bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantal tektin synthesis and ciliary length in sea-urchin embryos
Authors: Stephens R. E. 
Published: April 2021

ABSTRACT
Previous work using pulse-chase labelling of embryos from the sea-urchin Strongylocentrotus droebachiensis during ciliogenesis, regeneration or steady-state maintenance and elongation showed that a ciliary outer doublet microtubule-associated protein, originally termed component-20, was synthesized in a fixed or quantal amount. This suggested that the limited synthesis of component-20 might limit ciliary length, since the embryo has a large pool of most other ciliary components. Labelling experiments with S. purpuratus embryos now confirm quantal synthesis of component-20, while antibodies to S. purpuratus sperm flagellar tektins identify component-20 as the ciliary equivalent of the flagellar 55 ×lO3Mr tektin, tektin A. Sequential pulse-chase labelling at various times prior to isolation of cilia proves that the high specific activity of this protein truly reflects de novo synthesis of a structurally stable protein and not rapid protein turnover. Embryos may be animalized by growth in the presence of zinc ions, resulting in cilia averaging nearly twice the normal 20 pm length. When these embryos are pulse-chase labelled during ciliary growth and elongation, labelling of tektin A is proportional to the greater ciliary length, as is the pool of labelled but unincorporated tektins and other minor proteins. Deciliated animalized and control embryos, pulsechase labelled during their identical phases of ciliary regeneration, incorporate labelled tektin A to the same extent and have similar pools of unincorporated proteins. The correlation of enhanced tektin A synthesis with increased ciliary length and the coincidence of tektin A synthesis with ciliary elongation are observations consistent with the hypothesis that tektin A is a ciliary length-limiting structural element.
Biochemical characterization of the proteins of Paramecium secretory granules
Authors: Tindall Stephen H. , Devito Lynn D. , Nelson David L. 
Published: April 2021

ABSTRACT
The proteins of trichocysts (secretory granules) from Paramecium tetraurelia have been biochemically characterized. Two-dimensional electrophoresis revealed 34 major components and at least 120 minor components, most with molecular weights ranging from 14000 to 21000 and isoelectric points ranging from 4·8 to 5·2. Comparison of two-dimensional electrophoretic patterns of trichocysts before and after exocytosis revealed only minor changes in these patterns, although the protein matrix undergoes a striking change in morphology. To clarify the interrelationships among trichocyst proteins, two proteins from extruded trichocyst matrix were purified to homogeneity and sequenced at their N termini. Their sequences are distinct, but they share limited homology.
The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells
Authors: Rochlin M. William , Chen Qiming , Tobler Markus , Turner Christopher E. , Burridge Keith , Peng H. Benjamin 
Published: April 2021

ABSTRACT
Talin is involved in mediating the cytoskeleton-extracellular matrix interaction at focal contacts in cultured fibroblasts. Recently this protein has been localized at both the myotendinous junction (MTJ) and the neuromuscular junction (NMJ) in skeletal muscle. At the MTJ, talin may mediate the insertion of myofibrils into the plasma membrane, thus serving a function similar to that seen at focal contacts. However, the function of talin at the NMJ is unknown. In this study, we examined its distribution at both mature and developing acetylcholine receptor (AChR) clusters in Xenopus muscle cells both in vivo and in vitro with immunofluorescence. At the NMJs of both myotomal and submaxillaris muscles, talin was absent from the AChR clusters. In cultured myotomal muscle cells, it was absent from 40% of both the nerve-associated AChR patches and the spontaneously formed AChR clusters located on the top surface of the cells. We therefore conclude that this protein is not essential for maintenance of AChR clusters at the NMJ. In addition to MTJs, talin was invariably associated with AChR clusters induced by polyornithine-coated beads, and, to a large extent, with spontaneously formed clusters on the ventral side of cultured cells. A common feature of these talin-positive domains is the deep membrane infoldings, where bundles of actin filaments are inserted into the membrane. Thus, talin may be involved in the formation and maintenance of these structures. The deep membrane infoldings, though prominent at most NMJs, are absent from the two muscles under study in vivo. Our work thus suggests that the postjunctional membrane at the NMJ is heterogeneous, consisting of an AChR cluster domain and, often but not always, a domain for proteins involved in cytoskeleton-membrane linkage as exemplified by talin.
A rapid and convenient assay for counting cells cultured in microwell plates: Application for assessment of growth factors
Authors: Oliver Michael H. , Harrison Nicholas K. , Bishop Jill E. , Cole Peter J. , Laurent Geoffrey J. 
Published: April 2021

