bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5
Authors: Mardon Helen J. , Sebastio Gianfranco 
Published: April 2021

ABSTRACT
The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin 4 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5 ; CS1,CS5+ or CS1,CS5. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, premRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.
Capillary endothelial cells secrete a heparin-binding mitogen for pericytes
Authors: Swinscoe John C. , Carlson Edward C. 
Published: April 2021

ABSTRACT
The cells of the retinal microvasculature consist predominantly of mesodermally derived pericytes and endothelial cells, and the regulatory factors which govern their co-ordinated growth and define their phenotypic characteristics in vivo may be regarded as key elements of the angiogenic process. An investigation of these cells in co-culture experiments has led to the identification of a potent mitogen for pericytes in medium conditioned by retinal endothelial cells (EC-FBS). EC-FBS activity was shown to be non-dialyzable, and stable to both heat and acid treatment. EC-FBS was inactivated by passage over a heparin-Agarose column. The column-bound activity could be eluted as a single peak at ∼1.0 M NaCl. Stimulation of pericyte growth was also achieved with platelet-derived growth factor (PDGF), acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) and could be blocked by using the appropriate antiserum (anti-PDGF or anti-aFGF). Neither antisera, however, blocked the activity of EC-FBS. The EC-FBS mitogen markedly altered the phenotypic behavior of pericytes compared with PDGF and the FGFs; yet, unlike them, it failed to stimulate the growth of smooth muscle cells (SMC) and Balb/c 3T3 cells.
Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture
Authors: Fukamachi Hiroshi 
Published: April 2021

ABSTRACT
It has been a subject of controversy whether fibroblastic cells are necessary for the proliferation of intestinal epithelial cells in primary culture. To answer this question, we have developed a serum-free primary culture system which allows reproducible and quantitative assays of proliferation and differentiation of fetal rat intestinal epithelial cells in the absence of fibroblastic cells. Pure intestinal epithelial tissues were obtained from 16.5-day fetal rats without contamination of mesenchymal cells, and were successfully cultured on a collagen gel in a medium consisting of Ham’s F12, bovine serum albumin, epidermal growth factor (EGF), insulin, cholera toxin, transferrin and hydrocortisone. The epithelial nature of the cultured cells was confirmed by the presence of cytokeratin in the cells. Under optimal culture conditions, intestinal epithelial cells readily attached to the substratum in a day, and proliferated rapidly in vitro, increasing their number about 10 times in the first 5 days. EGF, insulin, cholera toxin, transferrin and hydrocortisone synergistically induced the epithelial proliferation, and lack of any one of them resulted in a significant reduction of the proliferation. In contrast, fetal bovine or horse serums, which have been widely used to supplement culture media, severely inhibited the epithelial proliferation.
Histological examination showed that the epithelial cells formed simple cuboidal epithelia with basallylocated nuclei when cultured on collagen gels. The intestinal epithelial nature of the cells was affirmed by the presence of villin on their luminal surface. Ultrastructurally, cells were connected by tight junctions and desmosomes at the subluminal region, and microvilli were projecting on the luminal surface, indicating that the cells in primary culture retained some characteristics of absorptive epithelial cells. Biochemical analysis showed that the cells expressed weak aminopeptidase activity while alkaline phosphatase or -glucosidase activities could not be detected, suggesting that they could not differentiate fully under these culture conditions. We concluded that the fetal intestinal epithelial cells can proliferate in the absence of heterologous cells, but that mesenchymal cells may be necessary for their full functional differentiation.
Growth requirements and expression of LDL receptor and HMG-CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium
Authors: Gherardi E. , Thomas K. , Cras T. D. Le , Fitzsimmons C. , Moorby C. D. , Bowyer D. E. 
Published: April 2021

ABSTRACT
A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2.
Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 -ethinylestradiol but not by epidermal growth factor (EGF).
On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells.
Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37°C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro.
The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.
Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads
Authors: Peng H. Benjamin , Chen Qiming 
Published: April 2021

