bluuebunny/crossref_metadata_2025_split huggingface dataset:
The membrane skeleton of Pseudomicrothorax: I. isolation, structure and composition
Authors: Peck Robert K. , Duborgel FranÇoise , Huttenlauch Irm , De Haller Gerard 
Published: April 2021

ABSTRACT
The epiplasm membrane skeleton of the ciliated protozoan Pseudomicrothorax dubius has been isolated and its three-dimensional structure and constituent proteins have been examined. The epiplasm functions as a cytoskeleton to define cell shape and the position of some cortical organelles. Scanning electron microscopy of the isolated epiplasm reveals a rococo cytoarchitecture in which basal bodies and trichocyst attachment sites are arranged in precise geometric arrays. SDS-PAGE reveals 40 bands, one of which is quantitatively the major band of the epiplasm and is composed of at least 3 different proteins and numerous isoelectric variants, as revealed by two-dimensional electrophoresis and peptide mapping. Polyclonal antisera were produced against native (antiserum 15) and SDS-denatured (antiserum 18) epiplasm. On immunoblots, antiserum 15 labels the hydrophilic proteins that are extracted from the epiplasm by treatment with dilute acid solution and that are predominantly glycoproteins, four of which are labeled with Concanavalin A on Western blots. On Lowicryl thin sections, antiserum 15 labels the epiplasm uniformly, except for the terminal plates, indicating that the glycoproteins are integral components of the epiplasm and are not membrane contaminants in the epiplasm fraction. Concanavalin A labeling of Lowicryl sections supports the latter result. On immunoblots, antiserum 18 labels the acid-insoluble epiplasm bands, the major structural elements of the epiplasm. One of the epiplasm bands at 52×103Mr is labeled by an anti-/) tubulin monoclonal antibody. Evidence is presented that this fi tubulin is not due to microtubule contamination of the epiplasm fraction.
Freeze substitution reveals a new model for sporangial cleavage inPhytophthora, a result with implications for cytokinesis in other eukaryotes
Authors: Hyde G. J. , Lancelle S. , Hepler P. K. , Hardham A. R. 
Published: April 2021

ABSTRACTRapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.
Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization
Authors: Escaig-Haye FranÇoise , Grigoriev Vladimir , Peranzi Gabriel , Lestienne Patrick , Fournier Jean-Guy 
Published: April 2021

ABSTRACT
Human mitochondrial transcripts have been examined at the ultrastructural level. After contact with ultrathin sections of a human lymphoid cell line (CEM) embedded in Lowicryl K4M, biotinylated mitochondrial probes yield specific hybrids identified by a colloidal gold immunocytochemistry marker that visualizes rRNA and mRNA coding for respiratory chain polypeptides CO II, CO III and ATPase-6. The mitochondrial transcripts are preferentially located close to the inner membrane, particularly the cristae, suggesting that intra-organelle protein synthesis is intimately associated with the mitochondrial membrane system. Quantitative analysis indicates that the mitochondria concentrate the labeling with intensities that vary with the type of RNA and that the nucleus induces a fight hybridization signal with each mitochondrial probe. The visualization of human mitochondrial DNA expression in correlation with the fine anatomy of the mitochondria constitutes a new approach for fundamental research on the organelle and for analyzing its behaviour in human mitochondrial diseases.
The timing of the formation and usage of replicase clusters in s-phase nuclei of human diploid fibroblasts
Authors: Kill Ian R. , Bridger Joanna M. , Campbell Keith H. S. , Maldonado-Codina Gabriela , Hutchison Christopher J. 
Published: April 2021

ABSTRACT
The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-ll-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-ll-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.
Dictyostelium discoideum mutants resistant to the toxic action of methylene diphosphonate are defective in endocytosis
Authors: Bof Mireille , Brénot Françoise , Gonzalez Carlos , Klein Gérard , Martin Jean-Baptiste , Satre Michel 
Published: April 2021

