bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from ebs patients
Authors: Morley S. M. , Dundas S. R. , James J. L. , Gupta T. , Brown R. A. , Sexton C. J. , Navsaria H. A. , Leigh I. M. , Lane E. B. 
Published: April 2021

ABSTRACT
Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex, in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma. We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex, the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture-induced keratins and in conditions where filament fragility is not otherwise obvious.
SV40-T antigen and HPV16 (E6/E7) immortalised keratinocyte cell lines were examined, established from control and epidermolysis bullosa simplex-affected individuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) forms of the disease. In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated. However after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those seen in skin from EBS patients. These aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes.
Differences were also seen in the cells during respreading after replating: cells containing mutant keratins were slower to respread than controls and fine aggregates were seen at the cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiological processes involving cytoskeleton remodelling may render the cells vulnerable to cytolysis in vivo if physically challenged during this time window. The steady reduction in the mitotic index of the epidermis during the first few years of life could then explain the clinical improvement which is frequently observed in growing children.
Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: Implications for the role of dynamic microtubules in cell locomotion
Authors: Liao Guojuan , Nagasaki Takayuki , Gundersen Gregg G. 
Published: April 2021

ABSTRACT
The role of microtubules (MTs) in cell locomotion is uncertain: while MTs are not essential for motility of certain cells, MTs are necessary for the directed translocation of large cells such as fibroblasts, endothelial cells and neuronal growth cones. Based on previous studies, we hypothesize that cell locomotion may involve MTs in two possible ways: (1) the rate of cell locomotion is proportional to MT level; or (2) cell locomotion is not proportional to MT level but requires a critical level of MTs to proceed. To test these hypotheses, we measured the rate of locomotion of NRK fibroblasts migrating into an in vitro wound, before and after treatment with different concentrations of nocodazole to generate cells with different levels of MTs. Locomotion of cells was monitored directly using timelapse recording and analyzed with an Image-1 image analysis program. Addition of nocodazole (≥50 nM) resulted in a rapid reduction in locomotion to a new rate that was maintained for &gt;2 hours. We found that addition of as little as 100 nM nocodazole decreased the rate of locomotion by more than 60%; and that 300 nM nocodazole completely stopped cell locomotion. Although 100 nM nocodazole decreased locomotion over 60%, we detected no qualitative change in MT distribution by immunofluorescence. Quantitative analysis of MT fluorescence in immunofluorescently stained preparations showed that 100 nM nocodazole had no detectable effect on MT levels and that 300 nM nocodazole only decreased MT levels to ∼40% of controls. Quantitative analysis of tubulin polymer levels by cell extraction and western blotting yielded results similar to those obtained by quantification of MT fluorescence. A comparison of the locomotion rate measurements with the MT level measurements indicated that over half of the cell locomotion rate could be blocked by nocodazole without significantly affecting MT levels in the cell; the remaining locomotion rate was reduced proportionally to MT levels. These results do not support the notion that a critical level of MTs is required for cell locomotion and suggest that only a portion (&lt;50%) of the speed of the cells is proportional to MT levels. Rather, by analogy with studies of MT antagonists on the mitotic spindle, they suggest a third possibility: that low concentrations of nocodazole interfere with MT dynamics and thus, MT dynamics are critical for the maximal speed of cell locomotion. This notion was further supported by analogous effects of taxol and vinblastine on cell locomotion: at concentrations that reportedly cause little change in the level of MTs, taxol and vinblastine also dramatically decreased the rate of locomotion of NRK cells. In summary, our results establish the relationship between microtubule levels and locomotion rate and suggest that dynamic MTs are rate-limiting for fibroblast locomotion.
Clustering of cx43 cell-to-cell channels into gap junction plaques: Regulation by camp and microfilaments
Authors: Wang Yingjian , Rose Birgit 
Published: April 2021

ABSTRACT
Cell-to-cell channels are often seen clustered at cell-cell contacts into the so-called gap junction plaques. The mechanism of this clustering is unknown. We show that the clustering of cell-to-cell channels composed of connexin43 is induced by elevation of cyclic AMP. The cAMP-induced clustering is enhanced by inhibition of glycosylation and abolished by disruption of microfilaments. Channel clustering thus seems to be regulated by cAMP and glycosylation and to involve microfilaments.
Characterisation of neurofilament protein nf70 mrna from the gastropod Helix aspersa reveals that neuronal and non-neuronal intermediate filament proteins of cerebral ganglia arise from separate lamin-related genes
Authors: Adjaye James , Plessmann Uwe , Weber Klaus , Dodemont Huub 
Published: April 2021

