bluuebunny/crossref_metadata_2025_split huggingface dataset:
A reversibly palmitoylated resident protein (p63) of an ER-Golgi intermediate compartment is related to a circulatory shock resuscitation protein
Authors: Schweizer Anja , Rohrer Jack , Jenö Paul , DeMaio Antonio , Buchman Timothy G. , Hauri Hans-Peter 
Published: April 2021

ABSTRACT
The recently identified 63 kDa membrane protein, p63, is a resident protein of a membrane network interposed in between rough ER and Golgi apparatus. To characterize p63 at the molecular level a 2.91 kb cDNA encoding p63 has been isolated from a human placenta gt10 cDNA library. Sequence analysis of tryptic peptides prepared from isolated p63 confirmed the identity of the cloned gene. The translated amino acid sequence consists of 601 amino acids (65.8 kDa) with a single putative membrane-spanning region and a N-terminal cytoplasmic domain of 106 amino acids. The human p63 cDNA exhibits a high level of sequence identity to the pig hepatic cDNA 3AL (accession number M27092) whose expression is enhanced after resuscitation from circulatory shock. An additional remarkable feature of p63 is that it becomes reversibly palmitoylated when intracellular protein transport is blocked by the drug brefeldin A. Overexpression of p63 in COS cells led to the development of a striking tubular membrane network in the cytoplasm. This suggests that the protein may be determinant for the structure of the p63 compartment.
Elevated cAMP levels induce multilayering of MDCK cells without disrupting cell surface polarity
Authors: Ullrich Oliver , Haase Winfried , Koch-Brandt Claudia 
Published: April 2021

ABSTRACT
The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer.
Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participitating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.
Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation
Authors: Pang Yuk-Ying Susana , Schermer Alexander , Yu Jun , Sun Tung-Tien 
Published: April 2021

ABSTRACT
Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfidecrosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments.
Defining E-cadherin-associated protein complexes in epithelial cells: plakoglobin β- and γ-catenin are distinct components
Authors: Piepenhagen Peter A. , Nelson W. James 
Published: April 2021

ABSTRACT
Ca2+-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include α-, β-, and γ-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either β- or γ-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and β-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of γ-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and β-catenin co-immunoprecipitate with E-cadherin. Laserscanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with γ-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for of the γ-catenin signal. Two-dimensional gel electrophoresis resolved the β-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI ∼ 6.1). The other protein comigrated with γ-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of ∼4.25; we refer to this protein as γ-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains α-, β-, and γ-catenin. Although plakoglobin shares sequence similarity with β-catenin, and comigrates with γ-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The asociation of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.
Fibronectin splice variants are differentially incorporated into the extracellular matrix of tumorigenic and non-tumorigenic hybrids between normal fibroblasts and sarcoma cells
Authors: Mardon Helen J. , Grant Richard P. , Grant Kate E. , Harris Henry 
Published: April 2021

ABSTRACT
Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.
Platelet shape change and protein phosphorylation induced by ADP and thrombin are not sensitive to short periods of microgravity
Authors: Schmitt Didier A. , Ohlmann Philippe , Gachet Christian , Cazenave Jean-Pierre 
Published: April 2021

ABSTRACT
Recent experiments have shown that the stimulation of animal cells in vitro by direct protein kinase C (PKC) activators is significantly reduced under microgravity (μg). Platelets undergo protein phosphorylation and morphological changes a few seconds after stimulation by agonists such as phorbol esters which activate PKC. Therefore, taking advantage of parabolic plane flight to obtain short periods of microgravity, we studied phosphorylation of myosin light chain (20K), specific PKC-dependent phosphorylation of a 40,000 Mr protein, pleckstrin (40K) and platelet shape change. SDS-PAGE analysis and electron microscopy were performed on platelets subjected to 20 seconds microgravity as compared to normal gravity (1 g) conditions. These investigations showed that neither Ca2+-calmodulin-mediated activation nor the PKC-dependent pathways are inhibited during short periods of microgravity.
Mitotic Golgi clusters are not tubular endosomes
Authors: Pypaert Marc , Nilsson Tommy , Berger Eric G. , Warren Graham 
Published: April 2021

ABSTRACT
HeLa cells were incubated with 15 nm BSA-gold for 1 or 2 hours to mark the endocytic pathway and mitotic cells were then isolated by shake-off. Thin, frozen sections were labelled with antibodies against two resident Golgi markers,β-(1,4)-galactosyltransferase and N-acetylglucosaminyltransferase I. Detection of the latter was aided by the use of a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. The secondary antibodies were coupled to either 5 or 10 nm gold so that the distribution of each of the three markers could be followed. Qualitative and quantitative stud-ies showed that there were two populations of clusters, those described by us earlier and termed Golgi clusters (Lucocq et al. (1987)J. Cell Biol. 104, 865-874), containing either or both Golgi markers, and clusters of tubular endosomes containing BSA-gold. There was very little overlap showing that Golgi clusters cannot be tubular endosomes as concluded by Tooze and Hollinshead (1992)Eur. J. Cell Biol. 58, 228-242.
An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays
Authors: Chandra Rashmi , Endow Sharyn A. , Salmon Edward D. 
Published: April 2021