ABSTRACT
Methylene diphosphonate is taken up in Dictyostelium discoideum amoebae by fluid-phase pinocytosis, and it inhibits growth through the production of methylene analogs of adenosine triphosphate and diadenosine tetraphosphate. Methylene diphosphonate resistance was thus used as the basis of a screening strategy for the isolation of endocytosis mutants. Fifteen Dictyostelium mutants, whose growth was resistant to 7.5 mM methylene diphosphonate, were obtained and three of them were characterized in more detail. They were partially defective in fluid-phase pinocytosis (both the rate and extent of FITC-dextran entry were reduced to 40-50% of the parent type activity) and they had smaller amounts of several lysosomal enzymes, such as acid phosphatase, N-acetylhexosaminidase, a-mannosidase (20-60% of the parent type activities). In contrast to the lysosomal hydrolases, the mutants had unchanged activities for enzyme markers selective for other compartments. They appeared phenotypically similar to the Dictyostelium mutant HMW570, which is defective in fluid-phase pinocytosis and oversecretes lysosomal enzymes. The methylene diphosphonate-resistant mutants were found to be unable to acidify fully their endosomal compartments and they have an increased endosomal pH, as shown by the use of the pH-sensitive fluorescence of FITC-dextran. Furthermore, the hypothesis proposing a defective acidification of the endosomal pathway was supported by the measurement of A TP-dependent vesicular acidification with acridine orange, and by in vivo 31P NMR spectroscopy with aminomethylphosphonate as a pH probe.
The β4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice
Authors: Kennel S. J. , Godfrey V. , Ch’ang L. Y. , Lankford T. K. , Foote L. J. , Makkinje A. 
Published: April 2021

ABSTRACT
More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The α6β4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the β4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAh to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region.
Sites of β4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAh 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space.
These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of β4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer’s patches, and portal vessels but not the central veins of the liver, are positive. The expression of the fβ4 integrin in blood vessels may indicate a specialized function for β4 at these sites that is distinct from its role in hemidesmo-some-mediated attachment.
Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos
Authors: Torpey N. P. , Heasman J. , Wylie C. C. 
Published: April 2021

ABSTRACT
We report the identity of a major component of Tritoninsoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.
Mesenchyme-mediated effect of dexamethasone on laminin in cocultures of embryonic gut epithelial cells and mesenchyme-derived cells
Authors: Simo P. , Simon-Assmann P. , Arnold C. , Kedinger M. 
Published: April 2021

ABSTRACT
Previous studies have shown that glucocorticoids accelerate intestinal maturation and that this process is mediated by the mesenchymal cells. The possible involvement of laminin (LN), a basement membrane component, in this mesenchymal mediation has been analyzed. For this purpose, the influence of dexamethasone (DX) on the synthesis of LN, its chain composition and its cellular distribution has been examined biochemically and immunocytochemically in two different mesenchyme-derived cell populations, fetal intestinal mesenchymal cells and fetal skin fibroblasts, as well as in cocultures of intestinal endodermal cells seeded on top of confluent fetal skin fibroblasts. Neither the amount of metabolically labeled LN purified by affinity chromatography (expressed per mg cell proteins), nor the A versus B chain ratio monitored after separation on gel electrophoresis and immunoblotting, showed significant differences after 5 days of DX treatment. However, glucocorticoids induced a shift from secreted to cell-associated LN molecules paralleling a striking difference in the immunostaining pattern of intracellular and surface LN in the mesenchyme-derived cell monocul-tures; the granular intracytoplasmic LN staining in the control cultures was replaced by a fibrillar organization of LN molecules concomitantly with an increased accumulation at the cell surface. In 2-day DX-treated cocultures, there was an acceleration of LN deposition at the epithelial-fibroblastic interface, which accompanied the enhanced expression of epithelial cell differentiation markers (brush border digestive enzymes). These DX-induced changes can be blocked by the addition of antiLN antibodies in the culture medium.
These findings further support the concept that glucocorticoid action on intestinal epithelial cells involves alterations in the extracellular microenvironment, assessed here for LN molecules, occurring at the level of the mesenchymal cell compartment. These changes may contribute to an accelerated organization of LN at the epithelial-mesenchymal interface and subsequently to epithelial differentiation.
Divalent cations and extracellular matrix receptor function during newt epidermal cell migration
Authors: Mahan James T. , Donaldson Donald J. 
Published: April 2021

