bluuebunny/crossref_metadata_2025_split huggingface dataset:
Coated vesicles from chicken liver bind ferritin
Authors: Passaniti Antonino , Roth Thomas F. 
Published: April 2021

ABSTRACT
The iron-storage protein ferritin was found to be associated with highly purified coated vesicles (CV) from chicken liver. Chicken liver ferritin was morphologically similar to ferritin from horse spleen and could be isolated using a specific anti-ferritin monoclonal antibody. This antibody recognized a 240 ×103Mr form of chicken ferritin but not the 22 ×103Mr ferritin subunit after protein transfer to nitrocellulose. CV purified by controlled-pore glass-bead chromatography also contained ferritin when assayed by monoclonal anti-ferritin antibody using a sensitive enzyme-linked assay. Ferritin remained associated with CV even after re-chromatography. Ferritin particles were observed to be associated with CV by electron microscopy. CV-associated ferritin could be quantitatively removed from CV by treatment of the CV with 0 · 5M-Tris-HCl + 2 M-urea at pH 8 · 5, conditions that also lead to dissociation of the clathrin lattice. Triton X-100 detergent treatment did not affect the association of ferritin with CV. These results indicate that purified CV from chicken liver contain ferritin in association with the clathrin lattice. The possible functional significance of this association is discussed.
Uneven distribution of surface antigens during antigenic variation in Paramecium primaurelia
Authors: Antony Claude , Capdeville Yvonne 
Published: April 2021

ABSTRACT
In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface distribution of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly than the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidie nature of the SAg membrane anchor and to a possible role of an endogenous Paramecium phosphatidylinositol phospholipase C.
A serum factor inducing neurite retraction of morphologically differentiated neuroblastoma × glioma NG108-15 cells
Authors: Ghahary A. , McPherson P. S. , Cheng K. W. 
Published: April 2021

ABSTRACT
It is well established that serum plays an important role in cell proliferation and differentiation. In this study, we have identified a serum factor that induces rapid neurite retraction of morphologically differentiated NG108-15 cells, cultured in serum-free medium containing 1 mM-dibutyryl cyclic AMP. The serum fraction of Mr &gt; 30×103 induces neurite retraction in a manner identical to that of the whole serum. The neurite retraction activity in serum appears to be acid- and heat-stable. The molecular weight of the serum neurite retraction factor (NRF) has been demonstrated to be approximately 70×103 by gel permeation on LKB-Ultrogel AcA-44. The neurite retraction activity is dosedependent, and the time required for half-maximal activity (t2) is 1-8 min. NRF is present in sera of various species studied, including human, cattle, sheep, rabbit and horse, but not in tissue extracts of kidney, heart, lung, skeletal muscle, and brain of the rat. However, rat spleen and liver homogenates, at a protein content of 1 mg ml−1, caused slight neurite retraction. It is noteworthy that NRF is not detectable in cerebral spinal fluid. Our data on the properties of serum NRF indicate that it differs from all of the well-established growth factors, namely, NGF, EGF, PDGF, FGF, NSILA, ECGF and TGF. Further studies on purified NRF will delineate the biological role(s) of this serum factor in the process of maturation and differentiation of developing neurones.
Glycosaminoglycans facilitate the movement of fibroblasts through three-dimensional collagen matrices
Authors: Docherty R. , Forrester J. V. , Lackie J. M. , Gregory D. W. 
Published: April 2021

ABSTRACT
The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.
Kinetochore size variation in mammalian chromosomes: An image analysis study with evolutionary implications
Authors: Cherry Lorraine M. , Faulkner Alexander J. , Grossberg Leigh Ann , Balczon Ronald 
Published: April 2021

ABSTRACT
The kinetochore, a proteinaceous plate that is the site for attachment of spindle microtubules to the metaphase chromosome, can be visualized using anti-kinetochore indirect immunofluorescence. We have used computer-assisted image analysis to measure the variation of kinetochore surface areas, as reflected by immunofluorescence areas, in cell lines derived from rat kangaroo, Chinese hamster and common rat, to determine if our size estimates correlate well with those obtained using measurements from electron micrographs. In addition, we used male and female human fibroblast cell lines, as well as a transformed human female cell line (HeLa), to examine kinetochore size variation among cells, between sexes, and between cell lines. We found that our system gave reproducible estimates of kinetochore size, and that these sizes correlated very well (r=0 ·95) with the electron micrograph measurements. In examining variation within humans, we observed measurable differences between cell lines. Despite this difference, all the human lines had size distributions that were leptokurtotic and positively skewed. The fact that very few chromosomes exhibited areas smaller than the mode gives support to the idea that mammalian chromosomes may require a specific, minimum amount of kinetochore material in order to maintain stable attachment to the mitotic spindle. On the other hand, the positive skewness seems to indicate that larger kinetochores, possibly the result of events such as Robertsonian fusions, are fully functional. The retention of this plasticity may allow the chromosomes to maintain an evolutionary adaptability that might otherwise be lost.
The transient division block in the D9ts mutant of Tetrahymena is inducible at every cell cycle stage
Authors: Cleffmann Günter 
Published: April 2021

