bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of glycosylation inhibitors on N-glycosylpeptides and on invasion of malignant mouse MO4 cells in vitro
Authors: Bruyneel Erik A. , Mets Marc De , Dragonetti Christian H. , Hooghe Robert J. , Virgilio Sergio Di , Mareeli Marc M. 
Published: April 2021

ABSTRACT
Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 µgml-1), between fucose-labeled and mannoselabeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of M04 cells with SW (O.4µgml-1). The shift induced by dNM (10mM)+SW (O.4µgml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; ImM) had major effects on both mannose and fucoselabeled structures and so did N-methyl-l-deoxynojirimycin (MdNM; 2mM) and castanospermine (CS; 100 µgml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucoselabeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells. After removal of sialic acid, control and SW-treated glycosylpeptides from both MO4 and embryonic chick heart cells had similar gel-chromatographic profiles, suggesting that a decrease in cell surface sialic acid accounts to a large extent for the difference between glycans from control and SW-treated cells. Additional biological experiments were done with dMM (1 mм)1 MdNM (2mM), CS (lOOµgml-1), 2,5-dihydroxymethyl-3,4-dihydroxyρyrrolidine (DMDP; 25Oµgml~i) and SW (0.4µgml-i) + dNM (10mM). All these compounds or combinations failed to inhibit invasion. The observation that inhibitors of N-linked glycan processing did not interfere with invasion, although they clearly modIfied the glycosylation of cell proteins, indicated that the integrity of glycans including those of the cell surface might not be a prerequisite for invasion of MO4 cells into living embryonic tissue in vitro.
Protein glycosylation in heat-sensitive and thermotolerance-deficient mutants of Chinese hamster ovary cells
Authors: Henle Kurt J. , Nagle William A. , Bedford Joel S. , Harvey Wells F. 
Published: April 2021

ABSTRACT
Chinese hamster ovary (CHO) cells are capable of developing a high degree of thermotolerance in response to appropriate heat conditioning. In this study we examined the relationship between thermotolerance development and protein glycosylation using four sublines of CHO cells. Two of these CHO sublines are characterized by an increased heat sensitivity and impaired cellular capacity for thermotolerance development. The data show that thermotolerance development after heat conditioning in the heat-sensitive, thermotolerance-deficient mutants was accompanied by reduced labeling of a Mr 50 000 glycoprotein (GP50), in both soluble and insoluble cell fractions. Similarly, activation of UDP-N-acetylgalactosaminyltransferase (Gal-NAcT) after hyperthermia was almost completely abolished in these cell lines. Both of these endpoints have been correlated previously with thermotolerance expression. The data are consistent with the glycosylation hypothesis that attributes increased heat resistance of thermotolerant cells, at least in part, to enhanced glycosylation and accumulation of endogenous glycoproteins, such as GP50.
DNA-RNA complexes that might represent transient attachment sites of nuclear DNA to the matrix
Authors: Patriotis Christos , Andreeva Mariana , Pascaleva Mary , Ivanov Veselin , Djondjurov Lalio 
Published: April 2021

ABSTRACT
In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The presence of such unusual structures is confirmed by the following evidence. When a preparation of matrix DNA is electrophoresed in agarose an RNA component always migrates together with DNA. There should be a close interaction between DNA and RNA in such a preparation because the presence of the RNA component causes resistance of DNA to DNase I and Exo HI. An intimate, hybrid-type association of part of the RNA component with DNA is indicated also by the fact that about 20% of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis with a-amanitin and actinomycin D it was shown that the bulk of associated RNA is transcribed by RNA polymerase III. Hybridization experiments showed similarity between the DNA sequences isolated from the complexes and those from the base of dehistonized DNA loops obtained by high-salt extraction of nuclei. This observation suggests that the complexes might represent attachment sites of nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA component. Experiments with induction of erythroid differentiation indicated that a profound reorganization of the nucleus, accompanying terminal differentiation, leads to a striking reduction in the number of complexes and thus in the number of attachment sites. This suggests that the complexes should function as transient attachment sites.
Laryngeal keratinocytes show variable inhibition of replication by TGF-β
Authors: Dilorenzo Teresa P. , Steinberg Bettie M. 
Published: April 2021

