bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plakophilin-3, a novel Armadillo-like protein present in nuclei and desmosomes of epithelial cells
Authors: Bonné Stefan , Hengel Jolanda van , Nollet Friedel , Kools Patrick , Roy Frans van 
Published: April 2021

ABSTRACT
We report on a novel Armadillo-like protein, termed plakophilin-3. The human protein, which is encoded by a 2.8 kb messenger RNA, has a predicted molecular mass of 87 kDa. The protein comprises 10 Armadillo-like repeats, preceded by an amino-terminal region of 293 amino acid residues and followed by a short carboxy-terminal region of 27 amino acid residues. Plakophilin-3 is classified as a member of the p120ctn/plakophilin subfamily of Armadillo proteins based on the number and organization of the Armadillo repeats and its high sequence similarity to other members of this family. CLUSTAL W alignment of p120ctn/plakophilin subfamily members showed the plakophilin-3 protein to be most similar to plakophilin-1 and -2. Western blot analysis of plakophilin-3 revealed expression in all epithelial cell lines tested but not in foreskin fibroblasts and various sarcoma-derived cell lines. This is unlike most other members of the p120ctn/plakophilin subfamily, which are widely expressed. By immunofluorescence, the plakophilin-3 protein was colocalized with desmoglein in desmosomes of epithelial cells. In addition, an intriguing speckle-like nuclear staining was observed. Hence, like plakophilin-1 and -2, plakophilin-3 displays a dual intracellular location, i.e. in the desmosomal plaque and in the nucleus. These results suggest the involvement of plakophilin-3 in both desmosome-dependent adhesion and signaling pathways. Furthermore, the human plakophilin-3 gene was mapped on the chromosomal locus 11p15 by fluorescent in situ hybridization.
Intracellular traffic of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells
Authors: Praetor Asja , Ellinger Isabella , Hunziker Walter 
Published: April 2021

ABSTRACT
Transfer of passive immunity from mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to MHC class I antigens. We have now expressed the human FcRn in polarized MDCK cells and analyzed the intracellular routing of the receptor. FcRn showed a predominant intracellular localization at steady state. Newly synthesized FcRn was delivered in a non-vectorial fashion to both the apical and basolateral surfaces of MDCK cell monolayers. Following internalization from the apical or basolateral domain, the receptor transcytosed to the opposite surface. These findings provide direct evidence for the transepithelial transport function of FcRn and indicate that the receptor undergoes multiple rounds of transcytosis.
Vinblastine induces an interaction between FtsZ and tubulin in mammalian cells
Authors: Yu Xuan-chuan , Margolin William , Gonzalez-Garay Manuel L. , Cabral Fernando 
Published: April 2021

ABSTRACT
The Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ.
Integrin α and β subunit contribution to the kinetic properties of α2β1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions
Authors: Masson-Gadais Bénédicte , Pierres Anne , Benoliel Anne-Marie , Bongrand Pierre , Lissitzky Jean-Claude 
Published: April 2021

ABSTRACT
The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for α and β chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds−1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-β1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the α2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-β1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of α2 chain function by inhibiting antibodies was corrected by anti-β1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of β1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-α2 chain antibodies.
Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-β1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.
A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles
Authors: Biederbick Annette , Rose Scott , Elsässer Hans-Peter 
Published: April 2021

ABSTRACT
Using antibodies against autophagic vacuole membrane proteins we identified a human cDNA with an open reading frame of 1848 bp, encoding a protein of 70 kDa, which we named lysosomal apyrase-like protein of 70 kDa (LALP70). Sequence analysis revealed that LALP70 belongs to the apyrase or GDA1/CD39 family and is almost identical to a human uridine diphosphatase, with the exception of nine extra amino acids in LALP70. Members of this family were originally described as ectoenzymes, with some intracellular exceptions. Transfected LALP70 fused to the green fluorescent protein localized in the cytoplasm with a punctate pattern in the perinuclear space. These structures colocalized with the autophagic marker monodansylcadaverine and the lysosomal protein lamp1.
Hydrophobicity analysis of the encoded protein revealed a transmembrane region at the N and C termini. Most of the sequence is arranged between these transmembrane domains, and contains four apyrase conserved regions. In vitro transcription/translation in the presence of microsomes showed that no signal sequence is cleaved off and that the translation product is protected from trypsin treatment. Our data indicate that LALP70 is a type III lysosomal/autophagic vacuole membrane protein with the apyrase conserved regions facing the luminal space of the vacuoles.
A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat
Authors: Kleene Ralf , Zdzieblo Joachim , Wege Konstanze , Kern Horst-Franz 
Published: April 2021

ABSTRACT
Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- andRER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent.
The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied.
Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5′end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.
Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion
Authors: Aballay Alejandro , Stahl Philip D. , Mayorga Luis S. 
Published: April 2021

ABSTRACT
Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.
Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2
Authors: Giuliani Roberta , Bastaki Maria , Coltrini Daniela , Presta Marco 
Published: April 2021

ABSTRACT
Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK1/2 phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel.
Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK1/2 in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27,30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and
R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK1/2-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant Δ27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK1/2 from its ability to stimulate uPA upregulation and/or in vitro angiogenesis.
In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells.
Chromatid cohesion during mitosis: lessons from meiosis
Authors: Rieder Conly L. , Cole Richard 
Published: April 2021

ABSTRACT
The equal distribution of chromosomes during mitosis and meiosis is dependent on the maintenance of sister chromatid cohesion. In this commentary we review the evidence that, during meiosis, the mechanism underlying the cohesion of chromatids along their arms is different from that responsible for cohesion in the centromere region. We then argue that the chromatids on a mitotic chromosome are also tethered along their arms and in the centromere by different mechanisms, and that the functional action of these two mechanisms can be temporally separated under various conditions. Finally, we demonstrate that in the absence of a centromeric tether, arm cohesion is sufficient to maintain chromatid cohesion during prometaphase of mitosis. This finding provides a straightforward explanation for why mutants in proteins responsible for centromeric cohesion in Drosophila (e.g. ord, mei-s332) disrupt meiosis but not mitosis.
Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade
Authors: Agnew Brian J. , Duman Joseph G. , Watson Charles L. , Coling Donald E. , Forte John G. 
Published: April 2021

ABSTRACT
Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H,K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H,K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H,K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.
Induced differentiation in HT29, a human colon adenocarcinoma cell line
Authors: Cohen Esther , Ophir Ilana , Shaul Yehuda Ben 
Published: April 2021

ABSTRACT
The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.
Metalloproteinase-mediated release of the ectodomain of L1 adhesion molecule
Authors: Beer Sandra , Oleszewski Matthias , Gutwein Paul , Geiger Claudia , Altevogt Peter 
Published: April 2021

ABSTRACT
The L1 adhesion molecule is an approx. 200-220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-α which is mediated by the membrane-bound metalloproteinase TNF-α converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein.
Functional characterisation of tetanus and botulinum neurotoxins binding domains
Authors: Lalli Giovanna , Herreros Judit , Osborne Shona L. , Montecucco Cesare , Rossetto Ornella , Schiavo Giampietro 
Published: April 2021

ABSTRACT
Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.
Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model
Authors: Hillmann Georg , Gebert Andreas , Geurtsen Werner 
Published: April 2021

ABSTRACT
The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques. The results were compared with monolayer cultures. Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy. Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions. The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation. Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system. Frequently, the fibers were oriented parallel to the long axis of the cells. Type VI collagen formed thin fibers and revealed a reticular pattern. In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers. Their shape and spatial distribution resembled that of cells in tissue biopsies more closely. The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information.