bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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->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.
Islands of acetylated histone H4 in polytene chromosomes and their relationship to chromatin packaging and transcriptional activity
Authors: Turner B. M. , Franchi L. , Wallace H.
Published: April 2021

The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labelled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic ‘puffs’, the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.
Mapping Map-2
Authors: Harrison Alistair , Hyams Jeremy S.
Published: April 2021

Microtubule-associated proteins (MAPs) are an important class of cytoskeletal proteins that are presumed to confer specific functional characteristics on microtubules. The best-characterised is the heat-stable brain MAP, MAP-2. MAP-2 consists of three subunits MAP-2a, MAP-2b (both having an apparent Mrof∼270K; K=103Mr) and MAP-2c (Mr 70K) (Vallee et al. 1981; Garner et al. 1988a). The first insight into the structure of the MAP-2 molecule emerged from the chymotryptic digestion of the protein (Vallee, 1980). Fragments with Mr values in the range of 32 to 39K were found to bind to, and promote the assembly of, microtubules, whilst a larger 240K fragment had no interaction and probably represented an arm domain that projected from the surface of the microtubule and interacted in some way with other cell components, or, indeed, other microtubules (Vallee and Borisy, 1977; Vallee, 1980).
The response of the centrosome to heat shock and related stresses in a Drosophila cell line
Authors: Debec Alain , Courgeon Anne-Marie , Maingourd MichÈle , Maisonhaute Claude
Published: April 2021

The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.
Fodrin in the human polymorphonuclear leucocyte: Redistribution induced by the chemotactic peptide
Authors: Fujimoto Toyoshi , Ogawa Kazuo
Published: April 2021

Fodrin, a membrane skeletal protein, was found to accumulate in the posterior portion of human neutrophils polarized morphologically after stimulation by the chemotactic peptide, IV-formylmethionyl-leucylphenylalanine (FMLP). In most (>90%) unstimulated neutrophils, the distribution of fodrin was found to be uniform by immunofluorescence microscopy. When FMLP (10−8M) was applied at 25°C, fodrin became polarized in about 40% of cells by Imin, about 70% by 2 min, and about 80% by 10 min. The cells with polarized distribution decreased thereafter to about 60% of the cells at 20 min and about 20% at 60 min. Using the under-agarose system, it was confirmed that the concentration of fodrin occurred in the region opposite to the direction of chemoattraction in moving cells.

By immunoelectron microscopy, most of the labeling for fodrin was observed in the filamentous cell cortex and not associated with the plasma membrane itself. In cells polarized morphologically by FMLP, the fodrin labeling became concentrated in the posterior portion of the cell; the labeling was found most densely in the granule-rich cytoplasm, while the filamentous tail region was not labeled intensely. The lamellipodium in the head region was also labeled only sparsely.

The results indicate that in human neutrophils fodrin exists as a cytoskeletal protein rather than as a membrane protein and that the protein accumulates in the endoplasm of the posterior portion in migrating cells. The rearrangement is likely to modulate the organization of the actin-rich cell cortex for cell locomotion.
Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin
Authors: Joseph L. B. , Kreutzer D. L. , Tanzer M. L.
Published: April 2021

The development of receptor-defective or -deficient mutants can be applied to the investigation of cellmatrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (PcO.OOl) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (α FN/β1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the β subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the α and β subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants. Furthermore, FACS analysis of antibody-labelled cells showed that the mutant cells had threefold less labelling of the fibronectin receptor by the 7E2 antibody than the wildtype cells. These data suggest that, in the mutant cells, the fibronectin receptor is present, but it is less abundant at the cell surface. This decrease in surface fibronectin receptors may be responsible for decreased cell spreading of the mutant cells on the fibronectin substratum. Thus, the linkage between adhesion and spreading potentially may be explored by studying these mutant cells.
Analysis of wound healing in an in vitro model: Early appearance of laminin and a 125×103M
r polypeptide during adhesion complex formation
Authors: Kurpakus Michelle A. , Stock E. Lee , Jones Jonathan C. R.
Published: April 2021

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K= 103Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.
Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes
Authors: Laird D. W. , Revel J.-P.
Published: April 2021

A 43 × 10 3 M r protein (designated connexin 43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluor-escent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence.
Failure of platelet-derived growth factor plus insulin to stimulate sustained proliferation of swiss 3T3 cells Requirement for hydrocortisone, prostaglandin E1; lipoproteins, fibronectin and an unidentified component derived from serum
Authors: Brooks Robert F. , Howard Mark , Leake David S. , Riddle Peter N.
Published: April 2021

Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for Swiss 3T3 cells made quiescent by growth to saturation density in high serum, and its activity is further potentiated by high levels of insulin, which alone have little effect. We show here that this is not the case for sparse 3T3 cells made quiescent by plating in low serum. Under these conditions, insulin alone is at least as effective as PDGF and frequently more so. Together, the response is no more than additive at best, and in many cases less than additive, the combined effect being no greater than for insulin alone. Instead, we find that optimal mitogenic stimulation requires the additional presence, besides PDGF and insulin, of hydrocortisone, prostaglandin Ei and an unidentified, non-dialysable component contained in serum treated with dithiothreitol (DTT) to inactivate endogenous growth factors. Interestingly, overnight pretreatment of the cells with hydrocortisone alone potentiates the subsequent response to PDGF+insu-lin, i.e. pretreatment induces a long-term memory that persists after the removal of the hydrocortisone from the medium. In short-term (24 h) thymidine incorporation assays, the combination of PDGF, insulin, hydrocortisone, prostaglandin E, and DTT-serum, is as effective as optimal levels of whole serum, but is unable to sustain longer-term proliferation (measured over 6 days). For this, high-and low-density lipoproteins, fibronectin and, to some extent epidermal growth factor (EGF), are also necessary.
Kinetic analysis of mitotic spindle elongation in vitro
Authors: Baskin Tobias I. , Cande W. Z.
Published: April 2021

Studies of mitotic spindle elongation in vitro using populations of diatom spindles visualized with immunofluorescence microscopy have shown that the two interdigitating half-spindles are driven apart by an ATP-dependent process that generates force in the zone of overlap between half-spindles. To characterize further the system responsible for spindle elongation, we observed spindle elongation directly with polarized light or phase-contrast video-microscopy. We report that the kinetics of spindle elongation versus time are linear. A constant rate of spindle elongation occurs despite the continuous decrease in length of the zone of overlap between half-spindles. The average rate of spindle elongation varies as a function of treatment, and rates measured match spindle elongation rates measured in vivo. When spindles elongated in the presence of polymerizing tubulin (from bovine brain), the extent of elongation was greater than the original zone of halfspindle overlap, but the rate of elongation was constant. No component of force due to tubulin polymerization was found. The total elongation observed in the presence of added tubulin could exceed a doubling of original spindle length, matching the elongation in the intact diatom. The linear rate of spindle elongation in vitro suggests that the force transducer for anaphase B is a mechanochemical ATPase, analogous to dynein or myosin, and that the force for spindle elongation does not arise from stored energy, e.g. in an elastic matrix in the midzone. Additionally, on the basis of observations described here, we conclude that the force-transduction system for spindle elongation must be able to remain in the zone of microtubule overlap during the sliding apart of half-spindles, and that the transducer can generate force between microtubules that are not strictly antiparallel.
Mitotic and meiotic spindles from two insect orders, lepidoptera and diptera, differ in terms of microtubule and membrane content
Authors: Wolf Klaus Werner
Published: April 2021

Spindles from the gonads of five insect species were examined after conventional preparation for electron microscopy. The aim of the study was to determine (1) the range of variation of the spindle membranes between mitotic and meiotic cells and (2) the correlation of possible differences with the microtubule content of the spindles. The study involved four moth species, Ephestia kuebniella, Pbragmato-bia fuliginosa, Orgyia tbyellina, Orgyia antiqua, and one fly, Megaselia scalaris. Somatic and gonial mitoses in all species examined showed a sparse spindle membrane inventory. In contrast, spermatocytes consistently had a multi-layered spindle envelope. In spermatocytes of all Lepidoptera species examined, but not in those of M. scalaris, diverse forms of intraspindle membranes existed in addition to the spindle envelope. Microtubule counts in serially cross-sectioned spindles of E. kuebniella revealed an about 6-fold increase in the mass of polymerized tubulin during the transition from spermatogonia to primary spermatocytes. The increase was 3.3-fold in O. tbyellina and less than 3-fold in Af. scalaris. The density of intraspindle membranes in E. kuebniella was higher than in O. tbyellina by fac tors of 1.8 to 3.0. The correlation between the amount of spindle membranes and the microtubule content of the spindle indicates a functional relationship. Spindle membranes are believed to influence microtubule stability via the regulation of the Ca2+ concentration within the spindle area. The high microtubule mass in spindles from Lepidoptera spermatocytes may result from the membrane-dependent lowering of the Ca2+ level within the spindles. Finally, an unconventional idea on the role of intraspindle membranes is offered. This concept is not intended to challenge the function of spindle-associated membranes as Ca2+-sequestrating compartments. Intraspindle membranes are considered as stuffing material in sheathed spindles. Membranous compartments reduce the free volume within the spindle. Thereby, monomeric tubulin is concentrated and the formation of microtubules is favoured.
Positional order and cellular handedness
Authors: Frankel Joseph
Published: April 2021

