numerous evaginations arise as cytoplasmic extensions from the tube cell and penetrate the component. The structural link between the cell and the evaginations is eventually lost and the isolated fragments of cytoplasm immediately break up to form discrete particles. These particles remain within the channels previously occupied by the evaginations.
At germination, the exine and intine layers of the pollen wall are disrupted and the tube cell emerges through the germinal aperture to establish the pollen tube. Polysaccharide deposition is resumed at about this stage, and consequently, a second microfibrillar layer begins to form beneath the first. The wall of the early pollen tube, therefore, has two distinct zones: the outer stratum is a loose superficial reticulum of fibrils and contains cytoplasmic inclusions; the inner stratum is structurally compact and is without inclusions.
Throughout the period of tube development, the presence of protein, including the enzyme acid phosphatase, can be demonstrated in the outer wall zone with cytochemical methods. Immunofluorescent staining using pollen antiserum immunoglobulin G in an indirect technique with fluorescein isothyocyanate-labelled secondary antibody shows that one component in the pollen tube wall is immunologically related to the male gametophyte, and results from immunoelectron microscopy support the conclusion that this consists of the inclusions rather than the polysaccharide fabric of the tube wall itself.
There are remarkable similarities between the cytoplasmic inclusions in the tube wall in Cycas and those found in the intine layer of flowering plant pollen. Both are of haplophase derivation, although the inclusions in the flowering plants are introduced via evaginations into the intine, the incipient pollen tube wall, at a much earlier stage in pollen development than they are in Cycas. The difference in the time of incorporation represents, perhaps, one more episode in the progressive evolutionary trend towards rapid pollen tube initiation and growth, and hence rapid fertilization, in spermatophyte pollen. This possibility is discussed, taking into account the view that the cycads and flowering plants are groups with a common ancestor.
Nucleic acid synthesis in microsporocytes of Lilium cv. cinnabar: events in the nucleus
Authors: Porter E. K. , Bird M.J. , Dickinson H. G. 
Published: April 2021

ABSTRACT
In an electron microscopic autoradiographic study of DNA and RNA synthesis during meiosis isolated Lilium microsporocytes were supplied with [3H]thymidine and [3H]uridine. DNA synthesis occurred in the nucleus during the zygotene and pachytene intervals of meiotic prophase. Most of the activity was associated with the chromatin, but some synthesis early in zygotene was located at the nucleolus. RNA synthesis occurred throughout prophase until diplotene, when all activity ceased until after division. The newly synthesized RNA was found mostly in association with the chromosomal peripheries or in the space between chromosomes. There was also a peak of [3 H] uridine incorporation at the nucleolus, which followed shortly after the synthesis of DNA at that site. The localization of DNA and RNA synthesis at the various stages of meiosis is discussed in relation to current concepts of chromosome pairing, crossing-over, ribosomal DNA amplification and cycles of RNA metabolism.
Cell-cycle kinetics and ultraviolet light survival in uv-1, a chinese hamster ovary cell mutant defective in post-replication recovery
Authors: Collins Andrew , Waldren Charles 
Published: April 2021

ABSTRACT
UV-1, an ultraviolet-sensitive mutant of CHO-K1, is abnormally slow to recover from the inhibition of DNA synthesis caused by u.v. irradiation. When synchronized UV-1 cells are irradiated in G1 their movement into S phase is unaltered, but thymidine incorporation is depressed (compared with that in the parent cell similarly treated). When irradiated in S phase, again incorporation is more depressed, and S phase suffers a greater delay in UV-1 than in the parent cell. UV-1 and its parent have similar capacities for excision repair of u.v.-induced damage inflicted in G 1 and so enter S phase with similar amounts of unrepaired damage. The single-cell survival was measured after irradiation at different points in the cell cycle. The mutant and parent cells have similar values of D0 (mean lethal dose) except in mitosis, when the parent cell shows markedly greater resistance to u.v. irradiation. Dq (quasi-threshold dose) is fairly constant for the parent cell, but in UV-1 it falls to a minimum in S phase. The responses of UV-1 to u.v. irradiation are generally consistent with its known defect in the process of post-replication recovery, i.e. the ability to join up the abnormally small DNA fragments synthesized on a u.v.-damaged template.
Nucleolar organizer structure and activity in a nucleolus without fibrillar centres: the nucleolus in an established Drosophila cell line
Authors: Knibiehler B. , Mirre C. , Rosset R. 
Published: April 2021