ABSTRACT
There is currently much interest in the role of mediators that regulate cell proliferation. Methods to assay proliferative effects of such mediators usually involve cell counting techniques, which are tedious to perform, or methods based on uptake of radiolabelled thymidine, which may be prone to errors caused by precursor pool artefacts. We describe here an assay for estimating the number of adherent cells present in a microculture and its application to the study of growth factors. The assay depends on the binding of Methylene Blue to the fixed monolayer at pH 8·5 and, after washing the monolayer, release of dye by lowering pH. The use of an elution solvent containing acidified ethanol ensures a linear correlation between absorbance of the dye and cell number, and enables the assay to be carried out in 96-well plates measuring absorbance with an automated vertical light-path microplate photometer. The assay is rapid, highly reproducible and easy to perform, making it ideal for screening large numbers of samples. It was shown to be applicable to a number of foetal and adult cell lines derived from man and experimental animals. It was also demonstrated to be useful for assaying purified growth factors and detecting growth promoting activity in cell and tissue extracts.
Glycosylation mutations of serine/threonine-linked oligosaccharides in low-density lipoprotein receptor: Indispensable roles of O-glycosylation
Authors: Kuwano Michihiko , Seguchi Tadashi , Ono Mayumi 
Published: April 2021

ABSTRACT
The low-density lipoprotein (LDL) receptor is a surface glycoprotein that mediates the cellular uptake of LDL, a cholesterol-carrying plasma protein (Goldstein et al. 1985). After receptor-mediated endocytosis of LDL, LDL receptor recycles to the cell surface from the acid compartment, the endosome, and LDL is then transported and degraded in lysosomes where the cholesterol ester core is hydrolysed and from which the unesterified cholesterol is released. The cholesterol molecules from LDL regulate de novo cholesterol biosynthesis and LDL receptor expression. Mutations in the receptor gene for the LDL receptor impair LDL uptake into cells and cause familial hypercholesterolemia (FH) because of the lack of normal regulation of cholesterol metabolism (Goldstein et al. 1985).
Involvement of transmembrane proteins in the association of cortical microtubules with the plasma membrane in tobacco BY-2 cells
Authors: Akashi Tomohiro , Shibaoka Hiroh 
Published: April 2021

ABSTRACT
Abundant microtubules (MTs) were present both in protoplasts isolated from tobacco BY-2 cells and on membrane ghosts prepared from such protoplasts. However, only a few MTs or none at all were observed on membrane ghosts prepared from protoplasts pretreated with protease, trypsin or chymotrypsin, although abundant MTs were present in protease-pretreated protoplasts. These observations suggest that the digestion of the extracellular portion of transmembrane protein(s) results in the dissociation of cortical MTs from the plasma membrane. Exogenously applied extensin or poly-L-lysine increased the cold-stability of cortical MTs in the control protoplasts, but not in protease-pretreated protoplasts. It appears that the transmembrane protein(s) is also involved in the stabilization of cortical MTs by extensin or poly-L-lysine. Cortical MTs in BY-2 cells were arranged parallel to one another and were resistant to cold. Treatment with protease rendered the MTs sensitive to cold and often disturbed the parallel array of cortical MTs. These results suggest that transmembrane protein(s) is involved in the arrangement and stabilization of cortical MTs in tobacco BY-2 cells.
Expression of a developmentally regulated cross-linking intermediate filament-associated protein (ifapa-400) during the replacement of vimentin for desmin in muscle cell differentiation
Authors: Cossette Louis J. , Vincent Michel 
Published: April 2021

