bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stabilization of intercellular contacts in MDCK cells during Ca2+ deprivation: Selective effects of monocarboxylic acids on desmosomes
Authors: Bogner P. , Skehan P. , Kenney S. , Sainz E. , Akeson M.A. , Friedman S. J. 
Published: April 2021

ABSTRACT
Short-chain monocarboxylic acids (MCAs) selectively protect desmosomal junctions of MDCK cells from disruption by chelating agents and low calcium medium. This effect occurs in the millimolar concentration range and increases inversely with carbon chain length (formate &gt; acetate = propionate &gt; butyrate &gt; isobutyrate &gt; isovalerate). The relative activity of MCAs does not correlate with their overall hydrophobicity or ability to chelate ions, or their effectiveness in lowering cytosolic pH. It exhibits chemical specificity and is dependent upon postconfluency culture age. MCAs also inhibit cell rounding produced by low concentrations of aminocarboxylate-chelating agents. Their effect on cell rounding, but not on desmosomes, can be antagonized by okadaic acid. The possibility is discussed that MCAs may produce their effects by binding specifically to protein(s) associated with the desmosome of mature, fully polarized MDCK monolayers.
Hemidesmosomes in the epithelial cell line 804G: Their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton
Authors: Riddelle K.S. , Hopkinson S.B. , Jones J.C.R. 
Published: April 2021

ABSTRACT
Recently, we identified a novel epithelial cell line, 804G, derived from rat bladder, which readily forms hemidesmosomes in vitro. One of the major structural components of the plaques of 804G cell hemidesmo-somes is a 230 kDa antigen recognized by autoantibod-ies in the sera of patients with bullous pemphigoid (BP). An additional polypeptide of 180 kDa also localizes to the hemidesmosome plaque of 804G cells as determined by immunoelectron microscopy. Using confocal fluores-cence/phase microscopy, we have employed both 230 kDa and 180 kDa antibody probes to monitor the fate of hemidesmosomes following closure of in vitro wounds, during mitosis, and following drug induced disruption of the cytoskeleton. The punctate cell-substratum asso-ciated staining generated by the hemidesmosomal anti-bodies in stationary unwounded 804G cell cultures is greatly diminished or even lost in cells which enter wound sites, presumably in response to enhanced cell motility. Few, if any, hemidesmosomes are observed at the ultrastructural level in cells which have migrated into the wound area. However, as closure of the wound becomes complete, staining along the substratum attached surface of cells returns. During mitosis, there is no obvious loss of hemidesmosomal antigens along the basal surface of 804G cells, and formed hemidesmo-somes can be observed in mitotic cells at the ultrastructural level. In 804G cells treated with colchicine, the typical subnuclear pattern of distribution of hemidesmosomal antigens is unaffected. In contrast, following treatment of 804G cells with cytochalasin D, hemidesmosomal antigens become concentrated at the cell periphery and no longer appear in the subnuclear region. Furthermore, formed hemidesmosomes are observed at the cell periphery of cytochalasin D-treated cells by electron microscopy. We suggest that hemidesmosomal plaques are mobile within the plasma membrane. We speculate that hemidesmosomal interactions with extracellular ligands are dynamic and we discuss a possible mechanism by which cytochalasin D induces reorganization of hemidesmosomes along the basal surface of 804G cells.
Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro
Authors: Arciniegas E. , Sutton A. B. , Allen T. D. , Schor A. M. 
Published: April 2021

ABSTRACT
α-Smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of αsmooth muscle actin (α-SM actin) may be induced to express the latter when exposed to TGF-β1. FVIII was detected by immunofluorescence, α-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing α-SM actin increased with time of incubation with TGF-β1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and α-SM actin after 5 days of incubation with TGF-β1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed α-SM actin.
Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-β1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-β1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and α-SM actin-negative. Other cells in the same cultures, however, remained ragged in morphology, FVIII-negative and α-SM actinpositive even after 20 days in control medium. This indicates that the inductive effect of TGF-β1 is partly reversible after 10 days incubation. Such reversibility was no longer apparent after 20 days incubation with TGF-β1.
Our results demonstrate that TGF-β1 induces the differentiation of aortic endothelial cells into a smooth muscle-like phenotype and suggest a novel role for TGF-β1 in atherogenesis.
Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in hep G2 cells
Authors: Moorby C. D. , Gherardi E. , Riddell D. , Bowyer D. E. 
Published: April 2021

ABSTRACT
Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen con-ditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, =48 and MDCK cells, were found to increase the bind-ing and internalisation of 125I-LDL at 37°C by Hep G2 cells (P &lt; 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibrob-lasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.
Axonal growth on astrocytes is not inhibited by oligodendrocytes
Authors: Fawcett James W. , Fersht N. , Housden L. , Schachner M. , Pesheva P. 
Published: April 2021

