bluuebunny/crossref_metadata_2025_split huggingface dataset:
Meiosis-specific cell cycle regulation in maturing Xenopus oocytes
Authors: Ohsumi Keita , Sawada Wako , Kishimoto Takeo 
Published: April 2021

ABSTRACT
Meiotic cell cycles differ from mitotic cell cycles in that the former lack S-phase in the interphase between meiosis I and meiosis II. To obtain clues for mechanisms involved in the cell cycle regulation unique to meiosis, we have examined changes in chromosomal morphology and H1 kinase activity during a meiotic period from metaphase I (MI) to metaphase II (MII) in Xenopus oocytes. Using populations of oocytes that underwent germinal vesicle breakdown (GVBD) within a 10 minute interval, we found that the kinase activity declined gradually during the 60 minute period after GVBD and then increased steadily during the following 80 minute interval, showing remarkable differences from the rapid drop and biphasic increase of the kinase activity in intermitotic periods (Solomon et al. (1990)Cell 63, 1013-1024; Dasso and Newport (1990)Cell 61, 811-823). We also found that the exit from MI lagged, by more than 30 minutes, behind the time of lowest H1 kinase activity, whereas the two events took place concomitantly at the end of meiosis II and mitosis. Conse quently, the H1 kinase activity was already increasing during the first meiotic division. When H1 kinase activation at MII was delayed by a transient inhibition of protein synthesis after GVBD, oocytes were able to support formation of interphase nuclei and DNA replication between the first meiotic division and the MII arrest, indicating that the cell cycle entered S-phase between meiosis I and meiosis II. These results strongly suggest that the machinery required for entering S-phase has been established in maturing oocytes by the end of meiosis I. The lack of S-phase in oocyte meiotic interphase, therefore, should be ascribed to cell cycle regulation that enables the transition from meiosis I to meiosis II without S-phase. The asynchrony between the inactivation of H1 kinase activity and the completion of meiosis I may be involved in the regulation of this unique feature of the meiotic cell cycle.
Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts
Authors: Hola M. , Castleden S. , Howard M. , Brooks R. F. 
Published: April 2021

ABSTRACT
Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.
Differential localization of myosin-II isozymes in human cultured cells and blood cells
Authors: Maupin Pamela , Phillips Carrie L. , Adelstein Robert S. , Pollard Thomas D. 
Published: April 2021

ABSTRACT
We used purified polyclonal antibodies to human cytoplasmic myosin-IIA and myosin-IIB directly labeled with fluorescent dyes to localize these myosin-II isozymes in HeLa cells, melanoma cells and blood cells. Both antibodies react strongly with myosin-II isozymes in HeLa cells, melanoma cells and blood eosinophils, but only anti-myosin-IIA antibodies stain platelets, lymphocytes, neutrophils and monocytes in smears of human blood. Both antibodies stain small spots along the stress fibers of interphase HeLa cells and melanoma cells, but double staining revealed that the detailed distributions of myosin-IIA and myosin-IIB differ. A low concentration of diffuse myosin-IIB is present in the cortex, both in lamellar regions around the periphery of the cell and over the free surface. Myosin-IIB is also concentrated in spots along perinuclear stress fibers. Myosin-IIA is absent from the cortex but is concentrated in spots along stress fibers located near the basal surface of cultured cells.
This population of peripheral stress fibers is highly enriched in myosin-IIA relative to myosin-IIB, but both are found together in centrally located stress fibers. In prophase and metaphase both isozymes are concentrated in the cortex in small spots less than 04.µm in size, similar to those in stress fibers. As the chromosomes begin the separate at anaphase, most of the myosin-II spots become concentrated in the outer 0.7 µm of the equatorial cortex in 100% of cells. This concentration of myosin-II isozymes in the cleavage furrow is maintained until the daughter cells separate. The superimposition of these small spots concentrated in the cleavage furrow produces the intense, uniform staining observed in conventional micrographs of whole cells.
Approaching microtubule structure with the scanning tunneling microscope (STM)
Authors: Maaloum M. , Chrétien D. , Karsenti E. , Hörber J. K. H. 
Published: April 2021

ABSTRACT
We demonstrate that the scanning tunneling microscope can be used to obtain information about arrangement of tubulin subunits in the microtubule wall. Long rows of subunits with a periodicity of 3.8±0.4 nm were clearly visible in the images of microtubules. The separation between the rows of subunits was 4.8±0.4 nm. Close inspection of two images revealed another periodicity of 7.8±0.4 nm in the contour levels of the protofilaments. This indicates that α and β tubulin monomers can be resolved. In these areas the monomers were arranged according to a ‘B-type’ lattice. Scanning tunneling microscope images confirm that the lateral contacts between tubulin monomers in adjacent protofilaments are compatible with a three-start, left-handed helix model. This study demonstrates that scanning tunneling microscopy can give direct information on the structure and organization of macromolecular assemblies and can complement the classical methods of electron microscopy and X-ray scattering.
A specifically apical sub-membrane intermediate filament cytoskeleton in non-brush-border epithelial cells
Authors: Rodriguez Marcelo L. , Brignoni Mirtha , Salas Pedro J. I. 
Published: April 2021

