bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells
Authors: Handel S. E. , Hendry K. A. K. , Sheterline P. 
Published: April 2021

ABSTRACT
Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D.
Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2×104 and 1.8×104 nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which colocalised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3×103 and 1.2 ×104 caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3× 103 caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15 min) implies a maximum time for the complete turnover of actin in the system.
Treatment of cells with low concentrations (3× 10−8 to 10−6m) of cytochalasin D, which prevents assembly at barbed-end assembly sites, also caused destruction of existing filament structures.
These data indicate that either substitution of endogenous assembly sites by exogenous nuclei, or blockage of existing endogenous assembly sites by cytochalasin, subverts the cycle of assembly and disassembly of normal actin filament architectures in PtK2 cells, leading to the assembly of aberrant structures.
Phenotypic modulation in sub-populations of human articular chondrocytes in vitro
Authors: Archer Charles W. , McDOWELL Jenny , Bayliss Michael T. , Stephens Myra D. , Bentley George 
Published: April 2021

ABSTRACT
Human articular cartilage has been separated into surface (approx. 15% of tissue depth) and deep zones (remaining tissue) and the constituent chondrocytes released by enzymic digestion. Subsequent culture either as a low density monolayer or as a suspension over agarose revealed distinct morphological and synthetic behaviour in the two populations. Whilst in monolayer these morphological differences disappeared with time in culture, over agarose they remained. Surface zone cells formed two types of cell cluster; one that was highly cellular with little extracellular matrix, and the other less frequent, which formed copious amounts of fibrillar matrix. Both types of cluster were surrounded by a layer of flattened chondrocytes. In contrast, deep cells formed a single cluster type that lacked a surrounding cell layer, but formed large amounts of sparse cartilage-like matrix and comprised morphologically typical chondrocytes. In monolayer, both populations gradually ceased to synthesise cartilage matrix components with the exception of link protein. In suspension, whilst the chondrogenic phenotype per se was preserved, there was, nevertheless, a loss in qualitative synthetic heterogeneity, which exists between surface and deep cells, that was not accompanied by changes in the differential rate of 35S incorporation into proteoglycan. Under these conditions, surface cells that normally do not synthesise keratan sulphate initiated de novo synthesis of this glycosaminoglycan. Consequently, it appears that the observed modulation in synthetic ability of the cell sub-populations is independent of the cluster morphology, which, once established, remains constant throughout the culture period.
The pattern of metalloproteinase expression by corneal fibroblasts is altered by passage in cell culture
Authors: Fini M. Elizabeth , Girard Marie T. 
Published: April 2021

ABSTRACT
We have examined the pattern of expression of four different matrix metalloproteinases (MMPs), collagenase, stromelysin, 92 kD gelatinase, and 72 kD gelatinase, by primary and passaged cultures of rabbit corneal fibroblasts. Primary cultures of this cell type have previously been shown to reproduce the normal tissue regulation of collagenase expression. We demonstrate qualitative and quantitative changes in the pattern of MMP expression as the cells are passaged in culture. Only a single MMP, 72 kD gelatinase, is constitutively expressed by primary fibroblast cultures. Phorbol myristate acetate (PMA) treatment upregulates expression of 72 kD gelatinase and turns on the expression of collagenase and stromelysin, as well as 92 kD gelatinase. However, the degree to which MMP expression is induced is minimal. Cells subcultured but a single time constitutively produce not only 72 kD gelatinase, but also collagenase and stromelysin. In addition, PMA treatment upregulates expression of collagenase, stromelysin and 92 kD gelatinase to high levels. In contrast, the expression of 72 kD gelatinase is repressed by treatment of passaged cell cultures with PMA. Our data indicate that the cell does not simply turn the MMP genes on or off, as a group, in response to various agents, but that it has the capacity for fine control over which MMPs are expressed and the degree to which each is expressed. Changes in MMP protein expression induced by PMA treatment are correlated with changes in specific mRNA levels in passaged cultures. The kinetics of mRNA accumulation suggest that the MMP genes can respond to multiple intracellular signals initiated in a temporal cascade by PMA. It is the combined effects of the individual signals on the accumulation of specific mRNAs that must determine the ultimate pattern of MMP protein expression. The distinct patterns of MMP expression produced by primary and passaged cell cultures may be analogous to patterns of expression that might occur under particular in vivo conditions.
Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo
Authors: Fujita Mamoru , Reinhart Frederick , Neutra Marian 
Published: April 2021

