bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells
Authors: Humbert C. , Santisteban M. S. , Usson Y. , Robert-Nicoud M. 
Published: April 2021

ABSTRACT
The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCI followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.
The pebble gene is required for cytokinesis in Drosophila
Authors: Lehner Christian F. 
Published: April 2021

ABSTRACT
Cytokinesis is developmentally controlled during Drosophila embryogenesis. It is omitted during the initial nuclear division cycles. The nuclei of the resulting syncytium are then cellularized at a defined stage, and cytokinesis starts in somatic cells with mitosis 14. However, cytokinesis never occurs in somatic cells of embryos homozygous or transheterozygous for mutations in the pebble gene. Interestingly, the process of cellularization, which involves steps mechanistically similar to cytokinesis, is not affected. Moreover, all the nuclear aspects of mitosis (nuclear envelope breakdown, chromosome condensation, spindle assembly and function) proceed normally in pebble mutant embryos, indicating that pebble is specifically required for the coordination of mitotic spindle and contractile ring functions.
The pebble phenotype is also observed, but only with very low penetrance, during the early divisions of the germ line progenitors (the pole cells). -Amanitin injection experiments indicate that these early pole cell divisions, the first cell divisions during embryogenesis, do not require zygotic gene expression. These divisions might therefore rely on maternally contributed pebble function. The maternal contribution from heterozygous mothers might be insufficient in rare cases for all the pole cell divisions.
Control of division plane in normal and griseofulvin-treated microsporocytes of Magnolia
Authors: Brown Roy C. , Lemmon Betty E. 
Published: April 2021

ABSTRACT
Meiotic cytokinesis in microsporocytes of Magnolia is an unusual form of the simultaneous type; phragmoplast expansion is not accompanied by a cell plate, wall deposition is centripetal, and infurrowing of the cytoplasm after first meiosis results in semicells connected by an isthmus. Dyad domains are further defined by interaction of extensive radial systems of microtubules emanating from the daughter nuclei and by a band of organelles polarized in the equatorial region. After second meiosis, phragmoplasts are organized in the interzonal regions between the sister nuclei in each semicell and also at the interfaces of microtubules forming secondary interzonals between non-sister nuclei. Wall deposition is not initiated until after phragmoplasts expand to the cell periphery and fuse in the isthmus. Centripetal wall deposition in boundaries of spore domains marked by radial arrays of microtubules results in simultaneous quadripartitioning of the microsporocyte into a tetrad of microspores. Treatment of microsporocytes with griseofulvin resulted in atypically placed nuclei and supernumerary nuclei. Abnormalities could be traced to displaced spindles and to spindles with multiple poles. Drug-induced multinucleate coenocytes were able to organize microtubules and initiate cytokinesis in altered patterns. The data suggest that spindle alignment and aggregation of spindle poles are two components of spatial control that are operative in determining the normal arrangement of nuclei, and that the final placement of walls is a function of the postmeiotic nuclear-based radial arrays of microtubules which define spore domains.
Very low cadmium concentrations stimulate DNA synthesis and cell growth
Authors: Zglinicki T. Von , Edwall C. , Östlund E. , Lind B. , Nordberg M. , Ringertz N. R. , Wroblewski J. 
Published: April 2021

ABSTRACT
Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death.
On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.
A monoclonal antibody study of protein distribution in the membrane skeleton of the ciliate Pseudomicrothorax
Authors: Curtenaz Sophie , Peck Robert K. 
Published: April 2021