ABSTRACT
Classical electron-microscopic techniques (enzymic digestion, EDTA regressive staining) allied with autoradiographic studies after [3H]uridine incorporation or after RNA synthesis initiated by an exogeneous RNA polymerase in the presence of tritiated GTP, enabled us to describe the fine structure and activity of the nucleolus in an established Drosophila cell line. This nucleolus is composed of a large central multilobed core containing proteins, RNA molecules and a DNA-containing component. This core is surrounded by and connected to large clumps of dense fibrillar nucleolus-associated chromatin, which are intermingled with fibrillogranular ramifications extending from the core towards the nuclear envelope. These ramifications are covered by granules of ribosomal ribonucleoprotein. As shown by EDTA regressive staining the nucleolar core contains a ribonucleoprotein network, which unravels and ramifies within a fibrous matrix. RNA synthesis takes place at the level of this network in the internal part of the core. The molecules synthesized are associated with proteins and are exported out of the core in the form of granules. Although it is composed of the same constituents as other nucleoli, the nucleolus of Drosophila cells seems to be less organized, in that it never displays fibrillar centres, which have been referred to as the nucleolar counterparts of the nucleolus-organizers in a wide variety of organisms.
The cytoskeleton and substratum adhesion in chick embryonic corneal epithelial cells
Authors: Billig D. , Nicol A. , Mcginty R. , Cowin P. , Morgan J. , Garrod D. 
Published: April 2021

ABSTRACTThe adhesions and cytoskeleton of 15-day chick embryonic corneal epithelial cells have been studied using interference reflection microscopy (IRM), electron microscopy and fluorescent staining with specific antibodies against actin, tubulin, prekeratin, fibronectin and laminin In vivo, desmosomes were the most frequent intercellular junctions and hemidesmosomes were prominent at the basal surface of the epithelium in contact with the basement membrane. The intact epithelium stained for actin, tubulin, and prekeratin and laminin, but not fibronectin, and the basement membrane for fibronectin and laminin.In monolayer culture intercellular desmosomes were reformed after dissociation with trypsin. However, we have concentrated on cell-substratum adhesions, because the relationship between substratum adhesions and the cytoskeleton has not been thoroughly investigated for epithelial cells. Most of our results apply to contacts with gelatin substrata but the situation was similar on glass or tissue-culture plastic. By IRM, focal contacts similar to those of fibroblasts were present, mainly beneath the leading lamellae of the peripheral cells of monolayered islands. Fluorescent antibody staining revealed that each focal contact was positioned at the end of an actin microfilament bundle. However, there was no correspondence between focal contacts and either prekeratin filaments or microtubules.Fibronectin fibrils were found principally beneath peripheral cells, but evidence is presented suggesting that fibronectin is not directly involved in cell-substratum adhesion of these cells. We suggest that the fibrillar fibronectin pattern arises because cells physically reorganize fibronectin which adsorbs or binds to the substratum from the medium.Electron microscopy suggested that these cells formed two types of contacts with the substratum : small dense plaque-like structures, probably hemidesmosomes, were present in addition to focal contacts. The former were associated with tonofilaments and were the same size (about 0 · 1 μ m in diameter) as the desmosomes and hemidesmosomes found in vivo. They were much smaller than focal contacts which were from one to several micrometers in length. These structures were not as well formed as the hemidesmosomes found in vivo, possibly because the substratum was not entirely appropriate. The significance of the two types of cell-substratum adhesions is discussed.
Ultrastructural changes in the surface layers of the newt’s egg in relation to the mechanism of its cleavage
Authors: Selman G. G. , Perry M. M. 
Published: April 2021