ABSTRACT
Myogenic and neurogenic tissues of the chick embryo transiently express IFAPa-400, a high molecular weight protein that colocalizes and is copurified with intermediate filaments. Using monoclonal antibody F51H2 to identify it, we carried out immunoelectron microscopy experiments on whole-mount chick embryo cells and showed that IFAPa-400 was localized at crossing points of intermediate filaments. Also, immunoblot experiments with F51H2, anti-vimentin and anti-desmin antibodies demonstrated the complete disappearance of IFAPa-400 in those muscle cell types that change their vimentin content for desmin during embryogenesis. During in vitro myogenesis, the expression of IFAPa-400 was shown to be concurrent with the progressive replacement of vimentin by desmin in myoblasts. When long-term myotube cultures were maintained on a fibroblastlike cell layer, we observed the complete replacement of vimentin by desmin, followed by the disappearance of IFAPa-400 from the myotubes. These results suggest that IFAPa-400 might be involved in the reorganization of the intermediate filament network during muscle differentiation.
Cellular invasion into matrix beads: Localization of fa integrins and fibronectin to the invadopodia
Authors: Mueller Susette C. , Chen Wen-Tien 
Published: April 2021

ABSTRACT
We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, α1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, α1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing α1 and several β chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, α1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with α1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of α1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.
Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts
Authors: Fox M. H. , Arndt-Jovin D. J. , Jovin T. M. , Baumann P. H. , Robert-Nicoud M. 
Published: April 2021

ABSTRACT
The temporal course of replication monitored by 2- or 5-min pulses of bromodeoxyuridine (BrdUrd) incorporation in synchronized 3T3 cells was mapped by high-resolution light microscopy employing a charge-coupled device (CCD) camera and a confocal laser scanning microscope (CLSM). The cells were labeled simultaneously with monoclonal antibodies directed against BrdUrd and nuclear lamin, and stained with the A+T-specific dye 4′,6-diamidino-2-phenylindole (DAPI). Stereoscopic reconstructions of cells showing both the lamin and BrdUrd distributions demonstrate that DNA replication occurs at discrete sites in the nucleus, the locations of which progress through a programmed sequence during S phase. Replication begins in a small number of sites in the interior of the nucleus exclusive of the nuclear membrane and proceeds rapidly in early S phase to encompass a relatively large number of small, discrete sites located throughout the nucleus, with the exception of the condensed heterochromatic regions. Replication is primarily confined to the condensed heterochromatic regions in mid-to-late S phase, and to the nuclear periphery at the end of S phase. These distinctive patterns demonstrate a programmed control of replication sites in the spatial domain in differentiated cell nuclei.
Pollen development in orchids 3. A novel generative pole microtubule system predicts unequal pollen mitosis
Authors: Brown R. C. , Lemmon B. E. 
Published: April 2021

ABSTRACT
Cytoplasmic preparation for the unequal first mitosis in non-vacuolate pollen of moth orchids (Pbalaenopsis) includes reorganization of the microtubular cytoskeleton and nuclear migration. Following meiotic cytokinesis, both microtubules and F-actin are unpolarized in microspores of persistent tetrads. Microtubules radiate from the centrally located nucleus and F-actin forms a reticulate pattern in the cytoplasm. Polarization of the microspores is marked by a dramatic reorganization of microtubules while the pattern of F-actin remains unchanged. We describe a novel system of microtubules at the generative pole (GPMS), which forms a polar structure at the distal surface and marks the path of nuclear migration prior to pollen mitosis. The GPMS consists of numerous microtubules that extend between the plasma membrane and nuclear envelope. The nucleus becomes displaced toward the generative pole and flattened in association with microtubules of the GPMS. Initiation of the GPMS is marked by a localized proliferation of ER and clearing of large organelles from the generative pole.
A common epitope is shared by ciliary rootlets and cell-cell adherens junctions in ciliated ependymal cells
Authors: Peraldi-Roux Sylvie , Klotz Catherine , Nguyen-Thanh-Dao Brigitte , Gabrion Jacqueline 
Published: April 2021