ABSTRACT
The response of secondary human laryngeal epithelial cells to transforming growth factor-β(TGF-β) was investigated, and this response was compared with that of epithelial cells derived from virally induced laryngeal papillomas. In most cases, both normal laryngeal epithelial cells and those derived from laryngeal papillomas exhibited growth inhibition in response to 10 ng ml-1 TGF-β. Response was not a function of cell density or proliferation rate when cells were in a low-calcium medium, but was reduced in high calcium. Using keratinocytes derived from several different tissue expiants, we found that cells grown from different expiants show marked variation in response to TGF-β.
Modulation of extracellular matrix biosynthesis by bovine retinal pericytes in vitro: effects of the substratum and cell density
Authors: Canfield Ann E. , Allen Terence D. , Grant Michael E. , Schor Seth L. , Schor Ana M. 
Published: April 2021

ABSTRACT
Bovine retinal pericytes plated on a two-dimensional substratum display a characteristic stellate morphology. In post-confluent cultures these cells aggregate spontaneously to form multicellular nodules. The same cells plated within a three-dimensional collagen matrix display an elongated sprouting morphology. Sprouting pericytes may be embedded within a gel either as individual cells or as multicellular aggregates. We have compared the nature of the matrix proteins synthesised by pericytes displaying these different phenotypes. Stellate pericytes cultured on plastic dishes synthesised predominantly type I collagen, some type III collagen and only traces of type IV collagen. The same collagen types were secreted when nodules had formed in postconfluent cultures on plastic, and by sprouting cells plated as single cells within the collagen gel. By contrast, sprouting pericytes plated as aggregates within the collagen gel secreted increased levels of type IV collagen and reduced amounts of type I collagen. Fibronectin was synthesised by pericytes under all experimental conditions examined; thrombospondin was produced in relatively large amounts by cells grown on plastic dishes, whereas only trace amounts could be detected in the medium when the cells were cultured within a collagen gel matrix. Transmission electron microscopy revealed that pericyte aggregates within a collagen gel contained cells in close apposition surrounded by a dense extracellular matrix. In contrast, cells in the centre of a nodule on plastic appeared to be separated from each other by loose extracellular material. These results suggest that the morphological and biosynthetic phenotypes of retinal pericytes are modulated by cell-matrix and/or cell-cell interactions.
Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane
Authors: Bloodgood Robert A. , Salomonsky Nancy L. 
Published: April 2021

ABSTRACT
The Chlamydomonas flagellar -surface exhibits a number of dynamic membrane phenomena associated with whole-cell gliding locomotion and the early events in fertilization. Crosslinking of a specific population of flagellar surface-exposed glycoproteins with the lectin concanavalin A or an anticarbohydrate mouse monoclonal antibody, designated FMG-1, results in a characteristic pattern of glycoprotein redistribution within the plane of the flagellar membrane. Recent evidence suggests that flagellar membrane glycoprotein movements are associated with both whole-cell gliding motility and the early events in mating. It is of interest to determine the transmembrane signaling pathway whereby crosslinking of the external domains of flagellar glycoproteins activates the intraflagellar machinery responsible for translocation of flagellar membrane glycoproteins.
The redistribution of flagellar membrane glycoproteins requires micromolar levels of free calcium in the medium; lowering the free calcium concentration to 10−7M results in complete but reversible inhibition of redistribution. Redistribution is maximal in the presence of 20 μM free calcium in the medium. Redistribution is inhibited in the presence of 20 pM free calcium by the calmodulin antagonists trifluoperazine, W-7 and calmidazolium, the calcium channel blockers diltiazem, methoxyverapamil (D600) and barium chloride, and the local anesthetics, lidocaine and procaine. The actions of all of these agents can be interpreted in terms of a requirement for calcium in the signaling mechanism associated with flagellar glycoprotein redistribution. In particular, the requirement for micromolar calcium in the external medium and the effects of specific calcium channel blockers suggest that flagellar membrane glycoprotein crosslinking may induce an increase in calcium influx, which may be the initial trigger for activating the flagellar machinery responsible for active movement of flagellar membrane glycoproteins.
Pulsed field gel electrophoresis and investigations into mammalian genome organization
Authors: Gardiner Katheleen 
Published: April 2021

ABSTRACT
Pulsed field gel (PFG) electrophoresis can resolve DNA molecules as large as several million base pairs (mbp) in size (Schwartz and Cantor, 1984; Carle and Olson, 1984; Gardiner et al. 1986; Carle et al. 1986; Chu et al. 1986; Clark et al. 1988; Anand, 1986; Gardiner and Patterson, 1988; Orbach et al. 1988). This is in contrast to conventional electrophoresis where the practical upper limit is 50–100 kb (lkb=103bp). This increased resolving power has especially important ramifications for the study of complex genomes, allowing new questions to be asked and providing faster solutions to older ones. It is now possible, for example, to examine gene organization, physically link and size mammalian genes, and search for translocation breakpoints by means that are far more rapid and reliable than conventional methods. PFG has made the cloning of large genes, or groups of genes, possible via the yeast artificial chromosome (YAC) method (Burke et al. 1987), and it also makes the mapping of the human genome a realistic endeavour. The purpose of this commentary is to discuss how this technique can be applied to the study of mammalian genomes, and to describe some of the insights into human genome organization that are beginning to emerge.
Identification and protein kinase c-dependent phosphorylation of α-adducin in human fibroblasts
Authors: Waseem Ahmad , Palfrey H. Clive 
Published: April 2021