ABSTRACT
The neuronal cytoplasmic intermediate filament (IF) protein HeNF70 of the gastropod Helix aspersa is identified by sequence analysis of the corresponding 4,600 bp cDNA isolated from a cerebral ganglion cDNA library. HeNF70 shares 60% sequence identity with the neuronal LoNF70 protein of the cephalopod Loligo pealei and only 36% identity with the Helix non-neuronal IF-A protein. All three molluscan IF proteins display the lamin-type extended coil 1b subdomain harbouring six additional heptads and all have long C-terminal sequences with sub-stantial homology to the tail domains of nuclear lamins. The lamin-like tail domains of the two neurofilament proteins share a unique motif comprising 13 residues, which is absent from Helix IF-A and all other known non-neuronal IF proteins. HeNF70 is encoded by a 9.5 kb RNA transcript. The very long 7.2 kb 3′-untranslated sequence contains a unique 26 nucleotide repeat extending over 0.5 kb in its 5′-region. The HeNF70 mRNA is expressed at low abundancy in cerebral ganglia but not in any of the 13 non-neuronal tissues tested. In contrast, all tissues express at fairly high levels the same 4.6 and 4.2 kb mRNAs encoding the Helix non-neuronal IF-A/B proteins. Blot hybridisation studies on genomic DNA and ganglion mRNA with subprobes from the cloned HeNF70 cDNA, as well as sequence analysis of an RT-PCR generated partial cDNA encoding a putative HeNF60 protein, indicate at least two different neuronal IF genes in Helix.
Characterisation of a laminarin sulphate which inhibits basic fibroblast growth factor binding and endothelial cell proliferation
Authors: Hoffman Richard , Paper Dietrich H. , Donaldson Jane , Alban Susanne , Franz Gerhard 
Published: April 2021

ABSTRACT
We have evaluated a series of laminarin sulphates with different degrees of sulphation (0.3-2.3) as antagonists of basic fibroblast growth factor (bFGF) and as inhibitors of the bFGF-dependent endothelial cell line FBHE. Inhibition of binding of bFGF by the laminarin sulphates increased with increasing degree of sulphation. Binding of bFGF to low affinity sites on BHK cells was inhibited more strongly than binding to high affinity sites. IC50 values for inhibi-tion of binding to low and high affinity sites by the most highly sulphated laminarin sulphate (LAM S5; degree of sulphation 2.31) were 12±8 µ g/ml and 69±66 µ g/ml, respec-tively. LAM S5 dissociated bFGF from low affinity sites on BHK cells but not from high affinity sites. LAM S5 increased the electrophoretic mobility of bFGF indicating that LAM S5 binds directly to bFGF. LAM S5 reduced uptake of bFGF by FBHE cells by 67%. Increasing the degree of sulphation of laminarin sulphates increased the inhibition of bFGF-stimulated DNA synthesis of the endo-thelial cell line FBHE (IC50 for LAM S5 approx. 1 µ g/ml). There was no inhibition of DNA synthesis of FBHE cells by LAM S5 in the presence of 1 µ g/ml bFGF indicating that bFGF antagonism is involved in the anti-proliferative activity of this compound. LAM S5 may be of value against diseases associated with bFGF-dependent cell proliferation.
Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in african trypanosomes
Authors: Brickman Marla Jo , Cook J. Michael , Balber Andrew E. 
Published: April 2021

ABSTRACT
We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal com-partments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37°C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4°C. When cells were incubated at 12°C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12°C. The morphology of collecting tubule membranes was specifically altered at 12°C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12°C were reversed 10 minutes after cells were returned to 37°C. We also used flow cytofluori-metric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value con-siderably higher than that of mammalian lysosomes. Flu-orescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when blood-stream forms were incubated in the presence of chloro-quine.
Investigation of the role of β1 integrins in cell-cell adhesion
Authors: Weitzman Jonathan B. , Chen Annabel , Hemler Martin E. 
Published: April 2021

