bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2a family
Authors: Kam Ephraim , Resing Katheryn A. , and Sue K. Lim , Dale Beverly A. 
Published: April 2021

ABSTRACT
The aggregation of cellular intermediate filaments is an important step in the terminal differentiation of keratinocytes. It has been shown that epidermal filaggrin can cause intermediate filaments to aggregate in vitro and may also have the same function in vivo. Filaggrin is derived via dephosphorylation and proteolysis from a highly phosphorylated precursor, profilaggrin, which is found in the granular layer of the epidermis. Using casein kinase II phosphorylated filaggrin as substrate, a profilaggrin phosphatase has been partially purified from rat epidermal homogenate by three chromatographic steps (DE52, hydroxylapatite and S200 gel filtration). Profilaggrin phosphatase activity eluted from the last column has a Km of 0.12 mM and a Vmax of 8 nmol/mg/min with respect to phosphofilaggrin. Results obtained by initial rate analysis showed that the enzymatic activity is not affected by phospho-tyrosyl phosphatase inhibitors and the active fractions preferentially dephosphorylate the alpha subunit of phosphorylase kinase which has been phosphorylated by cAMP-dependent kinase. These results suggest that epidermal profilaggrin phosphatase is not a phospho-tyrosyl phosphatase or a type 1 phospho-seryl/phospho-threonyl phosphatase. Dephosphorylation is not affected by EDTA, calcium or magnesium, but is very sensitive to okadaic acid inhibition (IC50 = 80 pM), suggesting that the enzymatic activity is related to that of the protein phosphatase 2A (PP2A). This characterization is further supported by two other results: (1) immunoblotting with a monoclonal antibody against the catalytic subunit of PP2A demonstrated the presence of a single band of ∼36 kDa across the peak of S200 cloumn fractions containing dephosphorylation activity; (2) immunohistochemistry experiments using anti-PP2A antibodies showed strong immunostaining of granular cells in the epidermis, as would be expected, if this phosphatase is responsible for the dephosphorylation of profilaggrin. The activity of profilaggrin phosphatase increases rapidly with decreasing concentrations of NaCl in the assay mix, an observation that is compatible with the idea that enzyme activation may be triggered by the decrease of sodium concentration when keratinocytes enter the granular layer. Further characterization of the profilaggrin phosphatase activity showed that it is distinct from other epidermal acid phosphatases because of the near neutral pH optimum at 6.5. It is also distinct from the iron-activated epidermal protein phosphatase, which has been suggested to be a candidate phosphatase for profilaggrin because the two enzymes have different substrate specificities. Future studies of pathological conditions that involve anomalous processing of profilaggrin will become more accessible by the identification of profilaggrin phosphatase as a member of the PP2A family in the work reported here.
Multiple structural types of gap junctions in mouse lens
Authors: Lo Woo-Kuen , Reese Thomas S. 
Published: April 2021

ABSTRACT
Gap junctions in the epithelium and superficial fiber cells from young mice were examined in lenses prepared by rapid-freezing, and processed for freeze-substitution and freeze-fracture electron microscopy. There appeared to be three structural types of gap junction: one type between epithelial cells and two types between fiber cells. Epithelial gap junctions seen by freeze-substitution were ∼20 nm thick and consistently associated with layers of dense material lying along both cytoplasmic surfaces. Fiber gap junctions, in contrast, were 1516 nm (type 1) or 17-18 nm thick (type 2), and had little associated cytoplasmic material. Type 1 fiber gap junctions were extensive in flat expanses of cell membrane and had a thin, discontinuous central lamina, whereas type 2 fiber gap junctions were associated with the ball- and-socket domains and exhibited a dense, continuous central lamina. Both types of fiber gap junction had a diffuse arrangement of junctional intramembrane particles, whereas particles and pits of epithelial gap junctions were in a tight, hexagonal configuration. The type 2 fiber gap junctions, however, had a larger particle size (∼9 nm) than the type 1 (∼7.5 nm). In addition, a large number of junctional particles typified the E-faces of both fiber types but not the epithelial type of gap junction. Gap junctions between fiber and epithelial cells had structural features of type 1 fiber gap junctions. These structural features suggest that the epithelial and the type 2 fiber gap junctions are of the common communicating type, whereas the type 1 fiber gap junctions may represent a new type of intercellular contact, not necessarily even derived from gap junction proteins, which serves an adhesive function in the lens.
Menadione-induced oxidative stress leads to a rapid down-modulation of transferrin receptor recycling
Authors: Malorni Walter , Iosi Francesca , Santini Maria Teresa , Testa Ugo 
Published: April 2021

