bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of epidermal differentiation by epiligrin and integrin α3β1
Authors: Symington Banu Erhan , Carter William Gene 
Published: April 2021

ABSTRACT
We previously reported that integrin α3β1 mediates epidermal intercellular adhesion as well as cell-substrate adhesion. P1B5, an anti-α3β1 specific monoclonal antibody, is a potent in vitro trigger of epidermal cell-cell adhesion and an inhibitor of cell-substrate adhesion. We now show that P1B5 specifically induces the intercellular localization of integrins α2β1 and α3β1, consistent with its role in inducing intercellular adhesion via these two integrins. P1F2, another anti-α3β1 antibody, does not induce either intercellular adhesion or intercellular accumulation of α3β1 and α2β1.
Growth of epidermal cells in high calcium, known to induce epidermal differentiation, also induces intercellular accumulation of α3β1 and α2β1 and increased cell-cell adhesion. We therefore asked whether P1B5 treatment induces epidermal differentiation. P1B5 treatment induces changes consistent with epidermal differentiation, including increased involucrin expression, stratification, and production of squames. P1F2 treatment has none of these effects. In vivo, epidermal basal cells are in close contact with the epithelial basement membrane component epiligrin. Growth of keratinocytes on purified epiligrin but not other matrix components specifically reduces involucrin expression by P1B5-treated keratinocytes. These results suggest that integrin α3β1 has a unique role in epidermal differentiation, that the epitope recognized by P1B5 is involved in triggering this differentiation, and that keratinocyte adhesion to epiligrin inhibits α3β1-mediated differentiation. Taken together, these data suggest that α3β1-mediated intercellular adhesion may be part of the terminal differentiation process in epidermal cells.
Overexpressing cell surface β1,4-galactosyltransferase in pc12 cells increases neurite outgrowth on laminin
Authors: Huang Qiuling , Shur Barry D. , Begovac Paul C. 
Published: April 2021

ABSTRACT
Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface β1,4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three-fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin.
Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation
Authors: Kiley Susan C. , Parker Peter J. 
Published: April 2021

ABSTRACT
U937 human promonocytic leukemia cells express PKC isozymes β1, β2, ε and ζ. Indirect immunocytofluorescence using affinity-purified PKC-specific antibodies indicates that each of the endogenous PKC isozymes in U937 cells display a unique compartmentalization within the intact cell. PKC-β1 is distributed between two identifiable pools: a cytoplasmic pool which redistributes to the plasma membrane upon activation with acute phorbol ester-treatment, and a membrane-bound pool associated with intracellular vesicles containing β2-integrin adhesion molecules, cd11b and cd11c. The vesicle-associated PKC-β1 translocates with the secretory granules to the plasma membrane upon agonist-stimulated activation. PKC-β2 is associated with the microtubule cytoskeleton in resting cells. PKC overlay assays indicate that PKC-β2 binds to proteins associated with microtubules, and not directly to tubulin. PKC-ε is associated with filamentous structures in resting cells and redistributes to the perinuclear region upon activation with phorbol esters. In differentiated U937 cells, PKC-β1 remains associated with vesicles translocat-ing from the trans-Golgi region to the plasma membrane and PKC-ε is primarily associated with perinuclear and plasma membranes. PKC-ζ, which does not respond to phorbol ester treatment, is primarily cytosolic in undiffer-entiated cells and accumulates in the nucleus of differenti-ated cells blocked in the G2 phase of the cell cycle. The data clearly demonstrate that individual PKCs localize to different subcellular compartments and promote the hypothesis that PKC subcellular localization is indicative of unique functions for individual PKC isozymes.
Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies
Authors: Rodriguez J. , Boulo V. , Mialhe E. , Bachere E. 
Published: April 2021

ABSTRACT
Various haemolymph components of the shrimp Penaeus japonicus were identified and characterised by monoclonal antibodies. Three groups of monoclonal antibodies were raised. Their reactivity to haemocyte types and/or secreted molecules was determined by immunofluorescence and the molecular masses of the antigens were analysed by western-blotting. A 170 kDa protein, in reducing conditions, was recognized by four panhaemocytic monoclonal antibodies from group 1. This protein was present both in the plasma and in the haemocytes from which it appears to be secreted. The shrimp haemocytes were separated by isopycnic cen-trifugation on a Percoll gradient and the different subpop-ulations were antigenically analysed using the two mono-clonal antibodies, 40E2-2A and 40E10-2B, from group 2. The granular cells were labelled by 40E2-2A which was specific for a protein of 142 kDa also present in plasma. By comparison, the 40E10-2B monoclonal antibody was assumed to be the marker for small hyaline and semi-granular cells since the granular ones were not labelled. Moreover, the antigen immunoprecipitated by this mono-clonal antibody was shown to have different molecular masses of 250, 150, 66 and 27 kDa under nonreducing con-ditions. It appeared to be secreted by the haemocytes. Some plasma proteins were recognized by the third group of monoclonal antibodies. The antibodies, designated 41D11-3A, 42C11-3B and 42E8-3C, all immunoprecipitated a protein with an apparent molecular mass of 180 kDa under reduced conditions. The 44E6-3D antibody was specific for a 75 kDa protein under reduced conditions and was shown to be immunoreactive against P. japonicus haemocyanin extract. An antigenic relationship was studied for the haemolymph components of P. japonicus and two other shrimp species, P. vannamei and P. indicus. Hypotheses about the possible nature of the proteins, antigenically identified, are advanced.
A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines
Authors: Hembrough Todd A. , Vasudevan Jayanand , Allietta Margaretta M. , Glass William F. , Gonias Steven L. 
Published: April 2021