ABSTRACT
Various β1 integrins (VLA-2, VLA-3, VLA-4) have been suggested to bind directly to themselves or to each other, thus mediating cell-cell adhesion. Here we expressed the human α2 and α3 subunits in three different cell lines (human erythroleukemia K562, human rhabdomyosar-coma RD and Chinese hamster ovary CHO cells). Although cell surface α2β1 and α3β1 in the transfectants mediated adhesion to matrix ligands (collagen or laminin 5, respec-tively), in no case did we observe enhanced cell-cell adhesion. In the presence of a range of different divalent cation concentrations, stimulatory anti-β1 antibodies or anti-α3 antibodies, VLA-2 and VLA-3 still did not appear to interact directly, through either heterophilic (i.e. VLA-3/VLA-2) or homophilic (i.e. VLA-3/VLA-3) mechanisms, to mediate cell-cell adhesion. Furthermore, in some but not all α3 transfectants we observed an unexpected decrease in cell-cell adhesion, suggesting a novel anti-adhesive function. This inhibitory effect was not observed for α2 transfection nor when the α3 cytoplasmic tail was exchanged with that of another integrin α subunit. Finally, no evidence for VLA-4/VLA-4 mediated cell-cell adhesion was observed using α4-transfected K562 and CHO cells. In conclusion, using many different combinations of cell lines, we found that cell-cell adhesion mediated by direct integrin/integrin interaction is not a widespread phenome-non, and is not observable in standard cell-cell adhesion assays. Furthermore, in some cell combinations, α3 expression may actually cause diminished cell-cell adhesion.
Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones
Authors: Blavier L. , Delaissé J. M. 
Published: April 2021

ABSTRACT
A key event in bone resorption is the recruitment of osteoclasts to future resorption sites. We follow here the migration of preosteoclasts from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture, and investigate the role of proteinases and demineralization in this migration. Our approach consisted in testing inhibitors of proteinases and demineralization on the migration kinetics. Migration was monitored by histomorphometry and the (pre)osteoclasts were identified by their tartrate resistant acid phosphatase (TRAP) activity. At the time of explantation, TRAP+ cells (all mononucleated) are detected only in the periosteum, and the core of the diaphysis (future marrow cavity) consists of calcified cartilage. Upon culture, TRAP+ cells (differentiating progressively into multinucleated osteoclasts) migrate through a seam of osteoid and a very thin and discontinuous layer of mineral, invade the calcified cartilage and transform it into a ‘marrow’ cavity; despite the passage of maturing osteoclasts, the osteoid develops into a bone collar. The migration of TRAP+ cells is completely prevented by matrix metalloproteinase (MMP) inhibitors, but not by a cysteine proteinase inhibitor, an inhibitor of carbonic anhydrase, or a bisphosphonate. The latter three drugs inhibit, however, the resorptive activity of mature osteoclasts at least as efficiently as do the MMP inhibitors, as assessed in cultures of calvariae and radii. Furthermore, in situ hybridizations reveal the expression of 2 MMPs, gelatinase B (MMP-9 or 92 kDa type IV collagenase) in (pre)osteoclasts, and interstitial collagenase (MMP-13) in hypertrophic chondrocytes. It is concluded that only MMPs appear obligatory for the migration of (pre)osteoclasts, and that this role is distinct from the one MMPs may play in the subosteoclastic resorption compartment. We propose that this new role of MMPs is a major component of the mechanism that determines where and when the osteoclasts will attack the bone.
Localization of myosin II A and B isoforms in cultured neurons
Authors: Rochlin M. William , Itoh Kazuyuki , Adelstein Robert S. , Bridgman Paul C. 
Published: April 2021

