bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs
Authors: Hutchison Christopher , Kill Ian 
Published: April 2021

ABSTRACT
The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.
Identification of proteins cross-reactive to phosphotyrosine antibodies and of a tyrosine kinase activity in boar spermatozoa
Authors: Berruti G. , Martegani E. 
Published: April 2021

ABSTRACT
We used two different anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins in a germ cell, the boar spermatozoon. Ejaculated spermatozoa presented three major polypeptides, of Mr 43000, 40000 and 36000, respectively, that were immunorecognized on Western blots. These proteins were selectively enriched in the Triton X-100-soluble fraction and were released neither after an A23187-induced acrosome reaction nor after sperm homogenization. These findings suggest the presence of the three proteins in plasma membrane regions not involved in the acrosomal vésiculation. When epididymal boar spermatozoa were investigated, Western blot analysis of the detergent-soluble fractions from caput sperm did not reveal any detectable 43000, 40 000 and 36 000 Mr proteins cross-reactive with phosphotyrosine antibodies, whereas the detergent-soluble fractions from cauda sperm yielded very strong immunoreactive signals.
Labelling of freshly ejaculated spermatozoa with [32P] orthophosphate yielded a wide range of labelled phosphoproteins, but we failed to identify specific tyrosine phosphorylation under the experimental conditions employed. Tyrosine phosphorylation occurred when specific synthetic polymers of tyrosine, commonly used for studying tyrosine protein kinases, were assayed as substrates against both the Triton-soluble and Triton-insoluble sperm fractions. This is the first immunological and biochemical report on the presence of phosphotyrosine-containing proteins and protein kinase activities that phosphorylate tyrosine residues in a mammalian mature spermatozoon.
Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field
Authors: Gaertig Jacek , Iftode Francine 
Published: April 2021

ABSTRACT
This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells. Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density. The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs. The course of further changes in cell organization depends on the polarity of fused cells. Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements. In contrast, cortical integration of heteropolar fused cells is limited. Cytoskeletal integration is particularly promoted if the cells are incubated in nongrowing conditions. Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.
Embryonic chicken fibroblast collagen binding proteins: distribution, role in substratum adhesion, and relationship to integrins
Authors: Ogle Roy C. , potts A. Jeannette , Yacoe Marshall , Little Charles D. 
Published: April 2021

ABSTRACT
Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dosedependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin β subunits; and that antibodies to avian integrins recognize a 100 000 MrCBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family.
Biochemical characterisation of polycation-induced cytotoxicity to human vascular endothelial cells
Authors: Morgan David M. L. , Larvin Victoria L. , Pearson Jeremy D. 
Published: April 2021

ABSTRACT
Pro-inflammatory effects of cationic proteins secreted by human granulocytes include induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. We have shown previously that a series of synthetic polycationic amino acids produce a dose-, time- and Mr-dependent inhibition of [3H] leucine or [3H] thymidine incorporation into macromolecules by human umbilical vein endothelial cells, and that the extent of inhibition was correlated with changes in cell morphology, with release of cytoplasmic constituents and was irreversible.
The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations. We have found that the extent of inhibition is related to both the identity of the monomer, for polymers of Mr 40 000 the order is ornithine&gt;lysine&gt;arginine, and to its configuration; poly-D-lysines are more potent inhibitors than poly-L-lysines of similar Mr. Only brief exposure to the agonist is required, 90% inhibition occurred after 10 min of exposure to poly-L-lysine (Mr 90000). Treatment of endothelial cells with neuraminidase, heparinase, hyaluronidase, chondroitinase or trypsin did not reduce their susceptibility to poly lysine. Inhibition of microtubule or microfilament formation also had no effect on polylysine cytotoxicity, indicating that internalisation of the polymer was not a prerequisite for the effect. Inhibition of protein synthesis or pretreatment with simple sugars likewise failed to block the effects of polylysine treatment. Natural cationic proteins exerted similar effects on endothelial cells, the extent of the effect apparently being related to the pI of the protein. We conclude that the extent of cytotoxicity induced by cationic polymers on proteins is related to the number of attachment sites to which an individual molecule binds, which in turn depends upon its size and conformation, and on the distribution of anionic domains on the cell surface.
Expression of anionic sites on tumour cells at different stages of tissue invasion in vivo: A comparative study by X-ray microanalysis
Authors: Evans Peter M. , Suker Dhamia K. , Gwynn Iolo ap 
Published: April 2021

ABSTRACT
Quantification of colloidal iron hydroxide (CIH) labelling by X-ray microanalysis was used to investigate anionic sites at the surface of Ehrlich carcinoma cells from different locations in the mouse host. Individual tumour cells from peritoneal ascites suspensions (pre-invasion stage) varied up to threefold in their ability to bind CIH and a similar degree of intra-tumour heterogeneity was observed in different experimental animals. Pre-treatment of the cells with neuraminidase confirmed that binding was at least partly due to surface sialic acid. Invasive cells isolated from mesenteric tumour nodules were also heterogeneous with regard to the availability of surface anionic sites, as were tumour cells adhering to the surface of the mesentery; however, in both these populations CIH binding was significantly greater on average than for free ascites tumour cells. The results suggest that surface anionic sites are determinants of the invasiveness of malignant cells in vivo, and that both the number and topography of these sites may be important in modulating tumour cell behaviour.
Dissociation of senescence-associated changes in differentiated gene expression and replicative senescence in cultured adrenocortical cells
Authors: Yang Lianqing , Hornsby Peter J. 
Published: April 2021