ABSTRACT
A protein of Mr∼120000, related to the human erythrocyte membrane skeletal protein α-adducin, has been identified by immunological criteria in human fibroblasts. Using similar methods, β-adducin (an Mr∼110000 protein that forms a dimeric complex with α-adducin in the erythrocyte) is not present in fibroblasts. Subcellular distribution studies reveal that fibroblast α-adducin is largely associated with the particulate fraction and is most effectively solubilized from that fraction by a combination of nonionic detergent and high salt. Immunocytochemistry of quiescent fibroblasts shows that α-adducin is clustered in large perinuclear arrays that may correspond to vesicular structures; weak staining was also found in the sub-plasma membrane region. As in erythrocytes, the phosphorylation of fibroblast α-adducin is elevated on exposure of cells to phorbol esters that activate protein kinase C (PK-C). In addition, various mitogens such as serum, bradykinin and vasopressin also stimulate α-adducin phosphorylation by a PK-C-dependent pathway. The elevation in α-adducin phosphorylation is maintained for up to 30 min after mitogen addition. Peptide maps of phospho-α-adducin from both fibroblasts and erythrocytes after PK-C-mediated phosphorylation showed multiple phosphorylated peptides but with dissimilar migration patterns, suggesting divergence of structure around the phosphorylation sites. Addu-cin appears to play an important role in the regulation of spectrin-actin interactions in the red cell and may play a role in cytoskeletal function in the fibroblast.
Activation of human keratinocyte migration on type I collagen and fibronectin
Authors: Guo Ming , Toda Ken-Ichi , Grinnell Frederick 
Published: April 2021

ABSTRACT
The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by kératino-cytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained β1inte-grin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, β1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In kéra-tinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of β1integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, β1integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of β1integrin subunits accompany development of migratory competence.
Size heterogeneity, phosphorylation and transmembrane organisation of desmosomal glycoproteins 2 and 3 (desmocollins) in MDCK cells
Authors: Parrish E. P. , Marston J. E. , Mattey D. L. , Measures H. R. , Venning R. , Garrod D. R. 
Published: April 2021

ABSTRACT
Metabolic labelling with [35S]methionine and immu-noprecipitation with specific antibodies to bovine desmosomal glycoproteins 2 and 3 (dg2 and dg3: desmocollins) reveals a triplet of polypeptides of Mr 115000, 107000 and 104000 in MDCK cells. Tunica-mycin treatment shows that this heterogeneity does not arise through differential N-linked glycosylation. Under conditions in which cells are actively forming desmosomes, the largest polypeptide, dg2, becomes phosphorylated on serine, but the two smaller polypeptides, dg3a and 3b, do not Controlled trypsinisation of intact cells yields three membrane-protected fragments (Mr 28000, 24000 and 23000) derived from these glycoproteins. The largest of these fragments is phosphorylated but the two smaller fragments are not A monoclonal antibody to bovine dg2 and dg3 stains MDCK cells cytoplasmically. In immunoblotting of MDCK cells the monoclonal antibody recognises dg2 strongly and shows a weaker reaction with a band of lower Mr corresponding to dg3a. It also recognises the immunoprecipitated 28000 Mr fragment from trypsinised cells and a smaller fragment of 24000 Mr.
The simplest interpretation of these data is that all three glycoproteins have a transmembrane configuration with a single membrane-spanning domain, and show heterogeneity of size and phosphorylation in their cytoplasmic domains.
The data are discussed in relation to the known structures of some cell adhesion molecules. Questions about the relative roles and distributions of the different polypeptides in desmosomal organisation are raised.
The interaction of Trypanosoma brucei with antibodies to variant surface glycoproteins
Authors: Webster P. , Russo D. C. W. , Black S. J. 
Published: April 2021