ABSTRACT
Skin expiants were placed in plastic dishes coated with fibronectin (FN), fibrinogen (FGN) or collagen. Explants were cultured for 16 h in serum-free medium containing calcium (Ca) and magnesium (Mg), or in medium containing either Ca-only, Mg-only or manganese (Mn)-only. In Ca/Mg, migrating kératinocytes on ail test substrata produced a sheet of contiguous cells that formed a robust halo around each explant. When Ca was the only divalent cation added, the halos in FN- and FGN-coated dishes were approximately 70-80% as large as in Ca/Mg. On collagen, however, the halos were significantly smaller than on the other two substrata. This substratum-specific response in Ca-only suggests that migration on collagen is fundamentally different than migration on FN and FGN. Halos as large or larger than those in Ca/Mg formed on all three substrata in Mg-only. In this case, the halos were not in the form of a sheet of contiguous cells, but were composed of dissociated cells that had migrated from the explant. Individual cells likewise migrated from explants cultured in medium containing Mn-only; however, these halos were never as large as in Mg-only. Thus, while exogenous Ca appears to be an absolute requirement for maintenance of cell-cell connections, the cell-substratum interactions that lead to migration can utilize either Ca, Mg or, to a lesser extent, Mn.
Additionally we found that migration on the generally nonpermissive protein, BSA, was not improved by the presence of Mn. Hence, even though Mn can mediate to some extent migration on FN, FGN and collagen, presumably by binding to integrin-like receptors on the cell surface, Mn does not alter receptor specificity in a way that permits migration on BSA.
To analyze the possibility that individual newt kératinocytes possess receptors for more than one ECM molecule, we cultured skin explants in Mg-only on a narrow zone of FN bordered on one side by collagen and on the other by uncoated plastic. Individual kératinocytes were able to migrate across the FN strip and continue on collagen but not on uncoated plastic, indicating that at least some kératinocytes have receptors for at least two matrix molecules.
Studies of muc-1 mucin expression and polarity in the mouse mammary gland demonstrate developmental regulation of muc-1 glycosylation and establish the hormonal basis for mrna expression
Authors: Parry Gordon , Li Jil , Stubbs John , Bissell Mina J. , Schmidhauser Christian , Spicer Andrew P. , Gendler Sandra J. 
Published: April 2021

ABSTRACT
Muc-1 is a major mucin glycoprotein expressed on the surface of mammary epithelial cells. It has attracted considerable attention as it is expressed in an aberrant form on many breast tumor cells. Here we describe studies using a recently obtained cDNA probe of Muc-1 expression during lactogenic development in the mouse. Northern blot analysis demonstrated that Muc-1 is expressed at all stages of lactogenic development but its levels are increased very significantly during midpregnancy and into lactation. The basis of this was examined using CID-9 mammary epithelial cell cultures. It was found that in the presence of insulin Muc-1 mRNA levels were increased by both hydrocortisone and prolactin, with the combination of the three hormones supporting maximum expression. Muc-1 mRNA levels were also modulated by culturing cells on a basementmembrane-like extracellular matrix that promoted mRNA levels 5-to 10-fold above levels in cells cultured on plastic tissue culture dishes. Immunocytochemical studies using monoclonal antibodies to carbohydrate epitopes on Muc-1 demonstrated that while Muc-1 was found at all developmental stages, it became increasingly sialylated during the course of pregnancy and into lactation. Additionally, we found that while Muc-1 is tightly polarized to the apical surface of the epithelium of lactating and pregnant mice it exhibited a less-polarized distribution on a small proportion of ductal cells in virgin mice. We conclude that the expression of Muc-1 is regulated at several different levels and by a number of different factors. We speculate that this may reflect different functional roles for Muc-1 at different stages of mammary development.
The blood-brain barrier regulates the expression of a macrophage sialic acid-binding receptor on microglia
Authors: Perry V. H. , Crocker P. R. , Gordon S. 
Published: April 2021

ABSTRACT
In vitro the expression of a sialic acid-binding receptor on murine macrophages, sialoadhesin, is regulated by exposure to an inducing agent present in serum. We have used immunocytochemistry to examine the macrophage populations of the nervous system in order to test whether this serum inducing agent (SIA) also regulates sialoadhesin expression in vivo and whether plasma proteins may influence the phenotype of macrophages of the nervous system. Microglia, the resident macrophages of the central nervous system, reside behind the blood-brain barrier and do not express sialoadhesin. Microglia and macrophage populations in the cicumven-tricular organs, choroid plexus and leptomeninges are exposed to plasma proteins and some macrophages express sialoadhesin at these sites. Injury to the CNS, which damages the blood-brain barrier, induces sialoadhesin expression on a proportion of macrophages and microglia within the parenchyma. The expression of sialoadhesin matches the temporal and spatial distribution of the plasma extravasation into the brain parenchyma.
These experiments show that exposure to SIA is necessary for sialoadhesin expression and lend further support to the idea that the phenotype of microglia is in part regulated by the presence of the blood-brain barrier.
The role of primer recognition proteins in DNA replication: association with nuclear matrix in Hela cells
Authors: Vishwanatha Jamboor K. , Jindalf Hitesh K. , Davis Randall G. 
Published: April 2021

ABSTRACT
Primer recognition proteins (PRP) enable DNA polymerase a to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin n, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin H and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin H colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin n is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin H from the nuclear matrix with octyl-β-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.
cdc2 kinase-induced destabilization of map2-coated microtubules in Xenopus egg extracts
Authors: Faruki Shamsa , Dorée Marcel , Karsenti Eric 
Published: April 2021

ABSTRACT
During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependant phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocod-azole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.