ABSTRACT
Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal anti-bodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organellerich central region and the thin, actinrich peripheral region. The staining colocalized with actin bundles proximal and distal to the marginal zone, though the staining was more prominent proximally. The trailing edge of growth cones and the distal portion of the neurite often had a rimmed appearance, but more proximal regions of neurites had cytoplasmic labelling. Localizing MHC-B in growth cones previously monitored during advance (using differential interference contrast microscopy) revealed a positive correlation with edges at which retraction had just occurred and a negative correlation with lamellipodia that had recently undergone protrusion. Cell bodies were brightly labelled for myosin heavy chain B. Myosin heavy chain A staining was dimmer and its colocalization with filamentous actin bundles in growth cones was less striking than that of myosin heavy chain B. Growth cones stained for both myosin heavy chain A and B revealed that the two antigens overlapped frequently, but not exclusively, and that myosin heavy chain A lacked the elevation in the marginal zone that was characteristic of myosin heavy chain B. The pattern of staining we observed is consistent with a prominent role for myosin heavy chain B in either generating tension between widely separated areas of the growth cone, or bundling of actin filaments, which would enable other motors to effect this tension. These data support the notion that conventional myosin is important in growth cone advance and turning.
The hepatocyte growth factor/scatter factor (HGF/SF) receptor, met, transduces a morphogenetic signal in renal glomerular fibromuscular mesangial cells
Authors: Kolatsi-Joannou Maria , Woolf Adrian S. , Hardman Patricia , White Stanley J. , Gordge Michael , Henderson Robert M. 
Published: April 2021

ABSTRACT
Previous studies have demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is secreted by mes-enchymal cells and that it elicits motility, morphogenesis and proliferation of epithelia expressing the met receptor. We now report that HGF/SF may act as an autocrine factor in fibromuscular renal mesangial cells. These cells mechan-ically support glomerular endothelia, control the rate of plasma ultrafiltration and are implicated in the pathogen-esis of a variety of chronic renal diseases. We detected met protein in the vascular stalk of metanephric glomeruli and in the mature mesangium. Mesangial lines from a mouse transgenic for a temperature-sensitive simian virus 40 T antigen expressed met mRNA and protein, and recombi-nant HGF/SF phosphorylated the met receptor tyrosine kinase. Cells were immortal in the permissive condition and HGF/SF enhanced proliferation in a defined medium. In the absence of the immortalising protein, division ceased and recombinant HGF/SF caused multipolar cells to become bipolar. The factor diminished stress fibres, their focal contacts and immunostaining for extracellular fibronectin, hence suggesting reduced substratum adhesion and enhanced motility. Mesangial lines also expressed HGF/SF mRNA and secreted bioactive factor; immuno-cytochemistry showed both ligand and receptor in individ-ual cells. HGF/SF blocking antibody aggregated the cells, suggesting that mesangial-derived factor affects basal cell conformation in an autocrine manner. We conclude that mesangial cells express both HGF/SF and met, and the factor induces morphogenesis of cultured mesangial cells. Therefore HGF/SF may have an autocrine role in mesangial biology but further studies are now required to investigate the potential importance of the factor in vivo.
Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology
Authors: Takegawa Kaoru , DeWald Daryll B. , Emr Scott D. 
Published: April 2021

ABSTRACT
We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S. pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34Δ strain. S. pombe Vps34p-specific antiserum detects a single protein in cells of ∼90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate, but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phos-phatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.
Identification of a Ca2+-binding light chain within Chlamydomonas outer arm dynein
Authors: King Stephen M. , Patel-King Ramila S. 
Published: April 2021

ABSTRACT
We describe here the molecular cloning of the Mr 18,000 dynein light chain from the outer arm of Chlamydomonas flagella. In vivo, this molecule is directly associated with the γ dynein heavy chain. Sequence analysis indicates that this light chain is a novel member of the calmodulin superfamily of Ca2+ binding regulatory proteins; this molecule is 42, 37 and 36% identical to calmodulin, centrin/caltractin and troponin C, respectively, and also shows significant similarity to myosin light chains. Although four helix-loop-helix elements are evident, only two conform precisely to the EF hand consensus and are therefore predicted to bind Ca2+ in vivo. In vitro Ca2+ binding studies indicate that this dynein light chain (expressed as a C-terminal fusion with maltose binding protein) has at least one functional Ca2+ binding site with an apparent affinity for Ca2+ of ∼3×10−5M. Within the Chlamydomonas flagellum, the transition from an assymmetric to a symmetric waveform (which implies an alteration in dynein activity) is mediated by an increase in intraflagellar Ca2+ from 10−6 to 10−4 M; this transition is altered in mutants that lack the outer arm. The data presented here suggest that a Ca2+-dependent alteration in the interaction of this dynein light chain with the motor containing heavy chain may affect outer arm function during flagellar reversal.