ABSTRACT
Axon growth in vitro may be inhibited by contact with oligodendrocytes, but most axons grow readily on the surface of astrocyte monolayers. Since both cell types are in close contact with one another in the damaged nervous system, we have examined the growth of axons on cultures which contain both astrocytes and oligo-dendrocytes. Cultures derived from neonatal rat fore-brain develop with a monolayer of large flat astrocytes attached to the culture dish, and with many smaller cells of the oligodendrocyte lineage on their surface. Dorsal root ganglia placed on these cultures grow axons readily, the overall extent of growth being unaffected by the presence or absence of oligodendrocytes, many of which express galactocerebroside and the inhibitory molecule janusin. A previous set of experiments had shown that growth of these axons is inhibited by oligodendrocytes by themselves. Scanning electron microscopy coupled with silver-intensified immunostaining reveals that the axons grow on the surface of the astrocytic layer, under-neath the oligodendrocytes, and are therefore in contact with both cell types as they grow. The presence of astrocytes therefore alters the results of axonal contact with oligodendrocytes.
Statistical evidence for a random commitment of pluripotent cephalic neural crest cells
Authors: Baroffio Anne , Blot Michel 
Published: April 2021

ABSTRACT
The neural crest (NC) of vertebrate embryos yields cell types belonging to the neural, melanocytic and mesec-todermal lineages. To test the possibility that the precursors of these lineages segregate from pluripotent cells by a process involving stochastic determinants, we have analyzed with statistical methods the associations between six differentiated cell types in 201 clones obtained in vitro from migrating cephalic NC cells. Our analysis suggests that neuronal, adrenergic and Schwann cells are not randomly associated, whereas these neural cell types differentiate in the clones independently of both melanocytes and cartilage. These results raise the possibility that pluripotent NC progenitors give rise to the precursors of the major NC-derived lineages (neural, melanocytic and mesectodermal) by a process involving stochastic restrictions of their developmental potentialities.
Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments
Authors: Dahlstrand Jonas , Zimmerman Lyle B. , McKay Ronald D.G. , Lendahl Urban 
Published: April 2021

ABSTRACT
Multipotential stem cells in the neural tube give rise to the different neuronal cell types found in the brain. Abrupt changes in intermediate filament gene expression accompany this transition out of the precursor state: transcription of the intermediate filament nestin is replaced by that of the neurofilaments. In order to identify human neural precursor cells, and to learn more about the evolution of the intermediate filaments expressed in the central nervous system, we have isolated the human nestin gene. Despite considerable divergence between the human and rat nestin genes, in particular in the repetitive parts of the carboxyterminal region, the positions of the introns are perfectly conserved. Two of the three intron positions are also shared by the neurofilaments, but not by other classes of inter-mediate filaments. This implies that nestin and the neu-rofilaments had a common ancestor after branching off from the other classes of intermediate filaments, and that nestin separated from the neurofilament branch before the different neurofilament genes diverged. The characterization of human nestin also facilitates the identification of human multipotential neural precursor cells. This will be of importance for central nervous system (CNS) tumor diagnosis and transplant-based clinical approaches to human neurodegenerative diseases.
Further characterisation of the talin-binding site in the cytoskeletal protein vinculin
Authors: Gilmore A.P. , Jackson P. , Waites G.T. , Critchley D.R. 
Published: April 2021

ABSTRACT
The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and a-actinin. In this study, we further characterise the talin-binding domain in vin-culin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gelblot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathioneagarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5 portion of the chicken vinculin gene, and by analysing vin-culin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.
The tight association of the tyrosine kinase substrate annexin ii with the submembranous cytoskeleton depends on intact p11- and Ca2+-binding sites
Authors: Thiel Carsten , Osborn Mary , Gerke Volker 
Published: April 2021

ABSTRACT
Annexin II, a member of the annexin family of Ca2+- and lipid-binding proteins, is a major substrate of the pp60src kinase. It is unique within the annexin protein family, since it can form a tight heterotetrameric complex with the cellular protein ligand p11, a member of the S100 protein family. Within the cell, the annexin II2p112 complex is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton. This intracellular localization is thought to be the consequence of a typical annexin II property observed in vitro, its Ca2+-dependent binding to phospholipids and cytoskeletal elements (F-actin, non-ery-throid spectrin). We employed site-directed mutagenesis to create mutant annexin II molecules with defects either in the p11-binding site or in the Ca2+-binding sites present in repeats 2, 3 and 4. The mutated annexin II derivatives were expressed in HeLa and RMCD cells by transfection of the appropriate DNA constructs in order to analyze the importance of p11- and Ca2+-binding for the intracellular localization of annexin II. Immunofluorescence microscopy with a monoclonal antibody that specifically detected the transfected annexin II derivatives indicated that the Ca2+-dependent incorporation of annexin II into the submembranous network depended on its ability to form the annexin II/p11 complex and on the presence of intact Ca2+-binding sites. Neither monomeric annexin II lacking an intact p11-binding site, nor the annexin II mutant with defects in the Ca2+- binding sites in repeats 2, 3 and 4 were associated with the Triton X-100-resistant network of the submembra-nous cytoskeleton.
Altered glycosylation and cell surface expression of β1 integrin receptors during keratinocyte activation
Authors: Kim Lawrence T. , Ishihara Shuichi , Lee Chong-Chou , Akiyama Steven K. , Yamada Kenneth M. , Grinnell Frederick 
Published: April 2021