ABSTRACT
The membrane skeleton, or epiplasm, of the ciliated protozoon Pseudomicrothorax dubius is a chemically and structurally complex layer. It is responsible for the cell shape and the positioning of some cortical organelles. One may expect that its possible morphogenetic role can be achieved only via a regional differentiation of the protein distribution in the epiplasm. We have tried to demonstrate such differentiation by preparing an epiplasm extract, which consists predominantly of concanavalin A (ConA)-positive glycoproteins. This fraction, either untreated or deglycosylated, was used to raise monoclonal antibodies (mAbs), whose specificity was tested on western blots of either untreated or deglycosylated epiplasm. The recognized polypeptides were then localized in situ by fluorescence and electron microscopic immunocytochemistry. Six mAbs are presented here. Four of them are directed against ConA-positive glycoproteins and show a localization of the latter on the outer surface of the epiplasm. The two others are directed against other epiplasmic polypeptides: one is specific for a common epitope shared by most of the epiplasmic proteins, but not by the glycoproteins, and labels the entire membrane skeleton, whereas the other recognizes three minor polypeptides, which seem localized to the inner part of the epiplasm.
Redistribution of a Golgi glycoprotein in plant cells treated with Brefeldin A
Authors: Satiat-Jeunemaitre Béatrice , Hawes Chris 
Published: April 2021

ABSTRACT
The fungal fatty acid derivative Brefeldin A (BFA), has been used to study the reversible distribution of a Golgi glycoprotein, the JIM 84 epitope, into the cytosol of higher plant cells. Treatment of both maize and onion root tip cells resulted in a rearrangement of the Golgi stacks into either circular formations or a perinuclear distribution. The Golgi cisternae became curved and vesiculated and in cells where the Golgi apparatus was totally dispersed the JIM 84 epitope was associated with large areas in the cytosol which were also vesiculated. On removal of the BFA the Golgi apparatus reformed and the JIM 84 epitope was again located in the cisternal stacks. This mode of BFA action is compared with that so far described for animal cells.
Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape
Authors: Sims John R. , Karp Seth , Ingber Donald E. 
Published: April 2021

ABSTRACT
Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 g/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Realtime videomicroscopic studies revealed that addition of 250 M ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 M. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors. GRGDSP had little effect on cell or nuclear shape in the absence of ATP and, hence, in the absence of cytoskeletal tension. These data suggest that large-scale changes in cell and nuclear shape result from the action of mechanical tension that is generated within the cytoskeleton via an actomyosin filament sliding mechanism, transmitted across integrin receptors and physically resisted by immobilized adhesion sites within the extracellular matrix. Rapid and coordinated changes of cell, cytoskeletal and nuclear form result when this cellular force balance is altered.
Co-localization and molecular association of dystrophin with laminin at the surface of mouse and human myotubes
Authors: Dickson George , Azad Ajith , Morris Glenn E. , Simon Horst , Noursadeghi Mahdad , Walsh Frank S. 
Published: April 2021

ABSTRACT
In Duchenne muscular dystrophy (DMD), deficiency of the protein dystrophin results in necrosis of muscle myofibres, associated with lesions in the sarcolemma and surrounding basal lamina. Dystrophin has been proposed to be a major component of the sub-sar-colemmal cytoskeleton involved in maintaining the integrity of the myofibre plasma membrane, and is known to associate with a group of sarcolemmal glycoproteins, one of which exhibits high affinity binding to the basal lamina component laminin. However, a direct or indirect transmembrane association of dystrophin in muscle cells with the myofibre basal lamina has not been demonstrated. To address this question we have examined dystrophin immunostaining and immunoprecipitation patterns in cultured mouse and human myotubes in comparison with that of the basal lamina component, laminin. Dual-immunolabelling revealed virtually complete co-localization of dystrophin on the inside surface of the muscle cell sarcolemma with plaques and veined arrays of laminin accumulating on the extracellular face. This pattern of laminin and dystrophin distribution was distinct from that of other cell surface molecules expressed in myotubes such as the neural cell adhesion molecule, NCAM, and the 1 integrin receptor, and immunoprecipitation of dystrophin from solubilized myotube extracts resulted in co-purification of laminin B1 chain confirming an association between these two components. The results thus provide the first direct cellular evidence of a transmembrane linkage between dystrophin in the sarcolemmal cytoskeleton with laminin in the overlying basal lamina. While the immunocytochemical distribution of laminin was apparently normal in dystrophin-deficient muscle cells, elevated levels of soluble laminin were present in extracts of mdx compared with normal mouse skeletal muscle. The absence of dystrophin may thus result in disruption at the molecular level of the cytoskeletal-basal lamina superstructure surrounding the sarcolemma in dystrophic myofibres.
Localization of ahe 6 and 4 integrin subunits in normal human nonlymphoid tissues
Authors: Natali P. G. , Nicotra M. R. , Bigotti A. , Martino C. De 
Published: April 2021

