bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure
Authors: Stolz Donna Beer , Jacobsonf Bruce S. 
Published: April 2021

ABSTRACT
In this report we describe a rapid, high-yield protocol for the isolation of apical (AP) and basolateral (BL) plasma membrane domains from monolayers of bovine aortic endothelial cells (BAECs) grown on tissue culture dishes as well as microcarrier beads. Using a modified cationic colloidal silica microbead membrane-isolation procedure, which deposits a uniform silica-polyacrylate pellicle over the entire AP membrane surface, a 4- to 9.6-fold relative enrichment of AP membrane and a 3.55- to 3.67-fold relative enrichment of BL membrane was obtained when the isolated domains were examined for silica and Na+/K+-ATPase, respectively. Immunoblotting of the isolated membrane domains displayed the presence of angiotensin-converting enzyme (ACE) exclusively in the AP domain and collagen receptors (CRs) highly enriched in the BL membrane domain when monolayers were grown on a gelatin substratum.
The Drosophila ncd microtubule motor protein is spindle-associated in meiotic and mitotic cells
Authors: Hatsumi Machiko , Endow Sharyn A. 
Published: April 2021

ABSTRACT
The nonclaret disjunctional (ncd) protein is required for normal chromosome distribution in oocytes and early embryos. Mutants of ncd cause frequent nondisjunction and loss of chromosomes, suggesting a role for the protein in spindle function or chromosome movement in meiosis and early mitosis. The ncd protein contains a region of predicted sequence similarity to the microtubule motor protein, kinesin. In vitro motility assays have demonstrated that ncd is a motor that unexpectedly moves toward the minus ends of microtubules, opposite to the direction of kinesin movement. Using antibodies directed against nonconserved regions of the protein, we have localized the ncd motor protein to the meiotic and early mitotic spindle, and to spindles in a mitotically dividing cultured cell line. Its presence in the spindle of meiotic and mitotic cells implies a role for the protein as a spindle motor. The motor may play an essential role in establishing spindle bipolarity in meiosis.
Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells
Authors: Rodrigo Rosa M. , Rendón M. Carmen , Torreblanca José , García-Herdugo Gregorio , Moreno Francisco J. 
Published: April 2021

ABSTRACT
We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying monoand two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.
Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines
Authors: Meikle Murray C. , Bord Sharyn , Hembry Rosalind M. , Compston Juliet , Croucher Peter I. , Reynolds John J. 
Published: April 2021

ABSTRACT
Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents.
In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2-to 3-fold more potent than either PTH or 1,25(OH)2D3.
Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis. Inhibitor gel zymography confirmed that TIMP-1 was constitutively synthesized and that its expression was increased by MCM treatment; TIMP-2 could not be detected.
These data confirm the findings of previous investigators that human osteoblasts constitutively produce gelatinase-A and TIMP-1, and show for the first time that they can also be stimulated to synthesize collagenase, gelatinase-B and stromelysin. The working hypothesis that osteoblast MMPs play an important role in bone resorption would therefore appear to be as valid for human as it is for rodent bone.
Human articular surface chondrocytes initiate alkaline phosphatase and type X collagen synthesis in suspension culture
Authors: Stephens Myra , Kwan Alvin P. L. , Bayliss Michael T. , Archer Charles W. 
Published: April 2021

ABSTRACT
The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10– 15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid
Authors: Thyberg Johan , Moskalewski Stanislaw 
Published: April 2021