ABSTRACT
The temperature-sensitive mutant D9 of Tetrahymena thermophila doubles its size at restrictive temperature. It does so by complete cessation of cell division for a limited time. After resumption of proliferation, division rate and specific growth rate are the same as at the permissive temperature, thereby maintaining the new cell size. In this study a detailed analysis of the process of controlling the new cell size is presented, by probing the temperature sensitivity of cell cycle phases. It will be shown that high temperature affects the size-controlling system immediately upon shift in temperature. Temperature pulses are effective at every stage in the cycle and are executed at the time of expected division. After return to the permissive temperature, cells gradually recover from the temperature pulse as seen by a decrease in division delay. Preparation for the next division is unaffected by the temperature pulse. It occurs at the same time as in untreated controls. The results allow us to describe some features of the division initiating system.
A monoclonal antibody raised against cytoplasmic fibrillar bundles from carrot cells, and its cross-reaction with animal intermediate filaments
Authors: Hargreaves Alan J. , Dawson Peter J. , Butcher Geoffrey W. , Larkins Audrey , Goodbody Kim C. , Lloyd Clive W. 
Published: April 2021

ABSTRACT
Carrot suspension cells contain cytoplasmic bundles of fibrils that are distinct from F-actin and microtubules and have some of the characteristics of intermediate filaments. In characterizing these fibrillar bundles further, we have raised a monoclonal antibody against them. This anti-fibrillar bundle antibody (AFB) immunoblots vimentin from a range of animal cells and tissues, as well as glial fibrillary acidic protein in brain and desmin in BHK fibroblasts, which are representatives of the type III intermediate filaments.
Immunofluorescence staining of PtK2 cells indicates that AFB recognizes a network co-distributing with cytoplasmic microtubules. When this co-alignment is disturbed with the anti-microtubule agent, colcemid, the AFB staining segregates with the collapsed perinuclear whorls of vimentin.
In carrot, AFB immunoblots the major bundle proteins but not plant tubulin. In plant as in animal cells, AFB immunofluorescently labels antigens that co-distribute with microtubules. In onion cells (which, unlike carrot, do not possess paracrystalline arrays of fibrils) AFB labels all four microtubule arrays throughout the cell cycle. The antigens do not, however, collapse around the spindle poles during mitosis.
Double immunofluorescence, using anti-dog brain tubulin, indicates that the FB antigen is more diffusely distributed than tubulin; it is patchy and co-alignment is not exact, particularly during early preprophase band formation.
Antigens in detergent-insoluble fibrils of carrot cells therefore exist both in animal type III intermediate filaments (IF), and in a more dispersed, microtubule-associated manner in onion meristematic cells. This constitutes an independent line of evidence for the existence of IF antigens in plants.
Retention of ciliary ninefold structure after removal of microtubules
Authors: Stephens R. E. , Oleszko-Szuts S. , Linck R. W. 
Published: April 2021

ABSTRACT
When axonemes of isolated gill cilia from the bay scallop Aequipecten irradians are heated at 45°C for a minimum of 8 min in a 10mM-Tris-HCl (pH 8), ImM-EDTA solution, nearly 80% of the tubulin is solubilized but most minor structural proteins are retained in a ninefold symmetrical configuration. This remnant consists of the junctional protofilaments, derived from outer doublet tubules, interconnected by nexin linkages, with radial spoke components still directed inwards. The remnant is of the same length as the original cilium, with the junctional protofilaments attached at the distal end to the ciliary tip and at the proximal end to the basal plate. Virtually identical fractionations can be achieved with blastula cilia isolated from both arctic and tropical sea-urchin embryos. The remnant is resistant to salt up to at least 1M concentration, judged by the constancy of protein composition. Immunoblotting with antibodies against sea-urchin sperm flagellar tektins indicates that the tektins remain within the ciliary remnant, supporting their location within the junctional protofilament domain. The fractionation is inhibited by low pH, by magnesium or calcium ions in the millimolar range, and by monovalent ions at 10-fold higher concentrations. About a quarter of the total ciliary calmodulin is bound to the axoneme at micromolar calcium levels but most is released upon thermal fractionation. Polymerization of tubulin in the presence of the remnant results in singlet microtubules, separate from the remnant proper, suggesting that doublet formation may require coordinate co-assembly of tubulin with skeletal proteins. These observations demonstrate the existence of a fibrous skeleton in the axoneme, composed largely of ciliary tektins, nexin linkages, and other structural proteins.
Properties of microtubule-free cortical residues isolated fromParamecium tetraurelia
Authors: Williams Norman E. , Honts Jerry E. , Stuart Kathleen R. 
Published: April 2021