ABSTRACT
The 6/ 4 integrin, of undefined receptor activity, has been shown to be expressed in a variety of murine epithelial cells. To gain information on the role of this heterodimer in tissue architecture as well as in malignant transformation we have performed an extensive immunohistochemical analysis of normal human tissues using monoclonal antibodies to 6 and 4 subunits. Because 6 is known to associate also with the 1 subunit to form a non-promiscuous receptor for laminin, the expression of 1 chain was also evaluated. The results of this study have shown that the 6 chain has a wide distribution in tissues, including small vessels and peripheral nerves. 6 colocalizes with 4 and 1 in most epithelial cells at the basolateral or basal aspect abutting the basement membrane. In a minority of tissues lacking 4, the 6 chain is coexpressed with 1. These findings demonstrate that the expression of 6/ 1 laminin receptor and 6/ 4 heterodimer is phylogenetically conserved, suggesting that they are likely to play an important role in cellular scaffolding through binding to laminin and to still uncharacterized ligand/s present in basement membranes.
The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly
Authors: Sanchez J. Aquiles , Marek Doreen , Wangh Lawrence J. 
Published: April 2021

ABSTRACT
Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg’s ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.
Selective interactions of cells with crystal surfaces Implications for the mechanism of cell adhesion
Authors: Hanein Dorit , Sabanay Helena , Addadi Lia , Geiger Benjamin 
Published: April 2021

ABSTRACT
In this study we have characterized the mode of cell adhesion to calcite and calcium (R,R)-tartrate tetrahydrate crystals. The use of crystals as adhesion substrata was motivated by their well-established chemical nature and structurally defined surfaces. We show that calcite binds A6 Xenopus laevis epithelial cells rapidly and efficiently, most likely via surface-adsorbed proteins. Surface topology had only a limited effect on the adhesive interactions. Calcium (R,R)-tartrate tetrahydrate crystals exhibits two chemically equivalent, yet structurally distinct faces that differ mainly in the surface distribution of their lattice water molecules and charges. However, despite the gross similarity between the two faces striking differences were noted in their adhesive behavior. One of the faces was highly adhesive for cells, leading to protein-independent attachment and spreading followed by cell death. In contrast, cell adhesion to the other surface of tartrate was slow ( 24 h) and apparently mediated by RGD-containing protein(s). It was further shown that the latter face of tartrate crystals could be “conditioned” by long (24 h) incubation with serum-containing medium, after which it becomes highly adhesive.
The results presented here indicate that crystal surfaces may serve as excellent, structurally defined, substrata for cell adhesion, that cell binding may occur directly or via RGD-containing proteins and that cell adhesion may be dramatically modulated by variations in surface structure. The implications of the results to the mechanism of cell-substratum adhesion are discussed.
Internal lamin structures within G1 nuclei of human dermal fibroblasts
Authors: Bridger Joanna M. , Kill Ian R. , O’Farrell Minnie , Hutchison Christopher J. 
Published: April 2021

ABSTRACT
The nuclear lamina is a mesh-like network of fibres subjacent to the inner nuclear membrane that is believed to be involved in the specific spatial reorganisation of chromatin after mitosis. To determine how the lamina might be involved in chromatin reorganisation, we have performed indirect immunofluorescence studies on quiescent and proliferating human dermal fibroblasts (HDF). Two monoclonal antibodies recognising human lamins A and C and three different fixation methods were employed. In indirect immunofluorescence studies, cultures of quiescent cells displayed a uniform perinuclear distribution of the antibodies. In proliferating cultures two distinct populations of cells were observed: one population displayed a typical perinuclear antibody distribution, while the second population displayed an unusual pattern consisting of a series of spots and fibres within the nucleus. By inducing cell-cycle synchrony in cultures we were able to determine that the unusual internal distribution of the lamin antibodies was restricted to cells in G1. Optical sectioning and 3-D reconstruction of the lamina structures in G1 nuclei was performed with a confocal laser scanning microscope (CLSM). This revealed that the internal lamin structures consisted of small foci and fibres proliferating throughout the nucleus. These structures were shown to be closely associated with areas of condensed chromatin but not nuclear membrane. As cells progress towards S phase the internal lamin foci disappear.
Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts
Authors: Messiaen J. , Read N. D. , Cutsem P. Van , Trewavas A. J. 
Published: April 2021