ABSTRACT
Using immunoblot, light and electron immunocytochemistry, we investigated the presence and the localization of polypeptides cross-reacting with the monoclonal antibody CC.310 (mAb CC.310), which is mainly directed against a 175K (K=103Mr) ciliary rootlet protein. In hypothalamic ependymal cultures, the unique antigen recognized by mAb CC.310 was associated with the Triton X-100-insoluble fraction in these cultures and electrophoretically migrated to 94K. mAb CC.310, which appears to be a very suitable marker for ciliated ependymocytes, allowed us to observe ciliogenesis during the growth of the ependymal cultures, from a single spot in each undifferentiated ependymal cell to a massive labeling in ciliated ependymal cells. In fully differentiated ciliated epen dymocytes, mAb CC.310 strongly reacted with fibrous structures corresponding to ciliary rootlets, as confirmed by ultrastructural observations. In addition, a weaker immunostaining was also found along the intercellular junctions, and showed that proteins sharing a common epitope are located in ependymal ciliary rootlets and near adherens-type junctional complexes. Immunofluoréscence studies confirmed the presence of positive labeling at the level of junctional complexes between cells in two epithelial lines, HeLa and PtK2, in which mAb CC.310 mainly reacted with one polypeptide of 85K.
Expression of lymphocyte perforin in the mouse uterus during pregnancy
Authors: Zheng Li Mou , Liu Chau-Ching , Ojcius David M. , Young John Ding-E 
Published: April 2021

ABSTRACT
In situ hybridization and immunofluorescence were used to study the expression of a lymphocyte poreforming protein (perforin) in the uterus of pregnant mice. Cells expressing perforin mRNA were detected as early as gestation day 5, whereas perforin protein was detected one or two days later. Although the number of cells expressing both perforin mRNA and perforin protein varied subsequently with time, they were consistently observed from the day of implantation until parturition. The highest levels of mRNA expression were observed sometime dining midgestation. The high abundance of this cytolytic protein in the metrial gland during pregnancy and the time course of its expression thus suggest that GMG perforin expression is tightly regulated, probably hormonally, by the uterus, and that GMG cell perforin plays an important role in the normal completion of pregnancy.
Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo
Authors: Aggeler Judith , Ward Jerome , Blackie Leslie Mackenzie , Barcellos-Hoff Mary Helen , Streuli Charles H. , Bissell Mina J. 
Published: April 2021

ABSTRACT
In the present study we provide evidence that the cytodifferentiation of primary mouse mammary epithelial cells within the alveolar-like structures formed after culture on a reconstituted basement membrane resembles development in vivo during late pregnancy and early lactation. During the first two days in culture on a basement membrane gel in the presence of lactogenic hormones, epithelial cells isolated from mid-pregnant mice are disorganized and central lumina are largely absent. Levels of mRNA for the milk proteins, β-casein and transferrin, are dramatically reduced. By the second or third day in culture, cytoplasmic polarization becomes evident and prominent apical junctional complexes are formed. Synthesis of both mRNA and milk protein is reinitiated at this time. By day 4, well-defined lumina appear, and abundant synthesis and secretion of casein and lipid is observed. A striking feature of this differentiation in culture is the specific localization of milk protein gene expression (β-casein mRNA) to luminal epithelial cells in the alveolar-like structures. At the ultrastructural level, increased milk protein synthesis and secretion are paralleled by a fourfold increase in rough ER that resembles the dramatic increase in the ER observed in vivo following parturition. One indication of tissuespecific differentiation observed in later cultures (days 4–11) is the synthesis and secretion of abundant casein micelles. A second characteristic of lactating mammary epithelial cells in vivo that has not previously been observed in culture is the secretion of milk fat globules. Taken together, these observations indicate that mammary epithelial cells plated onto a reconstituted basement membrane differentiate to the lactating phenotype in culture.
Morphological expression of cell transformation induced by c-Ha-ras oncogene in human breast epithelial cells
Authors: Russo J. , Tait L. , Russo I. H. 
Published: April 2021