ABSTRACT
Absorptive cells of the intestinal epithelium endocy-tose proteins from both apical and basolateral membrane domains. In absorptive cells of suckling rat ileum, luminal protein tracers first enter an apical tubulovesicular endosomal system, then enter larger apical endosomal vesicles and multivesicular bodies (MVB), and finally are delivered to a giant supranuclear lysosomal vacuole. To determine whether proteins endocytosed from the basolateral domain in vivo enter the same endosomal or lysosomal compartments as those taken up from the apical side, we simultaneously applied cationized ferritin (CF) apically (by intra-luminal injection) and horseradish peroxidase (HRP) basally (by intravenous injection), and examined absorptive cells after 3 min to 60 min using light, electron and fluorescence microscopy. At early times, CF and HRP entered separate endosomal compartments at apical and basolateral poles. At no time did HRP enter the apical tubulovesicular system, and CF never entered early basolateral endosomes. After 15 min, however, both tracers appeared together in large late endosomes and MVB located apically, above the giant vacuole. From 15 to 60 min both tracers accumulated in the giant vacuole. Membranes of some apical late endosomes, all apical MVB, the giant vacuole, and occasional sub-nuclear vesicles contained immunoreactive lgpl20, a glycoprotein specific to late compartments of the endosome–lysosome system. These results show that highly polarized intestinal epithelial cells have separate apical and basolateral early endosomal compartments, presumably to maintain distinct membrane domains while allowing endocytosis and recycling of membrane from both surfaces. Apical and basolateral endocytic pathways, and presumably vesicles delivering hydrolytic enzymes and lysosomal membrane components, converge at the apical late endosome.
Ehrlich ascites tumour cells show tissue-specific adherence and modify their shape upon contact with embryonic fibroblasts and myotubes
Authors: Wójciak Beata , Korohoda Włodzimierz 
Published: April 2021

ABSTRACT
Adhesiveness of Ehrlich ascites tumour (EAT) cells to glass, to mouse peritoneal membrane, living and aldehyde-fixed mouse embryo fibroblasts and chick embryo fibroblasts, myoblasts and myotubes was investigated. The ascitic EAT cells (and leukaemia L1210 cells) did not adhere to glass and peritoneum but readily adhered to embryo fibroblasts, myoblasts and myotubes. The attachment was followed by cell spreading and migration. Fixation of fibroblasts or myogenic cells with aldehydes did not prevent ascitic cells from attaching but reduced the rate of spreading. Only direct interaction of ascitic cells with embryo myoblasts or fibroblasts induced changes in tumour cell adhesiveness followed by cell spreading and locomotion. These results are discussed in relation to an observation that ascitic cells growing as a cell suspension intraperitoneally grow as a solid tumour when injected subcutaneously.
Calcium depletion of Dictyostelium cells selectively inhibits cyclic nucleotide phosphodiesterase synthesis at a post-transcriptional step
Authors: Coukell M. B. , Cameron A. M. 
Published: April 2021

ABSTRACT
We reported previously that Ca2+ deprivation of Dictyostelium discoideum amoebae inhibits both the induction by cyclic AMP of soluble cyclic nucleotide phosphodiesterase (PD) activity in wild-type cells and the constitutive production of this activity in mutant HC35 cells. In this study, the mutant strain has been used to characterize this phenomenon further and to attempt to identify the Ca2+-dependent step(s) in PD production. Incubation of HC35 cells with 0.9 mM EGTA increased the rate of 4SCa2+ efflux from preloaded cells and partially inhibited PD production. The effect of EGTA on both processes was enhanced considerably by the presence of 1 fiM A23187. This concentration of ionophore alone had little effect on either Ca2+ efflux or PD production. Addition of 1 pM free extracellular Ca2+ to amoebae incubated with EGTA and A23187 increased PD production two-to sixfold. Ca2+ depletion did not appreciably inhibit total protein synthesis or total protein glycosylation as determined by the incorporation of [3H]leucine or [3H]mannose, respectively. Also, the abundance of PD mRNAs was approximately the same in Ca2+-depleted and control cells. In contrast, immunoblot analysis of extracellular fluids and intracellular extracts of these cells revealed a dramatic reduction (up to 85%) in the levels of PD-related polypeptides associated with the Ca2+-depleted cells. These results suggest that Ca2+ deprivation selectively inhibits PD production by impairing a post-transcriptional process.
Computer modelling of Tetrahymena axonemes at macromolecular resolution: Interpretation of electron micrographs
Authors: Sugrue P. , Avolio J. , Satir P. , Holwill M. E. J. 
Published: April 2021