A major problem in thinking about asymmetrical patterns in organisms is that of obtaining ‘big hands from little hands’ (Harrison, 1979). The big hands are our own, whereas the little hands are asymmetrical organic molecules. Since the molecules that make up our left and right hands are of identical asymmetry, the different handedness of these big hands must be specified at some more complex level of organization. For Harrison (1979) and, more recently, Brown and Wolpert (1990), the link between the levels is a tethered macromolecule or macromolecular aggregate, implying a pre-existing structural system within which the tethering takes place.
Tailless keratins assemble into regular intermediate filaments in vitro
Authors: Hatzfeld Mechthild , Weber Klaus
Published: April 2021

To study the influence of the non a-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non α-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purifiy the protein by a two-step procedure.

The filament–forming capacity of the truncated K8 with wild–type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occuring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.
Senescent human diploid fibroblasts are able to support DNA synthesis and to express markers associated with proliferation
Authors: Kill Ian R. , Shall Sydney
Published: April 2021

The characteristic limited reproductive life-span of normal human fibroblasts in culture is due to a steadily decreasing fraction of cells able to proliferate in the standard rich growth media. We have observed that restricting the growth factor supply to old cells for variable lengths of time in culture increases the fraction of cells that can enter S-phase; although these cells do not go on to divide. Thus, it seems that there is a transient phase between the proliferating state and the irreversibly post-mitotic,senescent state. Perhaps a ‘quiescent-G0’ state, which can be maintained in the presence of growth factors, is a stage on the pathway to mortalization and senescence.
Structure of a Plasmodium yoelii gene-encoded protein homologous to the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum
Authors: Murakami Kenji , Tanabe Kazuyuki , Takada Suehisa
Published: April 2021

A cation-transporting ATPase gene of Plasmodium yoelii was cloned from the parasite genomic library using an oligonucleotide probe derived from a conserved amino acid sequence of the phosphorylation domain of the aspartyl phosphate family of ATPases. The complete nucleotide sequence was determined and it predicts a 126717Afr encoded protein composed of 1115 amino acids. Northern blot analysis revealed that the gene is transcribed during the asexual stages of parasite development. The P. yoelii protein contains functional and structural features common to the family of aspartyl phosphate cation-transporting ATPases. The parasite protein shows the highest overall homology in amino acid sequence (42%) to the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Homologies to other aspartyl phosphate cation-transporting ATPases including a plasma membrane Ca2+-ATPase were between 13 and 24 %. The structure predicted from a hydropathy plot also shows 10 transmembrane domains, the number and location of which correlated well with the sarcoplasmic reticulum Ca2+- ATPase. On the basis of these results, we conclude that the parasite gene encodes an organellar, but not plasma membrane, Ca2+-ATPase. The P. yoelii protein, furthermore, contains all six amino acid residues in the transmembrane domains that were recently identified as comprising a high-affinity Ca2+-binding site. It follows that organellar Ca2+- ATPases of rabbit and Plasmodium conserve functionally important amino acid residues, even though they are remote from each other phylogenetically.
Saccharomyces cerevisiae mutants lacking a functional vacuole are defective for aspects of the pheromone response
Authors: Dullo Vjekoslav , Riezman Howard
Published: April 2021

The endl mutant belongs to a group of four vacuolar protein sorting mutants (class C vps) that lack a morphologically distinguishable and functional vacuole. These mutants share several other phenotypes, such as the inability to grow at 37 °C or on nonfermentable carbon sources. We show that, as in the case of the endl mutant, vpsl6, vpsl8 and vps33 mutants all internalize but do not degrade α. In addition, all four mutants are defective for α- factor-induced projection formation to the same extent. A more detailed investigation of pheromone response in the endl mutant reveals that one aspect of the early response (induction of FUS1) is as defective as late responses (cell cycle arrest and projection formation). In contrast, another measure of the early response (induction of STE2) is normal. These data suggest that the biogenesis of a functional vacuole is necessary for optimal response to pheromone.