ABSTRACT
The distribution of dystrophin in Xenopus myotomal muscle cells was examined in conventional and confocal immunofluorescence microscopy. By labeling dissoci-ated single muscle fibers with a monoclonal or a poly-clonal antibody against dystrophin, we found that dys-trophin is ten times more concentrated at the myotendinous junction (MTJ) than at the extrajunc-tional sarcolemma. At the MTJ, dystrophin lines the membrane invaginations where myofibrils attach to the membrane. It is colocalized with talin, but is not related to the distribution of acetylcholine receptors (AChRs) which are clustered at the postsynaptic membrane in the vicinity of the MTJ in these fibers. We found that the localization of dystrophin can be induced in cultured Xenopus myotomal muscle cells by treating them with polystyrene latex beads. Dystrophin is discretely local-ized at the bead-muscle contacts. With electron microscopy, a sarcolemma specialization with all the salient features of the MTJ, including basal lamina-lined membrane invaginations along which myofibrils make attachment. Although these beads also induce cluster-ing of AChRs, the patterns of dystrophin and AChR localization are distinct. The appearance of dystrophin at the bead-contacted sarcolemma is coincident with the development of the membrane invaginations. This, together with its concentration along membrane invagi-nations at the MTJ in vivo, suggests a role for dys-trophin in the formation of this junctional specializa-tion. Since the signal for MTJ development can be presented to cultured muscle cells in a temporally and spatially controlled manner by beads, this system offers a simple model for analyzing the mechanism of this sar-colemma specialization.
Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: Studies with Tetrahymena thermophila
Authors: Leick Vagn 
Published: April 2021

ABSTRACT
Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD=4×10 −9 M to 1×10 −8 M). Moreover, Scatchard transformations of the binding data show that there are about 5×105 binding sites per cell on the cell surface.
Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdansLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl--endorphin, is internalized preferentially into small vesicles. The difference may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein.
Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).
Cell division in Aspergillus
Authors: Doonan John H. 
Published: April 2021

ABSTRACT
Amenable to sophisticated genetic and molecular analysis, the simple filamentous fungus Aspergillus nidulans has provided some novel insights into the mechanisms and regulation of cell division. Mutational analysis has identified over fifty genes necessary for nuclear division, nuclear movement and cytokinesis. Molecular and cellular analysis of these mutants has led to the discovery of novel components of the cytoskeleton as well as to clarifying the role of established cytoskeletal proteins.
Mutations leading to defects in the kinases (i.e. p34cdc2) and phosphatases (i.e. cdc25 and PP1), which are known to regulate mitosis in other eukaryotes, have been identified in Aspergillus. Additional, as yet novel, mitotic regulatory molecules, encoded by the nimA and bimE genes, have also been discovered in Aspergillus.
Developmental stage- and tissue-specific expression of shibire, a Drosophila gene involved in endocytosis
Authors: Chen M. S. , Burgess C. C. , Vallee R. B. , Wadsworth S. C. 
Published: April 2021

ABSTRACT
Dynamin, a microtubule-activated GTPase, has recently been identified as the product of the shibire gene in Drosophila. shits mutants are defective in synaptic vesicle recycling, which leads to rapid and reversible temperature-sensitive paralysis. In the present study, results from RNase protection assays and analysis of cDNA clones define a complex pattern of developmental- and tissue-specific alternative splicing at two sites within the coding region. This analysis is also supported by western blot analysis with two polyclonal antibodies. In situ hybridization data revealed a high concentration of shi transcripts in the central and peripheral nervous system throughout neuronal development. Other than the nervous system, shi transcripts are also expressed at a high level in early embryos, larval imaginal discs, and male and female gonads. These data provide a basis for interpreting the wide range of phenotypic effects of shi mutations in terms of the putative membrane-sorting properties of dynamin and for further functional study of different dynamin isoforms.
Localization of actin in the Tetrahymena basal body-cage complex
Authors: Hoey John G. , Gavin R. H. 
Published: April 2021