ABSTRACT
A computer-generated model of the structural arrangement of the complete 9+2 ciliary axoneme of Tetrahymena at macromolecular resolution (4 nm) is presented. The model reconciles detailed information about subcomponents from negative-stained, thin-section and freeze-fracture electron micrographs, integrating the images into a consistent three-dimensional picture. This illuminates problems such as the requirement for compaction of dynein to form the arm, difficulties in visualization of the circumferential links, construction of the central sheath, and the comparative periodicities of the inner and outer arms. The model is pragmatic in that it is flexible and easily changed, as new information becomes available. It is also useful in the development of dynamic concepts, such as a spatial description of the dynein cross-bridge cycle, which is illustrated, or relationships between adjacent doublets during sliding and bending.
The polarized epithelial phenotype is dominant in hybrids between polarized and unpolarized rat thyroid cell lines
Authors: Zurzolo Chiara , Gentile Raff Aele , Mascia Anna , Garbi Corrado , Polistina Claudio , Aloj Luigi , Awedimento Vittorio Enrico , Nitsch Lucio 
Published: April 2021

ABSTRACT
We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive.
Behaviour of nucleolar proteins in nuclei lacking ribosomal genes: A study by confocal laser scanning microscopy
Authors: Hernandez-Verdun D. , Robert-Nicoud M. , Geraud G. , Masson C. 
Published: April 2021

ABSTRACT
The behaviour of nucleolar proteins in cycling PtK1 cells and in micronuclei with or without NORs was investigated by immunofluorescence using antibodies from autoimmune sera and confocal laser scanning microscopy. These antibodies were shown by electron microscopy to recognize antigens confined to only one of the three basic nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). Serial optical sections allowed us to determine the three-dimensional organization of these components in the nucleolus of cycling cells. Furthermore, clear differences were found in the distribution of the various antigens in micronucleated cells. Three patterns could be observed: (1) the FC antigens were found mainly in the nucleoli, but also in varying amounts in the dots; (2) surprisingly, the DFC antigens were found to accumulate preferentially in the dots; (3) the GC-specific marker stained intensively the nucleoli as well the dots. The results are interpreted with regard to possible mechanisms for targeting nucleolar proteins to the site of nucleolar formation.
Nuclear localization of an o-glycosylated protein phosphotyrosine phosphatase from human cells
Authors: Meikrantz William , Smith Douglas M. , Sladicka Melissa M. , Schlegel Robert A. 
Published: April 2021

ABSTRACT
Histochemical staining of immunoprecipitates of p65, a component of human M phase-promoting factor, identified the molecule as having phosphatase activity. The enzyme, purified 3400-fold from mitotic cell extracts by (NH^SCL precipitation, DEAE chromatography, and immunoaffinity chromatography on immobilized anti-p65 IgG, was inhibited by Zn 2+ and Na 3 VO 4 but not NaF or β-glycerophosphate; 32 P-labeled poly(Glu, Tyr) was more efficiently dephosphorylated than phosphorylated histone or phosphorylase a. Indirect immunofluorescence showed most of the phosphatase to be localized in the nucleus of interphase cells, with a fine, granular distribution unaltered by detergent extraction; in mitotic cells, p65 was localized on chromosomes. ELISA of subcellular fractions confirmed this localization. Immunoreactive p65 was recovered from immobilized wheat germ agglutinin (WGA) upon elution with N-acetylglucosamine; similarly, WGA recognized immunoaffinity-purified p65 on blots. Alkaline hydrolysis of blotted protein prevented WGA binding, indicating that phosphatase p65, like a small group of other nuclear proteins, contains O-linked carbohydrate terminating in N-acetylgluco-samine.
A perichromosomal region contains proteins phosphorylated during mitosis in Xenopus Laevis cells
Authors: Dilworth Stephen M. 
Published: April 2021

ABSTRACT
An antibody that recognizes the phosphorylated form of nucleoplasmin has identified another nuclear protein whose antigenic form is regulated in a mitosis-specific manner, with a dramatic increase in binding occurring in all mitotic cells. The protein is localised around the periphery of condensed chromosomes during mitosis in a manner analogous to another nucleoplasmin-related polypeptide NO38. Mitosis-specific expression of the antigenic site is dependent on phosphorylation of the polypeptide; binding of the antibody is dramatically reduced byprior incubation of the polypeptide with phosphatases. Migration on SDS-PAGE suggests that the protein has an exceptionally large relative molecular mass, in excess of 400000. The probable mitosisspecific phosphorylation and location of this antigen suggests a subcell ular storage mechanism for proteins during mitosis.
Vanadate inhibits both intercellular adhesion and spreading on fibronectin of BHK21 cells and transformed derivatives
Authors: Edwards J. G. , Campbell G. , Grierson A. W. , Kenn S. R. 
Published: April 2021