ABSTRACT
Binding to Trypanosoma brucei of polyvalent IgMs and IgGs, monoclonal IgGs and Fabi fragments of monoclonal IgGs specific for exposed epitopes of T. brucei variant surface glycoproteins (VSGs) was monitored by both immunofluorescence and immu-nocytochemistry. All antibodies and antibody fragments, were uniformly distributed over the parasite surface after incubation with the organism at 0°C. Upon warming to 37 °C bound antibodies and fragments were detected in the flagellar pocket and intracellular organelles. Removal of single layers of bound antibody, or Fabi fragments, from the cell surface at 37 °C, as determined by immunofluorescence, was complete within 20 min and occurred in the presence or absence of protein synthesis. Para-sites that had shown an altered distribution of surface-bound antibody after warming remained fully covered with VSGs of the original antigen type as shown by immunocytochemistry.
Cycles of F-actin redistribution in the dermal glands of an insect relate to secretion
Authors: Delhanty Patrio , Locke Michael 
Published: April 2021

ABSTRACT
Cells of the dermal glands in Calpodes ethlius Stoll (Lepidoptera, Hesperiidae) survive without division but increase in size from moult to moult. The secretory cells become especially large, measuring up to 6 mm long in the fifth larval stadium. The nucleus in these giant cells has a sponge-like profile, with the nuclear envelope indented around cytoplasmic intrusions, each of which comes to contain several secretory vacuoles. The secretory vacuoles coalesce just before the glands discharge at ecdysis. Rhodaminyl phalloin labelling shows that F-actin redistributes in cycles corresponding to ecdysial secretion atlarval and pupal moults. Capsules of F-actin form around the vacuoles before ecdysis. After the vacuoles discharge, actin from the capsules survives in strands that aggregate into storage bundles during the intermoult as though the filamentous actin itself is redistributed and stored. The storage bundles disappear as the F-actin capsules re-form at the next moult
N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane
Authors: Jackson Robert C. , Modern Paul A. 
Published: April 2021

ABSTRACT
The exocytotic release of secretory products from fragments of sea urchin egg cortex has been shown to be inhibited by covalent modification of membrane sulfhydryl groups with N-ethylmaleimide (NEM). Exocytotically competent preparations of reconstituted cortex, formed by recombination of purified cortical vesicles (CVs) with fragments of egg plasma membrane (PM) were also inhibited by treatment with NEM. The cellular localization of sulfhydryl-containing constituents) responsible for inhibition was investigated by treating CVs and/or PM with NEM prior to reconstitution. Both native cortex and cortex reconstituted with NEM-treated components were challenged with calcium-containing buffers. Exocytosis was monitored by phase-contrast microscopy, and quantitated by light scattering. Evidence for CV-PM fusion was obtained with an immunofluorescence-based assay that permits visualization of the transport of CV content proteins across the PM. Cortex reconstituted by recombination of NEM-treated CVs with untreated PM or by recombination of untreated CVs with NEM-treated PM was exocytotically competent, whereas cortex formed by recombination of NEM-treated CVs with NEM-treated PM was inactive. These results: (1) support the hypothesis that the mechanism of exocytosis in native and reconstituted cortex is the same; (2) provide evidence that both CV and plasma membranes participate in the release of CV contents from reconstituted cortex; and (3) suggest that sulfhydryl-containing protein(s) present on the surface of purified CVs and plasma membrane are involved in exocytosis.
Direct visualization of cross-links in the primary plant cell wall
Authors: McCann M. C. , Wells B. , Roberts K. 
Published: April 2021

ABSTRACT
We have investigated the structure of the onion primary cell wall at high resolution, using shadowed replicas of rapidly frozen deep-etched specimens.
We have sequentially extracted polymers from the wall and have visualized both these and the remaining structures at each extraction step.
By viewing the structures in as near their native state as possible, an accurate three-dimensional picture of wall construction has been assembled, facilitated by viewing stereo pairs of micrographs. Our observations show that the physical links between cellulose microfibrils that we observe in the intact wall are generally shorter (20-40 nm) than the isolated molecules we extract (30-&gt;700nm), suggesting that lateral interactions must occur between linking polymers and cellulose in muro. These cross-links are hemicellulosic and we believe them to be xyloglucans: their removal allows increased lateral association of microfibrils. Na2CO3-extractable pectic fractions form a separate coextensive network, the removal of which does not affect basic cellulose/ hemicellulose architecture. Preliminary evidence for a lamellate model of wall construction has been obtained. In addition, we propose a positive role for hemicellulose in maintaining the ordered spacing of cellulose microfibrils, perhaps regulating wall porosity and strength. The basic wall parameters that we derive impose constraints on possible cell wall models.