ABSTRACT
Although many pieces of evidence support the notion of a role for the cytoskeleton in epithelial polarization, no cytoskeletal component has been found to be specifically apical, except for some actin-binding proteins. Here we report the apical distribution of a 53 kDa cytokeratin. Furthermore, this cytokeratin co-purified with biotinylated apical plasma membrane proteins in high density complexes. Differential biotinylation of the basolateral domain showed that the 53 kDa protein is mainly attached to the apical membrane, although a companion 58 kDa protein attaches to both apical and basolateral membrane proteins. Immunoprecipitation experiments indicated that a number of apical components are directly or indirectly linked to the 53 kDa protein. These results indicate the existence of a terminal web-like structure in non-brushborder cells, which attaches to the apical domain and may play a role in apical polarization, especially during the acquisition of polarity from non-polarized cellular stages.
Integrin α 6β 4 mediates dynamic interactions with laminin
Authors: Tözeren Aydin , Kleinman Hynda K. , Wu Stephen , Mercurio Arthur M. , Byers Stephen W. 
Published: April 2021

ABSTRACT
We present here a novel form of dynamic adhesion in which both the integrin receptor and the ligand supporting dynamic adhesion have been identified. Laminar flow assays showed that laminin supported attachment of α 6β 4-positive cells in the presence of fluid shear stress (τ≤2 dyn/cm2), indicating that these cells adhered to laminin within a fraction of a second. Further increases in flow rate (3.5 dyn/cm2≤τ≤100 dyn/cm2) initiated rolling of attached cells in the direction of flow, suggesting that rapidly formed adhesion is reversible and repeatable. Laminin fragment E8, which interacts with α6 integrins, supported dynamic attachment and rolling but extracellular matrix glycoprotein fibronectin did not. In cell lines that express α 6β 4 but not α 6β 1an anti-α 6 monoclonal antibody inhibited attachment to laminin in the presence of flow and following 5 minutes of static incubation. Infusion of this antibody onto cells adherent to laminin-coated slides led to rapid detachment of cells from the substratum. An anti-β1 monoclonal antibody diminished adhesion strength following static incubation but did not inhibit rapid attachment and flow-initiated rolling. These results indicate that in some α 6β 4-expressing epithelial and carcinoma cell lines, integrin α 6β 4mediates rapidly formed dynamic interactions with laminin.
Epican, a heparan/chondroitin sulfate proteoglycan form of CD44, mediates cell-cell adhesion
Authors: Milstone Leonard M. , Hough-Monroe Lynne , Kugelman Lisa C. , Bender Jeffrey R. , Haggerty John G. 
Published: April 2021

ABSTRACT
Epican is a heparan/chondroitin sulfate proteoglycan form of CD44 and is expressed on the surface of keratinocytes from the basal layer to the granular layer of the epidermis. To analyze the adhesive properties of epican apart from the influence of other adhesive molecules found on keratinocytes, mouse L cell fibroblasts were transfected with CD44Epican cDNA. The epican expressed on the surface of transfected L cells was predominantly a heparan or chondroitin sulfate proteoglycan.
The CD44Epican-transfected L cells acquired: (a) a selfaggregating phenotype that required hyaluronan but was calcium-independent; and (b) a new capacity to adhere to keratinocytes, a property that was blocked by an antiepican antibody. Both aggregation and adhesion of CD44Epican-transfected cells were completely prevented by pretreatment with hyaluronidase, but were totally restored by the addition of exogenous hyaluronan. Aggregation of transfected L cells was minimally influenced by other glycosaminoglycans, but adhesion of transfected L cells to keratinocytes was substantially inhibited by heparin.
The ability of epican to mediate adhesion between keratinocytes was evaluated in a newly developed adhesion assay. In the presence of 0.03 mM calcium, a monoclonal antibody against epican inhibited keratinocyte adhesion to keratinocyte monolayers by 80%.
These data demonstrate that epican causes adhesion and aggregation in the transfected L cell model system, that the adhesive function of epican is hyaluronan-dependent, and that epican could have an adhesive function in intact epidermis.
Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network
Authors: Gillard Baiba K. , Thurmon Lisa T. , Harrell Rhonda G. , Capetanaki Yassemi , Saito Megumi , Yu Robert K. , Marcus Donald M. 
Published: April 2021

ABSTRACT
Our previous observations on the immunocytochemical colocalization of intermediate filaments and glycosphingolipids led us to analyze the role of filaments in the biosynthesis and intracellular transport of glycosphingolipids. Cells with (vim+) and without (vim−) vimentin intermediate filaments were cloned from the adrenal carcinoma cell line SW13. There was no difference between vim+ and vim− cells in the proportion of newly synthesized C6-NBD-glucosylceramide transported to the plasma membrane. The vim+ cells synthesized glycosphingolipids, especially lactosylceramide and globotriosylceramide, and to a lesser extent GM3 ganglioside, more rapidly than vim− cells. The altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a vimentin network was responsible for the differences in biosynthesis, mouse vimentin cDNA was transfected into vim− cells. Transfected cells that expressed a mouse vimentin network demonstrated a twofold or greater increase in the rate of biosynthesis of neutral glycosphingolipids and gangliosides. There was no difference between vim+ and vim− cells in the synthesis of ceramide or sphingomyelin, or in their content of phospholipids or cholesterol. The nature of the biochemical defect(s) underlying the diminished incorporation of radiolabeled sugars into glycosphingolipids is unclear. Possibilities include alterations in the ultrastructure of the Golgi and/or abnormalities in a portion of the endocytic pathway.
Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation
Authors: Chrzanowska-Wodnicka Magdalena , Burridge Keith 
Published: April 2021