ABSTRACTWe have found that shape-preserving residues devoid of microtubules can be prepared from Paramecium using Triton X-100 at high ionic strength. These residues contain many proteins, including one showing antigenic relationship to chicken a-spectrin, and three showing antigenic relationship to Tetrahymena cortical proteins. These antigens have been localized by immunofluorescence microscopy, and the isolated cortical residues have been characterized ultrastructurally. These preparations should be useful in detailed studies of the structure, development and evolutionary relationships of cortical architecture in ciliated protozoa.
Identification and localization of major cortical proteins in the ciliated protozoan, Euplotes eurystomus
Authors: Williams Norman E. , Honts Jerry E. , Lu Qun , Olson Cheryl L. , Moore Kenneth C. 
Published: April 2021

ABSTRACT
Shape-preserving cortical residues have been isolated from Euplotes eurystomus cells by the application of Triton X-100 at high ionic strength. These integrated structures consist of articulated plates and widely interspersed cages that formerly contained basal bodies associated with the clusters of cilia characteristic of this cell type. Using SDS-PAGE and immunolocalization procedures, we have identified major subunit proteins of both the plates (116, 110 (×103)Mr, and the basal body cages (86× 103Mr). The potential for studies of these proteins in contributing to our understanding of cortical development and evolution in Euplotes is discussed.
Effect of hypoxia upon the growth and sprouting activity of cultured aortic endothelium from the rat
Authors: Smith Paul 
Published: April 2021

ABSTRACT
Endothelial cells were obtained from the aortas of Wistar rats by collagenase digestion. Cells were grown to confluence in medium 199 enriched with L-glutamine but without specific growth factors. Cells were subcultured into 35 mm dishes or 25 cm2 flasks coated with fibronectin. For cell growth studies, cells were seeded onto multiwell plates or 35 mm dishes. In two experiments the cells were grown in an hypoxic atmosphere of 5·3% O2 and in a third the level of oxygen was 2·5%. Control cultures for each experiment were grown in 5% CO2 and air. Cell populations were counted at 2-day intervals and at the termination of each experiment the cells were fixed and the area of each plate or flask occupied by sprouting cells was assessed by point counting.
Endothelial cells grown in 5·3% O2 grew more rapidly and attained confluence earlier than in the controls. An atmosphere of 2·5% O2 did not accelerate growth but neither did it inhibit it, so after 9 days there were as many hypoxic cells as there were controls. Hypoxia also stimulated sprouting activity to occur earlier and to become much more extensive than in control cultures. Under the influence of hypoxia, sprouting consisted of complex anastomotic or arborizing patterns forming syncytium-like masses beneath the monolayer of oval cells. This process appeared to originate from foci of altered endothelial cells that had become retracted, smaller, elongated and migratory, and which displayed increased immunoreactivity for factor VIII antigen. It was concluded that a level of hypoxia, similar to that in systemic veins, stimulates growth of arterial endothelium and provokes enhanced sprouting activity. This behaviour may be relevant to the process of vascularization of wounds or neoplasms.
Differences in the exposure of c- and n-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies
Authors: Dráber Pavel , Dráberová Eduarda , Linhartová Irena , Viklický Vladimír 
Published: April 2021

ABSTRACT
A panel of 11 monoclonal antibodies specific to α- or β-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated a-tubulin revealed that a set of antibodies against the N-terminal domain of the α-subunit (TU-01, TU-02, TU-03, TU-09, 6-11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.
NACM, A cytopathogen from naegleria ameba: purification, production of monoclonal antibody, and immunoreactive material in NACM-treated vertebrate cell cultures
Authors: Dunnebacke T. H. , Dixon J. S. 
Published: April 2021

