bluuebunny/crossref_metadata_2025_split huggingface dataset:
SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes
Authors: Alkemade J. A. C. , Molhuizen H. O. F. , Ponec M. , Kempenaar J. A. , Zeeuwen P. L. J. M. , Jongh G. J. de , Vlijmen-Willems I. M. J. J. van , Erp P. E. J. van , Kerkhof P. C. M. van de , Schalkwijk J. 
Published: April 2021

ABSTRACT
Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8–10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.
These findings demonstrate that SKALP is an inducible proteinase inhibitor and support the concept that SKALP acts as a regulatory molecule in cutaneous homeostasis.
Characterization of very acidic phagosomes in breast cancer cells and their association with invasion
Authors: Montcourrier P. , Mangeat P. H. , Valembois C. , Salazar G. , Sahuquet A. , Duperray C. , Rochefort H. 
Published: April 2021

ABSTRACT
Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 μm) in which endocy-tosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcour-rier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epiflu-orescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 μm diameter latex beads and fluorescent Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are het-erophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H+-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (&lt;4), lower than the lysosomal value (pH ∼5) in the same cells and in specialized phagocytotic cells such as macrophages.
We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.
Control of stable lamellipodia formation by expression of cell surface β1,4-galactosyltransferase cytoplasmic domains
Authors: Appeddu Paul A. , Shur Barry D. 
Published: April 2021

ABSTRACT
Mesenchymal cell migration on basal lamina is mediated, in part, by the binding of cell surface β1,4-galactosyltrans-ferase (GalTase) to specific N-linked oligosaccharides in the E8 domain of laminin. On migrating cells, surface GalTase is anchored to the cytoskeleton; when GalTase is prevented from associating with the cytoskeleton, lamellipodia formation and subsequent migration are inhibited. To define better the involvement of GalTase-cytoskeleton interactions in cell motility, we examined the lamellipodia formation, polarity and migratory behavior of stably trans-fected 3T3 fibroblasts expressing increased or decreased levels of GalTase capable of interacting with the cytoskele-ton. Initially, the motile behavior of individual cells was quantified in the absence of exogenous stimuli. Cells that overexpress GalTase binding sites for the cytoskeleton changed their polarity more frequently and translocated more erratically than did control cells when assayed on laminin substrata. These differences were not observed, however, when cells were plated on fibronectin, which does not contain binding sites for surface GalTase. GalTase-transfected cells were also assayed for their ability to polarize in response to a specific stimulus. In this case, the ability of a cell to reorient towards a gradient of platelet-derived growth factor was found to be directly proportional to the amount of GalTase associated with the cytoskeleton. Differences in response to platelet-derived growth factor were not due to differences in growth factor binding. Indirect immunofluorescence showed that altering the level of GalTase did not affect the ventrally distributed pool of GalTase stably associated with the cytoskeleton; however, stress fiber formation was inhibited. Thus, increasing surface GalTase binding sites for the cytoskele-ton leads to erratic, multipolar behavior in the absence of any vectorial stimulus, but the ability to form a functional lamellipodium in response to a stimulus is dependent upon the amount of surface GalTase associated with the cytoskeleton. Apparently, cells are able to regulate cytoskeletal assembly and lamellipodial stability by altering the expression and/or affinity of appropriate matrix receptors, such as GalTase, and their correspond-ing binding sites in the cytoskeleton.
The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum
Authors: Watson Ned , McGuire Vince , Alexander Stephen 
Published: April 2021

ABSTRACT
The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and noncovalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the assembly of specific extracellular matrices.
Elastin exhibits a distinctive temporal and spatial pattern of distribution in the developing chick limb in association with the establishment of the cartilaginous skeleton
Authors: Hurle Juan M. , Corson Glen , Daniels Karla , Reiter Rebecca S. , Sakai Lynn Y. , Solursh Michael 
Published: April 2021

ABSTRACT
In this work we have analyzed the presence of elastic components in the extracellular matrices of the developing chick leg bud. The distributions of elastin and fibrillin were studied immunohistochemically in whole-mount preparations using confocal laser microscopy. The association of these constituents of the elastic matrix with other components of the extracellular matrix was also studied, using several additional antibodies. Our results reveal the transient presence of an elastin-rich scaffold of extracellular matrix fibrillar material in association with the establishment of the cartilaginous skeleton of the leg bud. The scaffold consisted of elastin-positive fibers extending from the ectodermal surface of the limb to the central cartilageforming regions and between adjacent cartilages. Fibrillin immunolabeling was negative in this fibrillar scaffold while other components of the extracellular matrix including: tenascin, laminin and collagens type I, type III and type VI; appeared codistributed with elastin in some regions of the scaffold. Progressive changes in the spatial pattern of distribution of the elastin-postive scaffold were detected in explant cultures in which one expects a modification in the mechanical stresses of the tissues related to growth.
A scaffold of elastin comparable to that found in vivo was also observed in high-density micromass cultures of isolated limb mesodermal cells. In this case the elastic fibers are observed filling the spaces located between the cartilaginous nodules. The fibers become reoriented and attach to the ectodermal basal surface when an ectodermal fragment is located at the top of the growing micromass. Our results suggest that the formation of the cartilaginous skeleton of the limb involves the segregation of the undifferentiated limb mesenchyme into chondrogenic and elastogenic cell lineages. Further, a role for the elastic fiber scaffold in coordinating the size and the spatial location of the cartilaginous skeletal elements within the limb bud is also suggested from our observations.
Nucleolar localisation of three Hox homeoproteins
Authors: Corsetti Maria Teresa , Levi Giovanni , Lancia Flavia , Sanseverino Lorenza , Ferrini Silvano , Boncinelli Edoardo , Corte Giorgio 
Published: April 2021