ABSTRACT
In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify antichicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahy - mena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.
Differential regulation of βiii and other tubulin genes during peripheral and central neuron development
Authors: Jiang Yuan Qing , Oblinger Monica M. 
Published: April 2021

ABSTRACT
Mammalian peripheral and central neurons differ considerably in the composition and properties of their axonal cytoskeletons. Recent reports of the selective expression of a high molecular weight (HMW) tau protein in neurons with peripherally projecting axons have furthered the idea that the microtubules in central and peripheral neurons are disparate. In the present study, we examined the possibility that the various tubulin genes are differentially expressed in central versus peripheral neurons. To examine this, we compared the expression of four of the β-tubulin mRNAs (classes βI, βII, βIII, βIV) and the α1-tubulin mRNA in rat dorsal root ganglion (DRG) neurons with their expression in cerebral cortex during postnatal development (P5-90), using northern blots and in situ hybridization. We document both similarities and differences in tubulin gene expression in these two regions of the neuraxis during postnatal development. In both DRG and cortex, the expression of the class β1- and βII-tubulin mRNAs and the α1-tubulin mRNA was higher at earlier stages of postnatal development than in the adult. However, class βIV-tubulin mRNA levels increased during cortical development but decreased during DRG postnatal development. The opposite pattern was found for the neuron-specific class βIII-tubulin gene, the mRNA levels of which were high in cortex, at birth and then decreased with increasing postnatal development. in DRG, the pm-tubulin mRNA levels generally increased during postnatal development. βIII-tubulin protein levels were examined qualitatively at different developmental stages (5–90 days) by immunoblotting and immunocytochemistry. These studies showed a robust reduction in Pm-tubulin levels from P5 to adult stages in cortex, but a more complex pattern of change in the DRG. The results suggest that differential utilization of the tubulin gene products exists in central and peripheral nervous tissue and that this may play a role in specifying differences in the functional properties of neuronal cytoskeletons in these regions.
Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein
Authors: Kagami Osamu , Kamiya Ritsu 
Published: April 2021

ABSTRACT
Dynein was extracted from outer arm-less axonemes of the mutant odal and fractionated by high-pressure liquid chromatography on a MonoQ column into seven distinct subspecies (named a–g). Each subspecies contained one or two heavy chains and several mediumsized and light chains; by vanadate/UV-induced photocleavage and SDS-polyacrylamide gel electrophoresis, eight distinct heavy chains were identified. Analysis of the mutant axonemes indicated that the subspecies f (containing two heavy chains) is missing in the inner-arm mutant idal and the subspecies a, c and d are missing in the mutant ida4. Six subspecies (all but f supported microtubule translocation with the maximal rate ranging from 2 to 12 μm s−1 and the apparent Km for ATP ranging from about 10 to 100 μM. All the subspecies translocated microtubules with the plus end leading, indicating that all the inner-arm dyneins are minus end-directed motors. Five subspecies (all but b and f) displayed microtubule rotation during translocation at rates of up to about 10 Hz. Unexpectedly, the Km values for ATP for translocation and rotation did not always agree; because of this, the pitch of the movement was variable with some subspecies. These observations indicate that axonemes are equipped with several inner-arm subspecies and that torque generation is a feature common to many of them.
A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton
Authors: Woods Angela , Baines Anthony J. , Gull Keith 
Published: April 2021

ABSTRACT
The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210 000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.
Continuous growth of vimentin filaments in mouse fibroblasts
Authors: Coleman Thomas R. , Lazarides Elias 
Published: April 2021

ABSTRACT
We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives - roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (&gt;20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton.
Amphibian intestinal villin: isolation and expression during embryonic and larval development
Authors: Heusser S. , Colin S. , Figiel A. , Huet C. , Keller J.M. , Pornet P. , Robine S. , Vandamme J. , Vandekerckhove J. , DAUçA M. 
Published: April 2021

ABSTRACT
An actin-binding protein of Mr 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian Mr 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal Mr 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal Mr 105,000 protein is immuno - logically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.