ABSTRACT
The mode of action of cell wall elicitors in the induction of various plant cell responses, such as the activation of host defence mechanisms, is unknown, but signal transduction through cytosolic free calcium ([Ca2+]i) has been suggested. This paper shows that polygalacturonic acid or oligogalacturonides cause a prolonged increase in [Ca2+]i of carrot protoplasts within 20 min of induction. Our data support the view that a special conformation of the oligogalacturonides possessing &gt;9 residues is necessary to induce an elevation in [Ca2+]i. The localization of [Ca2+]i elevation around the periphery of protoplast cytoplasm and the inhibition of the response with verapamil suggest that exogenous Ca2+ is the major source for the rise in [Ca2+]i.
Aα and Bβ chains of fibrinogen stimulate proliferation of human fibroblasts
Authors: Gray Andrew J. , Bishop Jill E. , Reeves John T. , Laurent Geoffrey J. 
Published: April 2021

ABSTRACT
During blood coagulation and wound healing, fibrinogen polymerises to form a fibrin matrix, providing a substratum over which connective tissue cells migrate and proliferate. Although a number of growth factors have been implicated in this process, a possible role for the fibrin(ogen) molecules themselves has not been considered. In this study we have investigated the ability of the constituent chains of fibrin(ogen) to induce fibroblast replication. Fibrinogen chains (Aα1, Aα2, Bβandγ) were separated by cation exchange chromatography and their mitogenic activity was assessed before and after treatment with thrombin. The Aα1, Aβ2 and Bβchains where all found to stimulate fibroblast replication (23 2.9%, 29.2 5.3% and 31.4 5% stimulation above control, respectively) and on the addition of thrombin this activity was enhanced. No activity was observed in the γ chain before or after treatment with thrombin. These results indicate that growth promoting activity is inherent in fibrin(ogen) structure, suggesting a novel mechanism for fibroblast proliferation during wound healing.
Specific responses of axons and dendrites to cytoskeleton perturbations: an in vitro study
Authors: Lafont F. , Rouget M. , Rousselet A. , Valenza C. , Prochiantz A. 
Published: April 2021

ABSTRACT
Several factors can influence the development of axons and dendrites in vitro. Some of these factors modify the adhesion of neurons to their substratum. We have previously shown that the threshold of neuron-substratum adhesion necessary for initiation and elongation of dendrites is higher than that required for axonal growth. To explain this difference we propose that, in order to antagonize actin-driven surface tension, axons primarily rely on the compression forces of microtubules whereas dendrites rely on adhesion. This model was tested by seeding the cells in conditions allowing the development either of axons or of axons and dendrites, then adding cytochalasin B or nocodazole 1 hour or 24 hours after plating. The addition of cytochalasin B, which depolymerizes actin filaments and thus reduces actin-tensile forces, increases the length of both axons and dendrites, indicating that both axons and dendrites have to antagonize surface tension in order to elongate. The addition of nocodazole, which acts primarily on microtubules, slightly reduces dendrite elongation and totally abolishes axonal growth. Similar results are obtained when the drugs are added 1 or 24 hours after plating, suggesting that the same mechanisms are at work both in initiation and in elongation. Finally, we find that in the presence of cytochalasin B axons adopt a curly morphology, a fact that could be explained by the importance of tensile forces in antagonizing the asymmetry created by polarized microtubules presenting a uniform minus/plus orientation.