ABSTRACT
Homeoproteins encoded by genes of the Hox family are nuclear proteins believed to act as transcription factors and to participate in the determination of the body plan. Here we show that in several vertebrate cells, they exhibit a sub-nuclear localisation associated with the nucleolus.
We used monoclonal antibodies to study the distribution of three homeoproteins, namely HOXB7, HOXC6 and HOXD4. The immunoreactivity to antibodies against HOXC6 protein in Xenopus laevis embryonic tissues is restricted to one or two spots within the nucleus; this dis-tribution partially overlaps that of fibrillarin, a protein of the fibrillar zone of the nucleoli. Indirect immunofluorescence analysis of the distribution of HOXB7 protein in 3T3 cells, and of HOXD4 protein in human neuroblastoma and Raji lymphoma cell lines and activated lymphocytes, results invariably in a nucleolar localisation. Purified nucleoli from stimulated T lymphocytes, and Raji cells contain an activity capable of binding, in a gel retardation assay, to an oligonucleotide specifically recognised by the HOXD4 homeoprotein. This activity is specifically removed by anti-HOXD4 antibodies and is found associated in southwestern blots with a single band with an apparent Mr of 30,000, corresponding to that of recombinant HOXD4. The functional significance of the nucleolar localisation of Hox proteins remains to be determined.
Activation of the small GTP-binding proteins rho and rac by growth factor receptors
Authors: Nobes Catherine D. , Hawkins Phillip , Stephens Len , Hall Alan 
Published: April 2021

ABSTRACT
The small GTP-binding proteins, rho and rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibres, whilst rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane that results in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the rho and rac signalling pathways, we have used a potent inhibitor of this activity, wortmannin. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein.
In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor and insulin though not by bombesin, phorbol ester or microin-jected recombinant rac protein. We conclude that phos-phatidylinositol 3,4,5 trisphosphate mediates activation of rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected rho protein, we conclude that lysophosphatidic acid activation of rho is mediated by a tyrosine kinase.
A protein similar to the 67 kDa laminin binding protein and p40 is probably a component of the translational machinery in Urechis caupo oocytes and embryos
Authors: Rosenthal Eric T. , Wordeman Linda 
Published: April 2021

ABSTRACT
Oocytes of the echiuroid, Urechis caupo, contain an abundant maternal mRNA that encodes a protein very similar to LBP/p40, originally identified as a non-integrin 67 kDa laminin binding protein. We have sequenced the Urechis caupo mRNA for LBP/p40, and a similar mRNA from the Hawaiian sea urchin, Tripneustes gratilla. Both of the encoded proteins, as well as LBP/p40 proteins from other sources, share significant homology in the amino 2/3 of the proteins, but diverge extensively at the carboxyl ends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentration remains constant for the first 48 hours of embryogenesis and then begins to decline. In sucrose gradients run with homogenates from coelomocytes, oocytes, and early embryos, all of the LBP/p40 protein appears to be associated with either polysomes or free 40 S ribosomal subunits. In later embryos, an increasing proportion of the protein is found in the soluble fraction. Immunohistochemistry indicates that LBP/p40 is uniformly distributed in early U. caupo embryos, with no localization at the cell surface. In later embryos LBP/p40 is localized in specific parts of the embryo which may correspond to neural tissue.
Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus
Authors: Dargemont Catherine , Schmidt-Zachmann Marion S. , Kühn Lukas C. 
Published: April 2021

ABSTRACT
Primary transcripts in eukaryotic cells undergo several processing steps within the nucleus, and resulting mature RNA molecules are selectively exported to the cytoplasm. Nucleo-cytoplasmic mRNA transport is an active process that likely involves RNA-protein interactions. To identify specific RNA-binding proteins, we designed a novel approach, which allows the analysis of interactions between mRNAs and proteins along the transport pathway. The method consists of inducing in vivo a covalent binding between nuclear proteins and microinjected mRNAs. Using such a procedure, we were able to detect a direct interaction between nucleoporin p62 with mRNA during export. The formation of the mRNA-p62 complex was inhibited by wheat-germ agglutinin, an inhibitor of mRNA export. Antibodies directed against p62 caused a substantial reduction in the rate of mRNA export from the nucleus.
Identification of subcellular compartments containing peptidylglycine α-amidating monooxygenase in rat anterior pituitary
Authors: Oyarce Ana Maria , Eipper Betty A. 
Published: April 2021