ABSTRACT
Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immuno-electron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluores-cence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.
Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule A new model for parietal cell transformation
Authors: Pettitt John M. , Humphris Danielle C. , Barrett Simon P. , Toh Ban-Hock , Driel Ian R. van , Gleeson Paul A. 
Published: April 2021

ABSTRACT
The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic ‘tubulovesicles’ as the apical surface of the cell progres-sively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesi-cles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimu-lated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with α-and β-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progres-sive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; conse-quently, they cannot have a role in parietal cell transfor-mation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is com-patible with the localization of cytoskeletal components in these cells.
p80-coilin: a component of coiled bodies and interchromatin granule-associated zones
Authors: Puvion-Dutilleul Francine , Besse Sylvie , Chan Edward K. L. , Tan Eng M. , Puvion Edmond 
Published: April 2021

ABSTRACT
We investigated at the electron microscope level the fate of the three intranuclear structures known to accumulate snRNPs, and which correspond to the punctuate immuno-fluorescent staining pattern (the coiled bodies, the clusters of interchromatin granules and the interchromatin granule-associated zones) after exposure to either a low salt medium which induces a loosening and partial spreading of nucleoprotein fibers or a high ionic strength salt medium and subsequent DNase I digestion, in order to obtain DNA-depleted nuclear matrices. The loosened clusters of inter-chromatin granules and the coiled bodies could no longer be distinguished from surrounding nucleoprotein fibers solely by their structure, but constituents of the clusters of interchromatin granules could be detected by in situ hybridization with both U1 and U2 DNA probes, and con-stituents of the coiled bodies were detectable mainly with the U2 DNA probe. The interchromatin granule-associated zones, the electron-opacity and compactness of which were preserved despite the loosening treatment, remained labeled with the U1 DNA probe only. In DNA-depleted nuclear matrices, the snRNA content of the coiled bodies, the clusters of interchromatin granules and their associated zones, which were all easily recognizable within the residual nuclear ribonucleoprotein network, was unmodi-fied. The data indicate, therefore, that the loosening procedure as well as the high salt extraction procedure preserve the snRNA content of all three spliceosome component-accumulation sites and reveal that interchro-matin granule-associated zones are elements of the nuclear matrix. The p80-coilin content of coiled bodies was also preserved whatever the salt treatment used. An intriguing new finding is the detection of abundant p80-coilin within the interchromatin granule-associated zones, both before and after either low or high salt treatment of cells. Therefore, p80-coilin is an integral constituent of the inter-chromatin granule-associated zones.
Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes
Authors: Guénette Suzanne , Magendantz Margaret , Solomon Frank 
Published: April 2021

ABSTRACT
To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy sup-pressors of a conditional mutation in α-tubulin. Cells expressing the recessive allele tub1-729 as their sole α-tubulin gene grow normally at permissive temperature. However, at 15°C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures. Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type α-tubulin genes, TUB1 and TUB3; and BUB3. BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules. Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729. The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the ‘no microtubule’ phenotype. Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not. Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but dis-ruption of the BUB3 gene produces slow growth and benomyl supersensitivity. Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.
Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase
Authors: Clarke Paul R. , Klebe Christian , Wittinghofer Alfred , Karsenti Eric 
Published: April 2021

ABSTRACT
During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein, Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of Cdc2/cyclin B protein kinase through Ran. In this report, we present direct biochemical evidence for the post-translational control of Cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits Cdc2/cyclin B activation in the presence of repli-cating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of Cdc2 and appears to be distinct from other effects on nuclear import, nuclear formation and DNA replication. When extracts were supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of Cdc2/cyclin B by Ran T24N was relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked in the GTP-bound state has no inhibitory effect on Cdc2/cyclin B acti-vation. In the light of these results, we propose that gener-ation of the GTP-bound form of Ran is required for Cdc2/cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase.
Collagen matrices attenuate the collagen-synthetic response of cultured fibroblasts to TGF-β
Authors: Clark Richard A. F. , Nielsen Larry D. , Welch Marshall P. , McPherson John M. 
Published: April 2021

ABSTRACT
Transforming growth factor-β, a potent modulator of cell function, induces fibroblasts cultured on plastic to increase collagen synthesis. In 5- and 7-day porcine skin wounds, which have minimal to moderate collagen matrix, respectively, transforming growth factor-β and type I procollagen were coordinately expressed throughout the granulation tissue. However, in 10-day collagen-rich granulation tissue type I procollagen expression diminished despite persistence of transforming growth factor-β. To investigate whether collagen matrix attenuates the collagen-synthetic response of fibroblasts to transforming growth factor-β, we cultured human dermal fibroblasts in conditions that simulate collagen-rich granulation tissue. Therefore, human dermal fibroblasts were suspended in attached collagen gels and collagen and noncollagen production was assayed in the absence and presence of transforming growth factor-β. Although transforming growth factor-β stimulated collagen synthesis by fibroblasts cultured in the collagen gels, these fibroblasts consistently produced less collagen than similarly treated fibroblasts cultured on plastic. This phenomenon was not secondary to nonspecific binding of transforming growth factor-β to the collagen matrix. Fibroblasts cultured in a fibrin gel responded to transforming growth factor-β similarly to fibroblasts cultured on plastic. Using immunofluorescence probes to type I procollagen, we observed that transforming growth factor-β increased type I procollagen expression in most fibroblasts cultured on plastic, but only in occasional fibroblasts cultured in collagen gels. From these data we conclude that collagen matrices attenuate the collagen synthetic response of fibroblast to transforming growth factor-β in vitro and possibly in vivo.
Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro
Authors: Garcés Jorge A. , Hoey John G. , Gavin R. H. 
Published: April 2021

ABSTRACT
The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluores-cence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin anti-bodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin anti-bodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.