ABSTRACT
It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.
Molecular cloning of a 47 kda tissue-specific and differentiationdependent urothelial cell surface glycoprotein
Authors: Wu Xue-Ru , Sun Tung-Tien 
Published: April 2021

ABSTRACT
Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM) - a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells.
The globular head domain of titin extends into the center of the sarcomeric m band: cdna cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins
Authors: Vinkemeier Uwe , Obermann Wolfgang , Weber Klaus , Fürst Dieter O. 
Published: April 2021

ABSTRACT
Immunoelectron microscopical results have shown that the Z and M bands of the sarcomere are interconnected by the long titin molecules. Here we have characterized by monoclonal antibodies, cDNA cloning and immunoelectron microscopy the two titin-associated proteins (190 and 165 kDa proteins), which seem responsible for the formation of a head structure on one end of the 0.9 μm long titin string. The human 165 kDa (1465 residues) and 190 kDa (1451 residues) proteins have unique N-terminal domains some 110 residues in length. Both proteins show 12 repeat domains with strong homology to either fibronectin type III (motif I) or immunoglobulin C2 (motif II) domains, which are arranged in the order II-II-I-I-I-I-I-II-II-II-II-II. Over these repeat domains the two proteins share 50% sequence identity (70% similarity). Epitopes situated in the C-terminal 138 or in the preceeding 206 residues of the 165 kDa protein locate in immunoelectron microscopy to stripes situated 18 or 15 nm from the center of the M band. An epitope situated 277 to 129 residues prior to the C-terminus of the 190 kDa protein (i.e. repeats 10 and 11) locates to the center of the M band. Thus the head structure of the titin molecule extends into the center of the M band. Microsequence data on peptides from the titin-associated bovine 165 kDa protein and from conventionally purified bovine M-protein argue together with the reactivity of the antibodies that 165 kDa protein and M-protein are identical. The integrating structure of the sarcomere, which is based on titin and its side-on (C-protein and 86 kDa protein) or end-on (190 kDa protein and 165 kDa protein) associated proteins arises from muscle-specific members of the superfamily of immunoglobulin-like proteins.
The sequence of alignment of microtubules, focal contacts and actin filaments in fibroblasts spreading on smooth and grooved titanium substrata
Authors: Oakley C. , Brunette D. M. 
Published: April 2021

ABSTRACT
Contact guidance refers to the reactions of cells with the topography of their substratum. Current hypotheses on the mechanism of contact guidance focus on the dynamic behaviour of the cytoskeletal components, but most observations have been made on cells that have already become oriented with topographic features of the substratum. The purpose of this study was to examine the sequence in which microtubules, focal contacts and microfilament bundles become aligned to the substratum topography as fibroblasts spread on grooved substrata. Human gingival fibroblasts were trypsinized and seeded onto grooved titanium surfaces produced by micromachining, as well as onto control smooth surfaces. After observation and photography of the spreading cells at times up to 6 hours, the cells were fixed and exposed to one or more of the following antibodies or fluorescent stains: phallacidin to stain actin filaments, monoclonal anti-tubulin, monoclonal anti-vinculin, antimouse IgG labelled with Texas-Red or FITC, and/or an aldehyde-reactive stain to identify the cell outline. The cells were photographed and cell area, shape and orientation were calculated. Cells were also examined with confocal microscopy to obtain optical sections so that cell height as well as the precise locations of the cytoskeletal components with respect to the vertical dimension of the grooved substrata could be determined.
Microtubules were the first element to become oriented parallel to the direction of the grooves and were first aligned at the bottom of the grooves. This alignment of microtubules was evident as early as 20 minutes after plating and preceded the orientation of the cell as a whole. Aligned actin microfilament bundles were not observed until 40-60 minutes and were observed first at the wall-ridge edges. At early times, focal contacts were distributed radially, but only after 3 hours did the majority of cells demonstrate aligned focal contacts. If the first cytoskeletal component to become aligned is the prime determinant of cell orientation, then these data suggest that microtubules in human gingival fibroblasts may determine cell orientation on grooved titanium surfaces. By analogy with microtubule behaviour in other systems, we suggest that microtubule orientation on grooved substrata may occur as a result of the substratum establishing shear- free planes.
Dynamic control of cell-cell adhesion and membrane-associated actin during food-induced mouth opening in Beroë
Authors: Tamm Sidney L. , Tamm Signhild 
Published: April 2021