ABSTRACT
Both soluble and integral membrane forms of peptidylglycine α-amidating monooxygenase (PAM) are expressed in the rat anterior pituitary, making it an ideal model system for studying the routing of proteins into secretory granules. To identify the subcellular compartments involved in the routing of integral membrane PAM, we used subcellular fractionation, metabolic labeling and immunoblot analysis. Mature secretory granules were found to contain full-length integral membrane PAM along with a significant amount of soluble PAM generated by endoproteolytic cleavage. PAM proteins were not co-dis-tributed with tyrosylprotein sulfotransferase activity during sucrose gradient centrifugation, indicating that the trans-Golgi/TGN is not a major PAM-containing compart-ment at steady state. Fractionation of the 4,000 g and 10,000 g pellets obtained by differential centrifugation identified a significant amount of integral membrane PAM in a light fraction lacking soluble secretory granule proteins. Metabolic labeling experiments with primary anterior pituitary cells demonstrated that integral membrane PAM enters a light compartment with similar properties only after exit from the trans-Golgi/TGN. Com-parison of the metabolic labeling and immunoblot analyses suggests that PAM in this post-trans-Golgi/TGN compart-ment is in organelles involved in the intracellular recycling of integral membrane PAM. Small amounts of full-length integral membrane PAM were also recovered in fractions containing internalized transferrin and may be in an endosomal compartment following retrieval from the cell surface.
A dialysis culture system for the study of the production and modulation of growth-regulatory molecules: studies using the P388D1 macrophage cell line
Authors: Proudfoot D. , Parrott D. P. , Bowyer D. E. 
Published: April 2021

ABSTRACT
P388D1 macrophage-like cells have previously been shown to produce both mitogenic and inhibitory regulators of porcine smooth muscle cell (pSMC) growth. The mitogenic activity was shown to have a molecular mass of &gt;10 kDa while the inhibitory activity was in the range of 2-6 kDa. In the present study, we present a novel dialysis culture system where P388D1 cells were grown in dialysis membranes with a 12 kDa cut-off which allowed continu-ous production of fractions of the culture medium. Using pSMC as target cells, mitogenic activity was found to be retained by the dialysis membrane while the low molecular mass inhibitory activity passed freely through the membrane.
The effect of the macrophage-activators phorbol myristate acetate (PMA), concanavalin A (ConA) and interferon-γ in combination with lipopolysaccharide (IFNγ/LPS) were investigated in the dialysis culture system. PMA, ConA and IFNγ/LPS were found to enhance the production of mitogenic activity by P388D1 cells. PMA also increased the production of growth-inhibitory activity, while ConA abolished inhibitor production and IFNγ/LPS had no effect on the amount of inhibitory activity produced by P388D1 cells.
The experiments show that the balance of production of mitogenic and inhibitory activities by macrophages can be modulated by agents that alter the state of activation of the cells. This could be of profound significance in the influence of macrophages on smooth muscle cell growth during the development of atherosclerosis.
A mechanical function of myosin II in cell motility
Authors: Jay Patrick Y. , Pham Peter A. , Wong Scott A. , Elson Elliot L. 
Published: April 2021

ABSTRACT
Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell’s rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface.
In vivo expression of alternatively spliced forms of integrin-associated protein (cd47)
Authors: Reinhold Martina I. , Lindberg Frederik P. , Plas David , Reynolds Stacy , Peters Marion G. , Brown Eric J. 
Published: April 2021

ABSTRACT
Integrin-associated protein (IAP) is a 50 kDa plasma membrane protein physically and functionally associated with β3 integrins in a variety of cells. IAP has an extracellular immunoglobulin domain, five transmembrane domains and a short intracytoplasmic tail. IAP is recognized by anti-CD47 antibodies and is expressed on cells, such as erythrocytes and lymphocytes, which do not express β3 integrins. To learn more about potential functions of IAP we examined its expression in vivo. Using the polymerase chain reaction, we detected 4 alternatively spliced forms of IAP which differ from each other only at their intracytoplasmic carboxy termini. These alternatively spliced forms are generated by inclusion or exclusion of three short exons within 5 kb in the genome and are highly conserved between mouse and man. There is tissue specificity of expression of the alternatively spliced forms of IAP mRNA, with bone marrow-derived cells expressing pre-dominantly one form and neural tissue another. Using polyclonal antibodies which recognize the alternatively spliced bone marrow (form 2) and neural (form 4) forms of IAP, we found that in accord with the mRNA, form 2 protein was expressed in all tissues primarily on bone marrow-derived cells and endothelia, while form 4 was highly expressed in the brain and peripheral nervous system. The evolutionary conservation of IAP isoforms and their tissue-specific expression suggest an important role for these intracytoplasmic domains in IAP function.