ABSTRACT
The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg2 +-ATPase activity and binds microtubules in pelleting assays In contrast to near fulllength ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.
Lymphocyte migration across high endothelium is associated with increases in α4β1 integrin (VLA-4) affinity
Authors: Hourihan Helen , Allen Terry D. , Ager Ann 
Published: April 2021

ABSTRACT
The constitutive recirculation of lymphocytes between the widely distributed organs of the immune system is essential for host defence. We have developed an in vitro model of lymphocyte migration from the blood into lymph nodes which employs primary cultures of high endothelial cells (HEC). HEC-adherent lymphocytes adopt one of two distinct morphologies which correlates with their position in the endothelial layer; type I cells are bound to the surface of HEC and type II cells are underneath the endothelial layer. In a previous study we reported that the numbers of type I and type II cells are independently regulated, however the relationship between these two lymphocyte populations was not determined. In this study we have carried out detailed kinetic, phenotypic and functional analyses of type I and type II lymphocytes and determined their relationship. Using allotype marked lymphocytes from the PVG.RT7a and PVG.RT7b rat strains in a pulse-chase analysis, type I and type II lymphocytes were found to represent the same population of lymphocytes at different stages of interaction with the endothelial layer, rather than representing two independent lymphocyte populations. Migration was an irreversible event and the efficiency of migration (i.e. transition from type I to type II) was related to the concentration of lymphocytes plated on to the HEC layer. Following transmigration lymphocytes showed an increased ability to migrate across HEC layers and to bind to immobilised CS1 peptide. The increased binding to CS1 peptide was transient and fell to control levels over a 3 hour time period. The expression of 4 integrin subunit on lymphocytes was unchanged following migration which suggests that the affinity of the CS1 receptor, 4 1, is upregulated by interaction with HEC. Together these results suggest that transendothelial migration is regulated by increases in the affinity of 4 1 integrin on lymphocytes following contact with HEC.
Immunoelectron characterisation of the inter-endothelial junctions of human term placenta
Authors: Leach Lopa , Clark Peter , Lampugnani Maria-Grazia , Arroyo Alicia G. , Dejana Elisabetta , Firth J. Anthony 
Published: April 2021

ABSTRACT
The molecular constituents of the paracellular clefts in human placental microvessels were investigated using antibodies against PECAM-1, pancadherin, A-CAM (N-cadherin), cadherin-5 and two types of integrins (those recognised by antibodies to the α1 chain and v 3). Ultrastructural localisation of these molecules in ultrathin frozen sections of human term placentae was attempted using colloidal gold immunocytochemistry, after establishing their presence by indirect immuno-fluorescence.
At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse distribution in the endothelium and in the abluminal interstitial space. PECAM-1 and pan-cadherin were also seen in the cytoplasm and luminal surface of the endothelium. Immunoelectron studies revealed that the cadherins and PECAM-1 were present in the wide regions of the paracellular clefts, but not in tight junctional regions. Using immunocytochemistry, these wide junctional areas were found to be associated with the cytoskeletal linking molecules vinculin and α-actinin. These regions may therefore contain adherens-type junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen which was cleft-specific, the others also being seen in the cytoplasm of the microvascular endothelium. Cadherin-5 and pan-cadherin were colocalised in the same wide junction, but were usually seen to occupy different microdomains of, and different wide zones of, the same cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular spacing between endothelial cells, and may be part of a paracellular “fibre matrix” with permeability-restricting properties.
Independent modes of propagation of calcium waves in smooth muscle cells
Authors: G. Mahoney Mỹ , Slakey Linda L. , Hepler Peter K. , Gross David J. 
Published: April 2021

ABSTRACT
The purinergic agonist adenosine triphosphate (ATP) stimulates an initial transient followed by subsequent oscillations in cytosolic calcium ion concentration ([Ca2+]i) in individual porcine aortic smooth muscle cells. Using microinjection of fura-2 covalently coupled to dextran, we have analyzed in detail the spatial and temporal features of the oscillations. We have observed both cytoplasmic calcium waves and gradients within single cells. Single cells can contain multiple loci of initiation of oscillations. Independent oscillations in a single cell can have independent frequencies and these oscillations can propagate without interference across the same region of the cell, suggesting that they arise either from separately regulated stores of Ca2+ or a single Ca2+ store operated by two separate release mechanisms. The shape of the wave front and the manner of the wave’s decay can vary from one oscillation to the next. Ca2+ signaling in individual arterial smooth muscle cells thus displays complex spatial and temporal organization.
Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells
Authors: Alemany Ramón , Vilá M. Rosa , Francí Clara , Egea Gustavo , Real Francisco X. , Thomson Timothy M. 
Published: April 2021

ABSTRACT
Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.