ABSTRACT
We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered β1 integrin subunit glycosylation accompanied by an increase in cell surface 01 integrin receptors. Activated keratinocytes contained two forms of the β1 integrin subunit, ∼93 kDa and ∼113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Preactivated keratinocytes contained 01 integrin subunits ranging from ∼97 to 110 kDa. These β1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of β1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller β1 subunits found in pre-activated keratinocytes, like the precursor 01 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a ∼10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa 01 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte a subunits associated with 01. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.
Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts
Authors: Kaur P. , Carter W. G. 
Published: April 2021

ABSTRACT
Normal human foreskin keratinocytes (HFKs) and transformed HFKs (FEPE1L-8 cells) generated by the introduction of cloned human papillomavirus type 16 sequences were compared for the expression and function of a family of adhesion receptors termed integrins. Initially, cells were examined in conventional monolayer cultures. FEPE1L-8s expressed integrins α1β1, α2β1, α3β1, α5β1, α6β4 and β1 at comparable levels to HFKs. Further, these receptors were fully functional in mediating specific interactions with exogenously supplied ligands. However, FEPE1L-8s exhibited decreased synthesis of a number of extracellular matrix components, including laminin, fibronectin and epiligrin, compared to normal HFKs, which may be an alternate mechanism for regulating adhesion. Subsequently, organotypic cultures (OCs), which provide a suitable in vitro model system for the ordered stratification and differentiation of keratinocytes, were used to study the regulation of integrins and various epidermal markers in normal and transformed cells. OCs consisted of keratinocytes plated on a collagen gel containing primary human fibroblasts, grown at an air-medium interface. Unlike normal HFKs, the transformed FEPE1L-8 cells exhibited (a) disorganized stratification and limited differentiation capacity, (b) invasion into the collagen gel, and (c) unregulated expression of α3β1 and α2β1, and underexpression of α6β4 integrins. Ordered stratification and spatial regulation of integrin expression could be induced in the FEPE1L-8s by substituting Swiss 3T3 fibroblasts in the collagen gel. Further data indicate that the human fibroblasts induce the transformed HFKs to invade into the collagen gel. We conclude that stromal cells play an important role in regulating (a) the differential expression of integrins, (b) the normal stratification and differentiation, and (c) the invasive behaviour of epidermal cells.
The small gtp-binding protein rab6p is distributed from medial golgi to the Trans-golgi network as determined by a confocal microscopic approach
Authors: Antony Claude , Cibert Christian , Géraud Gérard , Maria Angelica Santa , Maro Bernard , Mayau Véronique , Goud Bruno 
Published: April 2021

ABSTRACT
A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exo-cytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20°C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.
Fluorescent phospholipids preferentially accumulate in sub-tegumental cells of schistosomula of Schistoxssoma mansoni
Authors: Furlong Stephen T. , Thibault Kathleen S. , Rogers Rick A. 
Published: April 2021

ABSTRACT
Schistosomes do not make sterols or fatty acids de novo and thus require host lipids for survival. The acquisition of host lipids may also be an important factor in the schistosome’s defense from host immunity; however, little is known about the regulation of this process. Here we have examined binding of radiolabeled and fluorescently labeled liposomes to schistosomula, and followed incorporation of fluorescent phospholipids into the worm by both morphological and biochemical methods. Saturable binding of radiolabeled phosphatidylcholine containing liposomes was observed and epifluorescence microscopy showed binding of C6-NBD-phosphatidyl-choline (C6-NBD-PC), C12-NBD-phosphatidylcholine (C12-NBD-PC) and C6-NBD-phosphatidyl-ethanol-amine (C6-NBD-PE) containing liposomes on the surface of the parasite. Following back-exchange with unlabeled liposomes, NBD-PC and NBD-PE were observed to be preferentially incorporated into specific cell types within the worm. Furthermore, cells which had accumulated the fluorescent lipid formed an interconnecting cellular network immediately below the tegument, identified as cytons. By contrast, fluorescein-PE was found only on the surface of the parasite and in the gut but not in the cytons. Biochemical analysis demonstrated that &gt;90% of the C6-NBD-PC and C12-NBD-PC remained as the intact molecule after a one hour incubation with the parasite, but that greater than 70% of the NBD-PE was converted to other lipids. These studies demonstrate that incorporation of phospholipid analogs into schistosomula can be followed morphologically and biochemically. As there was little localization of NBD-PE or NBD-PC in the gut, these analogs must be assimilated by crossing the tegument. Furthermore, specificity in the worm’s uptake processes are evident.