bluuebunny/crossref_metadata_2025_split huggingface dataset:
Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum
Authors: Schlatterer Christina , Gollnick Frank , Schmidt Eckhard , Meyer Rainer , Knoll Gerd 
Published: April 2021

ABSTRACT
Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+ concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+ indicator fura-2. Stimulation of cells with cAMP at the concentration required to induce gene expression (&gt; 100 μM) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a rise in [Ca2+]i, with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2′-deoxy-cAMP, which binds to the cAMP receptor, induced an increase in [Ca2+]i, whereas the membranepermeant analogue 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the change in [Ca2+]i is mediated by the cell surface cAMP receptor. Since HC85 mutant cells, which lack the Gα2 subunit of the G-protein that couples the receptor to phospholipase C, also responded to stimulation with cAMP, the Ca2+ influx does not seem to be triggered by the phosphoinositide signaling cascade.
In order to find out whether such changes in [Ca2+]i could be part of the differentiation process in vivo we determined the volume of the extracellular space in aggregates of cells as a basis for the estimation of the physiological cAMP concentration in the multicellular organism. Electron microscopy revealed only narrow gaps between the cells, with the extracellular space comprising roughly 1% of the cell volume. From the estimation of the resulting concentration range of cAMP we consider it highly probable that cells in an aggregate encounter doses of cAMP that elicit a global rise in [Ca2+]i; this could be part of the signal transduction system involved in the processes of differentiation.
Changes in annexin I and II levels during the postnatal development of rat pancreatic islets
Authors: Ohnishi Makoto , Tokuda Masaaki , Masaki Tsutomu , Fujimura Takashi , Tai Yuji , Matsui Hideki , Itano Toshifumi , Ishida Toshihiko , Takahara Jiro , Konishi Ryoji , Hatase Osamu 
Published: April 2021

ABSTRACT
The expression patterns and the dynamic changes in content of both annexin I and annexin II in the rat pancreatic islets during postnatal development were investigated by both western blot analysis and immunohistochemistry. Immunohistochemical methods clearly demonstrated the presence of annexins I and II exclusively in pancreatic islets, while exocrine tissues were not stained by anti-annexin antibodies. Pancreatic islets were diffusely stained with no specific differences in distribution between different cell types. The expression of annexin I in pancreatic islets gradually increased with postnatal development. A developmental study of annexins I and II by western blot analysis essentially supported the results obtained by immunohistochemistry. In addition, the increasing expression of two protein tyrosine kinases, epidermal growth factor-receptor/kinase and pp60sre, which phosphorylate annexin I and annexin II, respectively, and of protein kinase C, which phosphorylates both proteins, was also shown during postnatal development in rat pancreatic islets. Thus, a relationship between the expression of annexins I and II and the maturation of islet cell function is suggested.
Identification of a key integrin-binding sequence in VCAM-1 homologous to the LDV active site in fibronectin
Authors: Clements John M. , Newham Peter , Shepherd Maura , Gilbert Richard , Dudgeon Timothy J. , Needham Lindsey A. , Edwards R. Mark , Berry Linda , Brass Andrew , Humphries Martin J. 
Published: April 2021

ABSTRACT
The integrin adhesion receptor α4β1 binds two ligands, the extracellular matrix glycoprotein fibronectin and the immunoglobulin superfamily member VCAM-1. Ligandbinding sites are contained with the HepII/IIICS domain of fibronectin, and within the homologous immunoglobulin domains 1 and 4 of VCAM-1. Previous studies have shown that the binding of each ligand to α4β1 is mutually exclusive, suggesting that they may employ similar mechanisms to bind receptor. Fibronectin contains at least three distinct peptide sequences that are active sites for α4β1 binding, two homologous sequences Leu-Asp-Val-Pro (LDVP) and Ile-Asp-Ala-Pro (IDAP), and a third related to Arg-Gly-Asp (RGD). Using a combination of site-directed mutagenesis and synthetic peptide approaches in conjunction with VCAM-1-dependent cell adhesion assays, we now report the identification of a key α4β1-binding sequence in both domains 1 and 4 of VCAM-1 as the tetrapeptide Ile-Asp-Ser-Pro (IDSP). Mutagenesis studies also suggest that an additional sequence in domain 1, KLEK, participates in receptor binding. Since IDSP is homologous to the LDVP and IDAP fibronectin peptides, this therefore provides a molecular explanation for the promiscuity of ligand binding by α4β1 and has implications for the design of synthetic VCAM-1 antagonists. The extrapolation of these findings to other integrin-binding immunoglobulin ligands is also discussed.
Mammalian cells have two functional RCC1 proteins produced by alternative splicing
Authors: Miyabashira Junko , Sekiguchi Takeshi , Nishimoto Takeharu 
Published: April 2021