ABSTRACT
A combination of immunocytochemical and electron microscopic methods was used to study the effects of okadaic acid, a specific inhibitor of protein phosphatase types 1 and 2A, on the Golgi complex and the microtubule system of interphase CHO cells. At a concentration of 0.25 M and within 2-3 h of exposure, okadaic acid caused a reversible disorganization of the Golgi complex, observed as a disintegration of the stacks of cisternae and formation of clusters of tubules and vesicles dispersed in the cytoplasm. At the same time, staining for mannosidase II was shifted from the Golgi stacks to the endoplasmic reticulum, whereas the clusters of tubules and vesicles for the main part were negative. This change in localization of the enzyme was not blocked by cycloheximide and thus not dependent on ongoing protein synthesis. The changes in the morphology of the Golgi complex were coordinated in time with a remodelling of the microtubule system, observed as a reduction in the number of microtubules, a tendency of the remaining microtubules to arrange in an aster-like pattern, and an increased sensitivity to low concentrations of the microtubule-disruptive drug nocodazole. After removal of the drug, the microtubule system was rapidly normalized (1-2 h) and subsequently also the Golgi complex (4-8 h). The results suggest that okadaic acid induces a redistribution of the Golgi stacks into the endoplasmic reticulum, leaving the trans-most elements behind as tubules and vesicles. Altogether, the changes in the organization of the Golgi complex and the microtubule system caused by okadaic acid resemble the changes previously described to occur in these organelles at the onset of mitosis, possibly indicating a similar mechanism of action involving specific protein phosphorylation.
Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages
Authors: Russell David G. , Xu Songmei , Chakraborty Prasanta 
Published: April 2021

ABSTRACT
The continued success of Leishmania as an intra-macrophage parasite is dependent on its ability to survive within an acidic intracellular compartment, resist degradation by lysosomal hydrolases, exploit the host cell as a source of nutrients, and avoid the macrophage’s antigen-presenting capabilities. All these requirements are dependent on the properties of the parasitophorous vacuole in which Leishmania resides. This study shows that the vacuole possesses membrane proteins characteristic of a lysosome, and has MHC class II molecules. The trafficking of a variety of endocytic markers supports this finding. However, a temporal study up to 14 days post-infection indicates that, as it matures, the vacuole gains mannose 6-phosphate receptor, and becomes more accessible to endocytosed ligand, suggesting that the vacuole has functionally translocated from a lysosomal to late endosomal compartment. Endocytosed material was detected in the flagellar pocket and inside the amastigote, demonstrating parasite uptake of intra-vacuolar material. Careful analysis of amastigotes suggests that they avoid antigen presentation by their host cell by limiting the release of potential antigens. These findings significantly extend our understanding of the mechanisms employed by Leishmania to ensure its survival in the macrophage.
N-cadherin and N-CAM in myoblast fusion: compared localisation and effect of blockade by peptides and antibodies
Authors: Mege R. M. , Goudou D. , Diaz C. , Nicolet M. , Garcia L. , Geraud G. , Rieger F. 
Published: April 2021

ABSTRACT
The expression and distribution of two cell adhesion molecules, N-cadherin and N-CAM, at the surface of cultured leg muscle cells from 11-day-old chicken embryos were studied and compared. N-cadherin, which was expressed by fusing myoblasts, was down-regulated on old myotubes while N-CAM was still present. Both molecules, as viewed by confocal microscopy, appeared to have coaccumulated at the areas of contact between fusing myoblasts. However, immunogold electron microscopy did not reveal significant colocalization of N-cadherin and N-CAM, and their segregation after antibody-induced patching suggested the absence of direct interactions between N-cadherin and N-CAM. The role of the Ca2+ dependent cell adhesion molecule N-cadherin in myogenesis was investigated. Myoblast fusion was inhibited (1) with a synthetic peptide containing the H-A-V sequence and (2) with a monoclonal anti-N-cadherin antibody, demonstrating that N-cadherin-mediated cell adhesion is required for myoblast fusion. Under the same conditions no effect of anti-N-CAM antibodies was observed. Taken together these observations suggest that N-cadherin, acting independently from N-CAM, is a major cell adhesion molecule involved in embryonic myoblast fusion in vitro .
A chimeric N-cadherin/β 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions
Authors: Geiger Benjamin , Salomon Daniela , Takeichi Masatoshi , Hynes Richard O. 
Published: April 2021