ABSTRACT
An amebic component NACM (Naegleria ameba cytopathogenic material) is obtained from free-living amebae and acts as a cytopathogen in cultured avian and mammalian cells. NACM has been purified and partially characterized using a scheme that includes ammonium sulfate precipitation, liquid chromatographic separations and electrophoretic isolations. The purified NACM product is biologically active. Its properties, revealed by its behavior following treatment with enzymes and during purification, are those of a protein; its size is in the molecular weight range of 36 000 and it has an isoelectric point of pH 4·2. Monoclonal antibodies have been produced to NACM that prevent its cytopathic activity, and, in dot-blot procedures, identify purified NACM-containing fractions. When used as immunostains, NACM antibodies disclose the presence of immunospecific material both in the amebae and in vertebrate cells inoculated with NACM. In the amebae, the immunostain is located at the tips of the pseudopodia and in the peripheral cytoplasm; in the vertebrate cells, it is absent in uninoculated cells and is not observed in the inoculated cells for the first 3–4 days after the addition of the NACM to the culture. Subsequently, in a time course associated with the development of cytopathic changes observed in the light microscope, immunostaining material develops in the perinuclear cytoplasm where it condenses into a large mass before the cells undergo lysis. On the basis of these results, it appears that NACM represents an unprecedented system in which a protein material from an organism, in the course of destroying unrelated cells, causes the production of a cytoplasmic product that is immunologically recognizable as the inoculating amebic material.
Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles
Authors: Philpott Michael , Green Martin R. , Kealey Terence 
Published: April 2021

ABSTRACT
We have already shown that shearing can be used to yield large numbers of viable intact hair follicles. We now show that these follicles can be viably maintained on permeable supports for 7 days in vitro as determined by their adenine nucleotide contents, rates of [mefhyl-3Hlthymidine and [U-14C]leucine uptake, [methyl-H]thymidine autoradiography, patterns of keratin synthesis and light and electron microscopy. These studies, however, show that after 7 days maintenance the morphology of maintained follicles shows a closer resemblance to the telogen rather than the anagen follicle. We therefore conclude that the failure of previous attempts at maintaining hair growth in culture is due to hair follicles prematurely entering the resting stage of their hair growth cycle, possibly as a response to isolation.
On the distribution of microtubule-associated intermediate filament antigens in plant suspension cells
Authors: Goodbody Kim C. , Hargreaves Alan J. , Lloyd Clive W. 
Published: April 2021

ABSTRACT
Intermediate filament antigens are known to coalign, patchily, with cortical microtubules in plant cells, but nothing else is established about this relationship or the form the antigens take. This was studied further using cell suspensions, instead of root tip cells, since their cortex is accessible to antibodies without the rigours of cell separation. Both carrot and sweetcorn suspension cells were labelled with three antibodies known to recognize animal intermediate filaments. The mitotic and cytokinetic apparatus could be stained with these antibodies, which, in interphase cells, also labelled cortical microtubule-like arrays. One antibody (AFB), raised against cytoplasmic bundles of 7nm fibrils from carrot cells, immunostained the bundles but only under conditions of fixation that did not allow the finer, microtubule-associated staining pattern to be seen. By exploring various preparatory conditions it was concluded that these two forms of antigen co-exist: they are not experimentally interconvertible but require different conditions for exposure to different antibodies.
Double immunofluorescence established that the intermediate filament antigens do not parallel the actin network, nor did cytochalasin D affect their distribution. Taxol, however, bundled the intermediate filament antigens, whereas they are dispersed when microtubules are depolymerized, rather than collapsing in perinuclear whorls.
Under conditions permitting the microtubule-associated antigens to be stained by immunofluorescence, carrot protoplasts were cleaved on grids, exposing the cortical microtubules. Immunogold labelling then showed that the antibody raised against fibrillar bundles recognizes patches of electron-dense material, along and between the microtubules, rather than individual filaments. To confirm that the plant antigens are capable of forming filaments, a high salt, detergent-insoluble fraction was prepared from the maize line. By dialysing from urea, intermediate-sized filaments could be reconstituted and they immunoblotted with the broadly cross-reactive antibody to intermediate filament antigens (anti-IFA).
These studies underline the problems of visualizing in plants, conformations of intermediate filament antigens that are not directly comparable to the extensively studied animal models, and that appear to be sensitive to the way in which cells are manipulated.