ABSTRACT
The present work describes the morphological pattern of c-Ha-ras-transformed MCF-10A cells. This immortalized human breast epithelial cell line was transfected utilizing the calcium phosphate technique with pHo6 containing the neomycin-resistant gene alone, and identified as MCF-lOAneo, or with the normal Ha-ras proto-oncogene, MCF-lOAneoN, or with the human p24 mutated Ha-ras oncogene, MCF-lOAneoT cells. These three cell types were studied by scanning and transmission electron microscopy at passages 6 and 20 post-transfection. It was observed that transfection with the plasmid alone did not induce any morphological changes in MCF-10A cells. These two cell types exhibited those features that are characteristic of mammary epithelial cells in culture. Amplification of the normal c-Ha-ras oncogene by transfection induced significant morphological changes at the level of cell shape, from flat to cuboidal, and cytoplasmic changes suggesting a more metabolically active cell. These changes were made more prominent by transfection with the mutated ras oncogene, which induced stratification of a cuboidal epithelium and increase in cell size as well as a more pleomorphic nuclear and cytoplasmic appearance.
Distinctive features induced by the mutated c-Ha-ras oncogene were the lengthening and thickening of cell surface microvilli, formation of blebs and emission of filopodial projections. It induced cytoplasmic changes consisting of formation of intracellular lumina, and increased the number of lysosomes, mitochondria and glycogen content, significantly decreasing the number of intermediate filaments. This is the first report that describes the morphological characteristics of a human breast epithelial cell line transformed in vitro.
Cell surface oligosaccharides on Dictyostelium during development
Authors: Amatayakul-Chantler Supavadee , Ferguson Michael A. J. , Dwek Raymond A. , Rademacher Thomas W. , Parekh Raj B. , Crandall Ian E. , Newell Peter C. 
Published: April 2021

ABSTRACT
Developmental studies of the changes in protein glycosylation are useful in elucidating the role of oligosaccharides in biological events. We have used the chemical technique, hydrazinolysis, to release oligosaccharides from cell surface glycoproteins of Dictyostelium discoideum. Oligomannose type, xylose- and fucose-containing oligosaccharides were found to be present. The charged oligosaccharides contained sulphate and mannose 6-phosphate residues; no sialic acid was detected. The charged oligosaccharides also contained significant amounts of xylose, arabinose, fucose and galactose, as well as mannose and N-acetylglucosamine, which were the main constituents of the neutral glycans.
By monitoring the chemical characteristics of the liberated oligosaccharides, dramatic changes in both the charge and size distribution of cell surface oligosaccharides were observed throughout the 24 h period of cell development. A comparison, however, between the neutral glycan structures of prestalk and prespore cells, over the same time frame showed no dramatic differences.
Discoidin, a lectin present on the cell surface of 8 h cells, was found not to be glycosylated. Affinity chromatography using immobilised discoidin was used to probe a sugar library made from the cell surface glycoproteins of 8h cells. Discoidin was found to bind selectively an oligosaccharide with the structure Manα3(Manα6)(Xylβ2)Manβ4GlcNAc. This oligosaccharide lacks a conventional N,N′-diacetyl- chitobiose core and has only been previously observed in plant glycoproteins. Peptide-N-glycosidase F treatment of horseradish peroxidase released an identical structure, confirming that the oligosaccharide was not a degradation fragment of the hydrazine. The oligosaccharide was found to inhibit discoidin-mediated haemagglutination with a Ki of 0.75 mM, a concentration approximately 100 times lower than that for galactose.
The correlation between changes in the amoebal plasma membrane oligosaccharide structures and the biological events occurring at different stages of development such as cell-cell adhesion and cell-substratum attachment suggest an important role for sugars in these processes.