ABSTRACT
We used rhodamine-phalloidin and ultrastructural methods to follow dynamic changes in adhesive cell junctions and associated actin filaments during reversible epithelial adhesion in the mouth of the ctenophore Beroë. A cruising Beroë keeps its mouth closed by interdigitated actin-coated appositions between paired strips of cells lining the lips. The mouth opens rapidly (in 0.2-0.3 s) by muscular action to engulf prey (other ctenophores), then re-seals after ingestion. We found that the interlocking surface architecture of the adhesive cells, including the actin-coated junctions, rapidly disappears after food-induced opening of the mouth. In contrast, forcible separation of the lips in the absence of food rips the junctions, still intact, from the surfaces of the cells. The prey-stimulated loss of adhesive cell junctions and associated actin cytoskeleton is one of the most rapid changes in actin-based junctions yet observed. This system provides unique experimental advantages for investigating the dynamic control of reversible cell adhesions and membrane-associated actin filaments.
Acidosomes: recipients of multiple sources of membrane and cargo during development and maturation
Authors: Allen Richard D. , Ma Le , Fok Agnes K. 
Published: April 2021

ABSTRACT
Acidosomes are organelles that in Paramecium are responsible for the acidification of phagosomes before phagosomes fuse with lysosomes. Using a combination of (a) the quick-freeze deep-etch (QF-DE) technique, (b) monoclonal antibodies (mAbs) that label specific membrane pools including those of the acidosomes, and (c) horseradish peroxidase (HRP)-uptake studies, we followed the development of acidosomes from the Golgi complex as well as the rapid transfer of HRP into the acidosomes. We also studied some of the characteristics of the involved membrane pools. Morphologically, acidosomes were first detected in the cytosol near the ER and Golgi stacks as clumps of tubules and vesicles, which apparently coalesced to form larger spherical or elongated preacidosomes. These clumped vesicles and preacidosomes had a QF-DE morphology resembling that of the mature acidosomes and were specifically labeled with mAbs that also labeled mature acidosomes. Within 10 s HRP cargo could be internalized by acidosomes while they were docked at the nascent vacuole membrane. This rapid uptake of HRP along with membrane occurs by vesicle fusion, a conclusion supported by QF-DE images. Thus the acidosome obtains its membrane from at least two sources, from the trans-Golgi network, and from the small HRP-containing vesicles. Cargo can also be acquired from two sources, the Golgi apparatus and the transport vesicles. Since it acquires non-particulate exogenous marker we conclude that the acidosome is linked to the endocytic pathway.
Differential expression of plasminogen activators and their inhibitors in an organotypic skin coculture system
Authors: Chen Chih-Shan , Lyons-Giordano Bernadette , Lazarus Gerald S. , Jensen Pamela J. 
Published: April 2021