ABSTRACT
In bovine adrenocortical cells senescing in culture, the decline in expression of cyclic AMP-inducible 17 α-hydroxylase in mass cultures and clones of bovine adrenocortical cells results from a decrease in the number of cells in the population capable of expressing this gene. As the population of cells decreases in proliferative potential, the fraction of the population that expresses 17 α-hydroxylase after induction with cyclic AMP decreases, until the population comprises entirely nonexpressing cells. We used in situ hybridization to study the relationship between the loss of the ability to replicate and the loss of expression of this differentiated function gene. Colonies of cells from early passage cultures comprised complex mosaics of expressing and nonexpressing cells, indicating a high-frequency switching event. At an individual cell level, there was a complete independence of replicative senescence and loss of 17 α-hydroxylase expression. Groups of cells with a morphology characteristic of replicative senescence, occurring frequently in early-passage cultures, comprised both expressing and nonexpressing cells, as did cells that appeared to be in rapid growth. This was confirmed by prior incubation of cells with bromodeoxyuridine to label cycling cells; both cycling and noncycling populations comprised mixtures of expressing and nonexpressing cells. In contrast, replication was required for the loss of 17 α-hydroxylase expression, since cells rendered incapable of division by treatment with mitomycin C maintained 17 α-hydroxylase expression over long time periods. Thus, the process that results stochastically in replicative senescence and the process that results in loss of 17α-hyroxylase expression are independent, but they have similar time-courses and both require replication for phenotypic switching to take place.
Steps in the morphogenesis of a polarized epithelium : I. uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts
Authors: Wang Allan Z. , Ojakian George K. , Nelson W. James 
Published: April 2021

ABSTRACT
The development of cell polarity in Madin-Darby canine kidney (MDCK) cells has been analyzed under conditions in which cells are induced to form multicellular epithelial cysts in stages that mimic the ontogeny of epithelial tissues and organs in vivo. The morphogenesis of MDCK cysts in suspension culture or in a collagen gel proceeds in distinct stages involving the initial aggregation of cells followed by development of a closed monolayer of polarized epithelial cells that surrounds a central lumen. The polarity of cells was determined at each stage by analyzing the distributions of marker proteins of the apical (gpl35) and basal-lateral (Na+,K+-ATPase) domains of the plasma membrane, the tight junction (ZO-1) and proteins involved in cell-cell (uvomorulin) and cell-substratum contact (type IV collagen). We show that cells have a distinctive and opposite polarity in cysts formed in suspension culture compared to those formed in collagen gels. In suspension culture, the basal-lateral membrane faces the central lumen and the apical membrane faces the outside, whereas in collagen gel, the basal-lateral membrane faces the outside collagen and the apical membrane faces the central lumen. Detailed analysis of the distributions of marker proteins during the morphogenesis of these three-dimensional structures indicates that: (1) cell-cell contact is sufficient to trigger the segregation of marker proteins of the apical and basal-lateral membrane domains to distinct regions of the membrane; (2) cell-cell contact induces association of the tight junction protein ZO-1 with the contact zone between cells; (3) localization of the tight junction protein ZO-1 to the apex of the lateral membrane and the establishment of the epithelial axis, however, requires the formation of a basal lamina and cell-substratum contact; (4) in suspension culture, MDCK cysts secrete and establish a basal lamina in the central lumen. These results show that cell-cell and cell-substratum contact have distinct roles in the morphogenesis of polarized epithelia. We suggest that the mechanisms involved in triggering cell polarity may be common to different polarized epithelia in vivo.
Control of fluid transport in human rectal adenocarcinoma cells (hra-19) in monolayer and collagen gel cultures
Authors: Kirkland Susan C. 
Published: April 2021