ABSTRACT
Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phos-phorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phos-phorylation is important, other tyrosine kinases may also be involved.
Assembly of the cadherin-catenin complex in vitro with recombinant proteins
Authors: Aberle Hermann , Butz Stefan , Stappert Jörg , Weissig Helge , Kemler Rolf , Hoschuetzky Heinz 
Published: April 2021

ABSTRACT
The cytoplasmic domain of classical cadherins is tightly associated with three proteins termed α-, β- and γ-catenin. These accessory proteins are of central importance for the adhesive properties of this class of cell adhesion molecules. In order to examine the molecular architecture of the cadherin-catenin complex in more detail we have expressed the catenins and the cytoplasmic domain of E-cadherin as fusion proteins in Escherichia coli, and analyzed the interaction of purified recombinant cadherin and catenins in combinatorial protein-protein interaction experiments. The cytoplasmic domain of E-cadherin cannot directly associate with α-catenin but interacts with high affinity with β-catenin, whereas the binding of γ-catenin (plakoglobin) to E-cadherin is less efficient. α- and β-catenin assemble into a 1:1 heterodimeric complex. The analysis of various truncated β-catenins revealed that an α-catenin binding site in β-catenin is localized between amino acid positions 120 and 151. The central role of β-catenin for the assembly of the heterotrimeric E-cadherin/α-catenin/β-catenin complex in mixing experiments with all components was demonstrated. The reconstitution in vitro of the cadherin-catenin complex should allow the study of the interaction with signalling molecules and with the actinbased cytoskeleton.
Coiled bodies in the nucleolus of breast cancer cells
Authors: Ochs Robert L. , Stein Thomas W. , Tan Eng M. , Keck W. M. 
Published: April 2021

ABSTRACT
Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992)Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immuno-electron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.
Heparin stimulates the proliferation of intestinal epithelial cells in primary culture
Authors: Flint N. , Cove F. L. , Evans G. S. 
Published: April 2021

ABSTRACT
Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminogly-cans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysac-charides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.
The effects of age on the response of rabbit periosteal osteoprogenitor cells to exogenous transforming growth factor-β2
Authors: Critchlow Matthew A. , Bland Yvette S. , Ashhurst Doreen E. 
Published: April 2021

ABSTRACT
Additional bone and cartilage are formed if transforming growth factor-β is injected into the periosteum of calvariae or long bones. To investigate this further, transforming growth factor-β2 was injected into the periosteum of the tibia of 3-day-old, 3-month-old and 2-year-old rabbits. In all instances, there was an increase in proliferation of the cells of the cambial layer of the periosteum, that is, the osteoprogenitor cells, and breakdown of the fibrous layer. Oedema was induced in the surrounding connective tissues.
Over the experimental period the normal neonatal tibia is undergoing rapid growth; there is periosteal bone formation and endosteal resorption. In the experimental neonatal tibiae, an increase in periosteal bone formation is seen after three injections of 20 ng of transforming growth factor-β2, which is accompanied by cartilage after five injections; the amounts of induced bone and cartilage increase with the number of injections. The chondrocytes hypertrophy after 4 days and the cartilage is replaced by bone endochondrally.
In contrast, after seven injections of 20 ng transforming growth factor-β2, there is only a small amount of new bone on the 3-month-old tibia and none on the 2-year-old tibia. One day after seven injections of 200 ng transforming growth factor-β2, there is a small amount of bone formation, while seven days after cartilage is found as small discrete nodules on the 3-month-old tibia, but as small areas within the bone on the 2-year-old tibia.
It is concluded that the primary effect of transforming growth factor-β2 in this experimental model is to increase the proliferative rate of the osteoprogenitor cells in the periosteum. It is argued that transforming growth factor-β2 does not initiate osteoblastic or chondrocytic differen-tiation of osteoprogenitor cells. It is suggested that their differentiation is controlled by the local environment, in particular, the vascularity and locally circulating growth factors.
Lamellipod extension and K+current in osteoclasts are regulated by different types of G proteins
Authors: Arkett Stuart A. , Dixon S. Jeffrey , Sims Stephen M. 
Published: April 2021

ABSTRACT
Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5′-O-(3-thiotriphosphate) or 5′-guanylyl-imidodiphosphate, but not the control compounds adenosine 5′-O-(3-thiotriphos-phate) or guanosine 5′-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5′-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5′-O-(3-thio-triphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5′-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Flu-oroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5′-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectify-ing K+ current is regulated by a large molecular mass, het-erotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.