ABSTRACT
Previously we cloned two human RCC1 cDNAs that differed in their noncoding region. In this study, we have found new human and hamster RCC1 cDNAs, which have an even more different coding region from that of the previously cloned RCC1 cDNAs yet can complement the RCC1 mutation in the tsBN2 cell line. The newly found RCC1 cDNAs encode a protein (designated as RCC1-I) that has an insertion of 31 (human) and 13 (hamster) amino acids at valine25 in the N-terminal region outside the RCC1-seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence that had already been determined, and was found to be located between the 6th and 7th exons, designated as the 6′ exon. Both the 5′ and 3′ ends of the 6′ exon correspond to the GT-AG rules for splicing, indicating that human RCC1-I mRNAs are produced by alternative splicing. The finding that both humans and hamsters have the insertion at the same RCC1 site suggests that the pattern of alternative splicing in the RCC1 gene has been conserved through evolution.
Expression of prelamin A but not mature lamin A confers sensitivity of DNA biosynthesis to lovastatin on F9 teratocarcinoma cells
Authors: Sinensky M. , McLain T. , Fantle K. 
Published: April 2021

ABSTRACT
The role of inhibition of prelamin A processing in the inhibition of DNA synthesis by lovastatin was examined by expressing prelamin A in F9 teratocarcinoma cells. These cells, normally lacking expression of the A/C lamins, were transfected with constructs expressing either prelamin A or mature lamin A and the effect of lovastatin on DNA biosynthesis was assessed. It was found that expression of prelamin A specifically conferred sensitivity to inhibition of DNA biosynthesis by lovastatin on F9 cells.
Isolation and characterization of a sea urchin zygote cortex that supports in vitro contraction and reactivation of furrowing
Authors: Walker Gary R. , Kane Robert , Burgess David R. 
Published: April 2021

ABSTRACT
The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATPyS), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.
Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy
Authors: Pietrasanta L. I. , Schaper A. , Jovin T. M. 
Published: April 2021

ABSTRACT
Scanning force microscopy (SFM) was used for imaging subcellular structures of cultured rat mammary carcinoma cells dried in air. Identification of cellular substructures was achieved by immunofluorescence and specific fluores-cence probes. Cells grown attached to a glass support exhibited submicrometer thickness in the dried state. Inside the nuclear domain the nucleoli appeared as prominent conical protrusions. Membrane extensions, microspikes and microvilli were well preserved at the cell periphery after fixation in glutaraldehyde vapor and air-drying and were distinguishable either as isolated elements or intercellular communications. The plasma membrane and soluble proteins were selectively removed with non-ionic detergent in a buffer system. The mitochondria were concentrated primarily in the perinuclear space and exhibited a well defined filamentous shape. Their identity was confirmed by specific fluorescence staining with rhodamine 123. In the membrane-free system achieved by dry-cleaving of the sample surface, the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. The smallest fibrous substructures revealed by SFM with the scanning tips used to date were ∼8 to 10 nm in height and 80 nm in width.
Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line
Authors: Rochwerger Leonora , Dho Sascha , Parker Linda , Foskett J. Kevin , Buchwald Manuel 
Published: April 2021

ABSTRACT
We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogencoated filters was permeabilized with nystatin, a forskolinstimulated Cl− permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogenregulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.
A novel role for E- and P-selectins: shape control of endothelial cell monolayers
Authors: Kaplanski Gilles , Farnarier Catherine , Benoliel Anne-Marie , Foa Colette , Kaplanski Solange , Bongrand Pierre 
Published: April 2021

ABSTRACT
The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked ‘rounding up’ of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.
Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells
Authors: Campochiaro Peter A. , Hackett Sean F. , Vinores Stanley A. , Freund John , Csaky Carl , LaRochelle William , Henderer Jeff , Johnson Mike , Rodriguez Ignacio R. , Friedman Zvi , Derevjanik Nancy , Dooner James 
Published: April 2021