ABSTRACT
The surface and cortical layers of an uncleaved newt egg have a characteristic ultrastructure which remains unaltered during cleavage; ultrastructural changes are confined to the region of the furrow. At the onset of cleavage there is a dipping inwards of the rough heavily pigmented animal surface to form a groove. Along the bottom of the groove the surface irregularities are reduced and a dense band (0·1 μm thick and 16μm wide) is formed immediately below the plasma membrane. Within this band there are parallel filaments, 8–10 nm in diameter, oriented in the direction of the future furrow. No structural modifications were observed below the cortical layers of the leading part of the furrow apart from accumulations of granules and the mid-bodies of the spindle remnant. It is proposed that the dipping-in of the groove is due to contraction within the filamentous band, rather than contraction in a sheet of subcortical gel as proposed previously. The filamentous band persists below the furrow during the later stages of cleavage.
The new unpigmented surface first forms as a strip across the animal surface and begins to grow at the bottom of the groove. Over most of its area, it is much smoother than the pigmented surface and has less material on the outside of the plasma membrane. There are microvilli along the bottom of the groove. The join between the new unpigmented and the old pigmented surface is abrupt. As the new unpigmented surface grows in extent, a narrow furrow forms below the lowest part of the groove and progresses towards the vegetal surface. For most of its length the furrow is between 10 nm and 0·5μm wide, but at its leading edge it is 2μm wide with microvilli on its surface and 10-nm filaments below the plasma membrane. It is concluded that the progressive formation of the furrow is due to active growth of new unpigmented cell surface.
At late cleavage a ridge 10μm high forms at the join between the new and old surface. After cleavage the ridges approach and meet to form the intercellular junction by which daughter blastomeres are held together along the animal surface.
The mechanism of cell cleavage in the newt egg and in other forms is discussed in the light of the present observations.
The three-dimensional arrangement of intergranal lamellae in chloroplasts
Authors: Paolillo D. J. 
Published: April 2021

ABSTRACT
The intergranal lamellae, or frets, are helically arranged around each granum. All helices within a plastid are co-directional. The helices wind in the same direction from plastid to plastid, from cell to cell, and from species to species, and are right-handed. The continuity of the intergranal membranes in 3-dimensional space is facilitated by the fact that all the helices within a plastid wind in the same direction. Because there are multiple frets around each granum, the fretwork consists of a highly interlocked series of membranes. The incompleteness of the integration of the fretwork suggests that continuity of the membranes from granum to granum is obtained, to a large degree, by fusions of membranes rather than by continuous growth and ramification of a simpler membrane system.
The formation of filopodium-like processes by Trypanosoma (Trypanozoon) brucei
Authors: Wright K. A. , Lumsden W. H. R. , Hales H. 
Published: April 2021

ABSTRACT
Bloodstream forms of Trypanosoma (Trypanozoon) brucei were observed in vitro to possess long filaments arising from both the posterior end and the tip of the flagellum, and also to become attached to various substrates, including themselves. The filaments were examined by negative staining and thin-section techniques and found to have a structure resembling filo podia. However, as their functional significance is still questionable, their designation as filopodia must be considered provisional. The possible functions of these structures, such as mediation of attachment, and release of antigenic material, are discussed.
Cytochemical localization of transferase activities: carnitine acetyltransferase
Authors: Higgins Joan A. , Barrnett R. J. 
Published: April 2021

ABSTRACT
Two methods for the cytochemical detection of free CoA and their utilization in the fine-structural localization of carnitine acetyltransferase in rat heart are described. The first utilizes the reducing property of the SH group of CoA to reduce potassium ferricyanide to potassium ferrocyanide, which in the presence of uranyl ions forms an electron-dense precipitate of uranyl ferrocyanide. The second utilizes the mercaptide-forming property of the free SH group of CoA, which forms a precipitate with cadmiumions.
Using the uranyl-ferrocyanide method, reaction product due to endogenous enzymic activity was found on and between the cristae and between the inner and outer membranes of the mito- chondria in fresh heart muscle. In aldehyde-fixed tissue activity was recorded only between the inner and outer membranes. Endogenous activity was removed by preincubation of the tissue in a solution of ferricyanide. On addition of acetyl CoA and carnitine to the incubation medium, fresh tissue, which had been preincubated in ferricyanide, showed reaction product between and on the cristae and between the inner and outer membranes of the mitochondria, while fixed tissue showed reaction product in the latter position only. In both cases the activity between the outer and inner mitochondrial membranes was dependent on both acetyl CoA and carnitine, while the cristae reaction occurred in the absence of carnitine, but required acetyl CoA. All activity was inhibited by mercuric chloride. Acetyl carnitine reduced the activity in the fixed tissue and had severe effects on the structure of fresh mitochondria. These results suggest the presence of carnitine acetyltransferase, which survives aldehyde fixation, on the inner surface of the outer mitochondrial membrane and/or the outer surface of the inner mitochondrial mem- brane. A second enzyme which released CoA from acetyl CoA occurred in relation to the cristae of unfixed mitochondria.
The cadmium method was less satisfactory than the uranyl-ferrocyanide method but with fixed tissue gave confirmatory results.