ABSTRACT
To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.
Nuclear proteins of the bovine esophageal epithelium: I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells
Authors: Tang Tang K. , Hong Tse-Ming , Lin Chen-Yong , Lai Mei-Ling , Liu Catherine H. L. , Lo Hsiu-Jong , Wang Ming-Eng , Chen Lan Bo , Chen Wen-Tien , Ip Wallace , Lin Diane C. , Lin Jim J.-C. , Lin Shin , Sun Tung-Tien , Wang Eugenia , Wang John L. , Wu Reen , Wu Cheng-Wen , Chien Shu 
Published: April 2021

ABSTRACT
Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro.
Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of Mr ∼33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of Mr ∼22,000 and ∼11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of ∼11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.
Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease
Authors: Oberhammer Franziska , Fritsch G. , Schmied M. , Pavelka M. , Printz D. , Purchio T. , Lassmann H. , Schulte-Hermann Rolf 
Published: April 2021

ABSTRACT
A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-β1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-β1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca2+-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-β1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.
Shapes of cells spreading on fibronectin: measurement of the stellation of BHK21 cells induced by raising cyclic AMP, and of its reversal by serum and lysophosphatidic acid
Authors: Edwards J. G. , Campbell G. , Carr M. , Edwards C. C. 
Published: April 2021

ABSTRACT
In common with many other animal cells in culture, BHK21, CHO and NIH-3T3 cells adopt bizarre stellate or arborized shapes when exposed, in the absence of serum, to agents which increase cytoplasmic cyclic AMP (cAMP). Dibutyryl cAMP, 3-isobutyl-1-methylxanthine, 5′-deoxy-5′-methylthioadenosine, cholera toxin and the invasive adenylate cyclase from Bordetella pertussis all induce similar shapes. Time lapse video recording of BHK21 cells spreading on fibronectin shows that stellate shapes are generated by outgrowth of neurite-like processes led by small fans of ruffling membrane. These structures stain strongly for F actin, and their out-growth is completely inhibited by cytochalasin D. Thus if stellation is caused by microfilament depletion, this must be selective for subsets of microfilaments.
We have quantified the shape changes of BHK21 cells using the parameter dispersion. They are prevented by low concentrations (1% by volume and below) of bovine sera. The inhibitory component of foetal bovine serum acts humorally, behaves as a macromolecule and is itself inhibited by suramin, but platelet-derived growth factor, insulin, vasopressin and bradykinin are inactive. The inhibitory activity of serum may be due to phos-pholipids, since it can be replaced by lysophosphatidic acid in the presence of serum albumin.
EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes
Authors: Suprenant Kathy A. , Dean Kathleen , McKee Julie , Hake Shelly 
Published: April 2021

ABSTRACT
The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acidlabile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypeptide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles.
Mycelial and syncytial growth in Schizosaccharomyces pombe induced by novel septation mutations
Authors: Sipiczki M. , Grallert B. , Miklos I. 
Published: April 2021

ABSTRACT
Mutation in the gene sep1+ of the unicellular fission yeast Schizosaccharomyces pombe impairs cell separation after cytokinesis and confers a branching mycelial mor­phology. The mutant is not defective in cell wall β-glu- canase activity but shows increased sensitivity to Ca2+ and Mg2+, and increased resistance to the microtubule inhibitor benomyl. The mycelial growth of sep1-1 pro­vides a convenient method for the examination of the polar growth pattern and for pedigree analysis as demonstrated by the segregation of mating types in the homothallic microhyphae. sepl is closely linked to adel (0.94 cM) on the right arm of chromosome II. The ts mutation spl1-1 confers a bent cell shape and causes aberrant septum formation at the restrictive tempera­ture. sep1+ and spl1+ perform closely related functions as their mutant alleles interact with each other and with another septation mutant cdc4-8. These functions may overlap with certain cytoskeletal processes and with the determination of cell polarity because the triple mutant forms huge multinucleate syncytia with promiscuous branching and rare septum formation.