ABSTRACT
Using immunohistochemistry and in situ hybridization, we have characterized the expression and localization of components of the plasminogen activator proteolytic cascade in an organotypic coculture system which consists of a “dermal” portion (human dermal fibroblasts throughout a collagen matrix) and a stratified, well-differentiated epidermal portion. Specifically, the following components were examined: the enzymes urokinasetype plasminogen activator and tissue-type plasminogen activator and their type 1 and type 2 inhibitors. Urokinase plasminogen activator mRNA and antigen were found predominantly in the least differentiated, basal keratinocytes; in some fields there was also faint deposition of antigen beneath the basal cells. The distribution of plasminogen activator inhibitor type 1 was similar to that of urokinase, except that inhibitor type 1 antigen deposition beneath the basal cells appeared more intense and uniform. In contrast to the results with urokinase plasminogen activator and inhibitor type 1, tissue plasminogen activator mRNA and antigen were localized focally in the suprabasal, i.e. more differentiated, keratinocytes. Plasminogen activator inhibitor type 2 mRNA and antigen were detected in most epidermal layers, but were more intense suprabasally and often spared the basal layer. These studies demonstrate that the same type of cell, i.e. the keratinocyte, can express different components of the plasminogen activator cascade depending on its state of differentiation. The change in expression of plasminogen activator cascade components with keratinocyte differentiation suggests distinct epidermal functions for these components, related to cell-matrix interaction and epidermal differentiation.
Phorbol ester-induced scattering of HT-29 human intestinal cancer cells is associated with down-modulation of E-cadherin
Authors: Fabre Myriam , García de Herreros Antonio 
Published: April 2021

ABSTRACT
The effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth characteristics of the colon cancer cell line HT-29 M6 were studied. TPA induced the scattering of proliferative HT-29 M6 cells: in the presence of the phorbol ester, HT-29 M6 colonies scattered and the cells acquired a flatter aspect with diminished cell-cell contacts. This effect of TPA required a persistent activation of PK-C and was acompanied by a slight decrease (30%) in the growth rate. Modifications by TPA of two scattering associated properties of these cells were also detected: TPA decreased cell-to-cell aggregation and enhanced the cellular attachment to matrix substrata (collagen, laminin). The decrease in cell-to-cell adhesion was correlated with a loss of cellular E-cadherin as evidenced by immunofluorescence or immunoblotting with a specific monoclonal antibody. Cell scattering was dependent on the extra-cellular concentration of Ca2+; an increase from 1.6 to 10 mM in the concentration of this ion completely blocked the morphological effects of TPA as well as its action on cell aggregation. This high concentration of Ca2+ also prevented the down modulation of E-cadherin as determined by immunofluorescence. However, the TPA-induced increase in cell attachment to the matrix was not affected by high calcium. These findings support the importance of altered cell-cell adhesion in the process of scattering and provide a good system for the study of down modulation of E-cadherin, a protein involved in the control of cell growth, differentiation and invasion of epithelial cells.
Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora Crassa are mediated by cytoplasmic microtubules
Authors: Steinberg Gero , Schliwa Manfred 
Published: April 2021

ABSTRACT
The cellular basis of organelle transport in filamentous fungi is still unresolved. Here we have studied the intra-cellular movement of mitochondria and other organelles in the fungus Neurospora crassa. Four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. Organelle movements were visualized by DIC optics and computer-enhanced video microscopy. In all cell models the transport of organelles was vectorial and saltatory in nature. The mean velocities for mitochondria, particles and nuclei were 1.4, 2.0, and 0.9 m/s, respectively. Treatment with 10 M nocodazole for 30 minutes caused a complete disappearance of microtubules and reversibly blocked directed transport of virtually all organelles, whereas cytochalasin D up to 20 M was without effect. Correlative video and immunofluorescence microscopy of small fragments of wall-less mutant cells revealed a clear match between microtubule distribution and the tracks of moving organelles. We conclude that organelle movement in the filamentous fungus Neurospora crassa is a microtubule-dependent process.
α7β1 integrin is a component of the myotendinous junction on skeletal muscle
Authors: Bao Z. Z. , Lakonishok M. , Kaufman S. , Horwitz A. F. 
Published: April 2021

ABSTRACT
Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an anti-body directed against the avian 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat 7 cytoplasmic domain. On sections of adult skeletal muscle the 7 integrin was enriched in the myotendinous junc-tion (MTJ). This localization was unique as neither the 1, 3, 5, 6 and v subunit localizes in the myotendi-nous junction. The distribution of the 7 subunit in the MTJ was examined during embryonic development. 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the devel-oping MTJ at this stage. 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the 7 sub-unit in the MTJ correlates with the insertion of myofib-rils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the 7 inte-grin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the 7 integrin recognizes a preformed cytoskele-tal structure. The presence of the 7 subunit in the myotendinous junction and the 5 subunit in the adhe-sion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Dif-ferences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.