ABSTRACT
HRA-19a1.1. cells, derived from a primary human rectal adenocarcinoma, form polarised monolayers when grown on tissue-culture plastic. HRA-19 monolayers have a heterogeneous morphology even after 150 passages in vitro or single cell cloning. The morphological changes observed in HRA-19 monolayers were postulated to be the result of vectorial fluid transport leading to accumulation of fluid between cells. To test this hypothesis, a variety of agents that control ion transport in colorectal epithelium were tested for their effect on HRA-19 morphology.
Forskolin, cholera toxin and prostaglandin E2 all markedly changed HRA-19 monolayer morphology, with the rapid disappearance of intercellular spaces. These agents all stimulate Cl− secretion in colorectal epithelium, i.e. transport from basolateral to apical surface, and therefore would be expected to reduce fluid accumulation at the basolateral side of the cell. Conversely, vasopressin, which stimulates absorption of Na+ and water across colorectal epithelium, leads to a small increase in intercellular spaces in the monolayer.
In collagen gel cultures, addition of cholera toxin, forskolin or prostaglandin E2 resulted in a large increase in colony size. In such treated cultures, thecolonies were ‘bubble-like’, often composed of a single rim of flattened cells, which appeared to encompass a fluid-filled space. Similar morphological changes were observed when HRA-19 cells were co-cultured with 3T3 cells. This effect was probably due, at least in part, to prostaglandin production by the 3T3 cells, as the effect could be markedly reduced by the addition of indomethacin to these cultures.
These results strongly suggest that the morphological changes observed in HRA-19 monolayers are generated as cells change their fluid transport characteristics. Therefore, it would appear that vectorial fluid transport can lead to marked morphological changes in monolayer cultures, which differ from the more usually reported dome formation. In addition, these studies show that HRA-19 cells, derived from a primary human rectal adenocarcinoma, retain the ability to respond to many of the agents active in controlling transport in normal colorectal epithelium.
Phosphorylation-related accumulation of the 125k nuclear matrix protein mitotin in human mitotic cells
Authors: Zhelev N. Z. , Todorov I. T. , Philipova R. N. , Hadjiolov A. A. 
Published: April 2021

ABSTRACT
The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI 6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named ‘mitotin’. Here, we show by short-term [35S] methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.
Lysosomal sorting mutants of coronavirus E1 protein, a Golgi membrane protein
Authors: Armstrong John , Patel Shaila , Riddle Peter 
Published: April 2021

ABSTRACT
As a model for the intracellular sorting of Golgi membrane proteins, we are studying the El protein of the coronavirus Mouse Hepatitis Virus A59. The wild-type protein, when expressed from synthetic RNA, is localised in the Golgi complex. When the second and third of the three predicted membranespanning sequences were deleted from the protein, the resulting mutant was retained in the endoplasmic reticulum. In contrast, removal of the first and second membrane-spanning sequences allowed the protein to pass through the Golgi complex and reach the lysosomes. Likewise, when 40 amino acids were deleted from the C-terminal cytoplasmic part of El, the truncated protein was transported to the lysosomes. We discuss the implications of these results for the structure of the El protein and the mechanism by which it is localised in the cell.
Microtubule-associated protein 3 (MAPЗ) expression in non-neuronal tissues
Authors: Huber Gerda , Matus Andrew 
Published: April 2021

ABSTRACT
Microtubule-associated protein 3 (MAPЗ, Mr 180 000), which in previous studies has been shown to be associated with glial processes and neurofilament-rich axons in rat brain, was examined in various non-neuronal rat tissues, immunoblots of adult rat tissues (brain, liver, heart, spleen, adrenal medulla and kidney) showed that MAPЗ is present in all organs tested. In addition we demonstrated that MAPЗ is a heat-stable protein. Using immunohistochemistry, we established the localIsation of MAPЗ in various cell types. MAPЗ-containing cells appeared to have in common an asymmetric morphology with long processes that need structural support. In kidney MAPЗ is limited to epithelial podocytes and in liver to Kupffer cells. In the adrenal gland, the cells of the cortex are devoid of MAPЗ compared to the cells of the medulla. High concentrations of MAPЗ are also found in cardiac muscle along the Z-disc and in the smooth muscle cells of the digestive tract. In spleen MAPЗ is found in cells of the white pulp surrounding central blood vessels. A co-distribution of MAPЗ with microtubules and intermediate filaments but not with microfilaments was found in each cell type examined. The widespread distribution pattern of MAPЗ together with its molecular size and heat-stability indicate that MAPЗ might be a member of the recently postulated family of homologous 200 000Mr mammalian tissue MAPs. Potential functions for MAPЗ in specific cell types are discussed.
The differentiation process of intestinal epithelial cells is associated with the appearance of statin, a non-proliferation-specific nuclear protein
Authors: Bissonnette Robert , Lee Menq-Jer , Wang Eugenia 
Published: April 2021

ABSTRACT
The expression of statin, a 57 000 Mr nuclear protein specifically present in non-proliferating cultured fibroblasts, was studied in vivo in the differentiating epithelial cells of the rat intestine. Using immunofluorescence microscopy we found that undifferentiated, proliferating crypt enterocytes are statin negative, whereas the differentiated nonproliferating villus enterocytes are statin positive. The epithelial cells of the intestine were isolated according to different stages of differentiation and the expression of statin was studied biochemically by immunoblotting assays. The prominent band (57 000 Mr) was present in abundance in villus cell fractions but undetectable in crypt cell fractions. These findings were also confirmed by immunofluorescence microscopy on individual intestinal epIthelial cells of the different isolated fractions. The results presented here, which are similar to observations made in cell cultures, suggest that statin is a unique protein associated with the non-proliferative state of differentiated cells in tissue.