ABSTRACTBoth intercellular adhesion and spreading on fibronectin of BHK21 hamster cells are inhibited by vanadate at concentrations that cause specific regulatory effects rather than general metabolic inhibition. Inhibition of aggregation of these cells in suspension (half-maximal in 10−5M vanadate) is rapid and reversible. The extent of inhibition, and its decline with culture age parallel inhibition by agents that depolymerize microtubules.Vanadate also reversibly inhibits spreading of both BHK cells and transformed derivatives on fibronectin. If 10−4 M vanadate is added to BHK cells that have spread in its absence, they remain spread,but transformed derivatives are sensitive to rounding by vanadate at 10−6M.The mechanisms by which vanadate inhibits both intercellular adhesion and spreading are unknown, and may be different for the two phenomena. Possible sensitive targets include cytoplasmic dynein for the former, and protein tyrosyl phosphatase for the latter.
Production of scatter factor by ndk, a strain of epithelial cells, and inhibition of scatter factor activity by suramin
Authors: Adams Josephine C. , Furlong Robert A. , Watt Fiona M. 
Published: April 2021

ABSTRACT
ndk are a strain of human epidermal kératinocytes that do not undergo terminal differentiation and which grow as single cells rather than compact colonies. We show that ndk are motile and secrete an epithelial scatter factor that has the same biochemical and immunological properties as the scatter factor previously purified from ras-transformed 3T3 fibroblasts. We have found that suramin, a polyanionic detergent, will reverse the activity of scatter factor from either cell type in the standard MDCK activity assay. When added to ndk cultures, suramin causes the cells to grow in coherent patches. This morphological change is accompanied by alterations in the distribution of actin and integrins, but not by stratification or terminal differentiation. The effect is reversed upon removal of suramin. We propose that the motile phenotype of ndk is due, at least in part, to autocrine production of scatter factor and that suramin may be useful for further studies of scatter factor binding to the cell surface.
Addition of protease inhibitors to culture medium of neuroblastoma cells induces both neurite outgrowth and phosphorylation of microtubule-associated protein MAP-1B
Authors: Diaz-Nido J. , Armas-Portela R. , Avila J. 
Published: April 2021

ABSTRACT
The addition of two synthetic peptides with antiprotease activity to the culture medium of mouse neuroblastoma cells results in the promotion of neurite outgrowth. One of these peptides has a sequence corresponding to the reactive center of protease nexin-1 and inhibits both trypsin and thrombin. Its effect on neuroblastoma cells is similar to that found on serum withdrawal from the culture medium, giving rise to cells with one or two long neurites, and is reversed upon the addition of thrombin to the culture medium. The sequence of the other peptide is present in one of the precursor proteins of the main component of the amyloid plaques of Alzheimer’s disease patients’ brains, and corresponds to protease nexin-2. It can inhibit trypsin but fails to inhibit thrombin at low doses. Its effect on neuroblastoma cells is slightly different from that observed after serum deprivation, as a significant proportion of stellate cells, with short and branched neurites, is observed. An increase in the phosphorylation of microtubule-associated protein MAP-1B, which accompanies neurite outgrowth induced by serum deprivation, is also observed upon addition of the two antiprotease synthetic peptides, although the nexin-2 (amyloid) peptide induces a less marked increase in phosphorylated MAP-1B than does the nexin-1 peptide. These results may be correlated with the different antiprotease activities of both synthetic peptides, thus suggesting a role for a balance between trypsin-like and thrombin-like proteases and their inhibitors in eliciting neurite outgrowth under normal and pathological conditions.
Differential behavior of centrosomes in unequally dividing blastomeres during fourth cleavage of sea urchin embryos
Authors: Holy Jon , Schatten Gerald 
Published: April 2021

ABSTRACT
The fourth cleavage in sea urchin embryos is unequal and represents an initial step in cell differentiation: vegetal blastomeres divide to produce micromeres, which are precursors of the skeleto- genic mesenchyme, and macromeres. The mitotic spindles of these unequally dividing cells are peripherally located and lie orthogonal to the vegetal pole plasma membrane; the aster of the micromere pole is closely apposed to the plasmalemma and presents a characteristic flattened profile. In order to investigate the role of centrosomes in the generation of the asymmetric vegetal spindle at fourth cleavage, structural dynamics of centrosomes in both equally and unequally dividing blastomeres were compared using immunofluorescence methods. Quantitation of immunofluorescence and three-dimensional reconstruction techniques demonstrate that micromere centrosomes differ from macromere centrosomes in two respects: (1) micromere spindle poles contain less centrosomal material than macromere poles, and (2) micromere centrosomes undergo a specific filiform elongation during late anaphase and telophase. The behavior of micromere centrosomes suggests that a unique spindle pole event, involving interactions of the microtubular cytoskeleton, centrosome and cell cortex, occurs during the process of unequal cleavage of vegetal blastomeres.