ABSTRACT
The retinal pigmented epithelium (RPE) plays a major role in normal and exaggerated retinal wound repair; the latter can result in epiretinal membrane formation and loss of vision. The RPE forms a stable monolayer of highly differentiated cells that proliferates only during wound repair. The mechanism underlying the change to the pro-liferating phenotype is unknown.
When grown on a plastic substratum, cultured RPE cells mimic the proliferating phenotype in situ; they escape density arrest and proliferate in serum-free medium. In this study, we have demonstrated that a platelet-derived growth factor (PDGF) autocrine loop is involved in RPE growth in serum-free medium, because: (1) RPE cells secrete PDGF into their media and express PDGF receptors; (2) the PDGF receptors on RPE cells are autophosphorylated in serum-free medium and suramin, an agent that displaces PDGF and other growth factors from their receptors, blocks the autophosphorylation; and (3) a neutralizing antibody to PDGF significantly decreases RPE growth in serum-free medium. When a linear scrape is made in an RPE monolayer, the cells migrate and pro-liferate to fill in the gap mimicking wound repair in situ. Cells along the edge of the scrape show increased expression of PDGF and PDGF-β receptors, and increased staining for proliferating cell nuclear antigen.
Immunohistochemistry and in situ hybridization demon-strate expression of PDGF in ganglion cells and cells of retinal blood vessels. PDGF is not detected in the outer retina or RPE in untreated eyes, but is detected in RPE participating in wound repair, either adjacent to laser burns or underlying retinal detachment. PDGF and PDGF receptors are also expressed in RPE in epiretinal membranes removed during vitreous surgery.
These data suggest that PDGF is an autocrine stimulator of growth in RPE that plays a role in retinal wound repair and epiretinal membrane formation.
Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid
Authors: Winter Gerhard , Fuchs Manuela , McConville Malcolm J. , Stierhof York-Dieter , Overath Peter 
Published: April 2021

ABSTRACT
Amastigotes of the protozoan parasite Leishmania prolif-erate in phagolysosomes of macrophages. They abundantly express glycoinositol phospholipids (GIPLs), which are considered necessary for parasite survival by providing a shield at the surface against lysosomal hydrolases and by serving as receptors for the interaction with host cells. The structures of four GIPLs of L. mexicana amastigotes were characterized by a combination of gas-liquid chromatog-raphy-mass spectrometry, methylation linkage analysis and enzymatic treatments. They contain the glycan struc-tures Manα1-3Manα1-4GlcN (iM2), Manα1-6(Manα1-3)Manα1-4GlcN (iM3), Manα1-2Manα1-6(Manα1-3)-Manα1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Manα1-6(Manα1-3)Manα1-4GlcN (EPiM3), which are linked to alkylacyl-phosphatidylinositol. The predominant amastigote GIPL, EPiM3 (∼2×107 molecules/cell), is located at the parasite cell surface, in the flagellar pocket and in lysosomal membranes, but not on host cell structures as shown by immunofluorescence and immunoelectron microscopy. In addition, amastigotes in infected Balb/c mice contain a glycolipid with similar distribution as EPiM3, which has the same characteristics as the Forssman antigen of mammalian cells. In contrast to EPiM3, there is strong evidence that this glycosphingolipid is not synthe-sized by amastigotes but by macrophages in the lesion. This suggests a mechanism of lipid transfer from the macrophage to the parasite.
Ezrin has properties to self-associate at the plasma membrane
Authors: Andréoli Christophe , Martin Marianne , Borgne Roland Le , Reggio Hubert , Mangeat Paul 
Published: April 2021

ABSTRACT
Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363-2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmuno-precipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129-139), both domains interacted syner-gistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin’s self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.
Analysis of a cortical cytoskeletal structure: a role for ezrin-radixin-moesin (ERM proteins) in the marginal band of chicken erythrocytes
Authors: Winckler Bettina , González Agosti Charo , Magendantz Margaret , Solomon Frank 
Published: April 2021

ABSTRACT
We are studying how the cytoskeleton determines cell shape, using a simple model system, the marginal band of chicken erythrocytes. We previously identified a minor component of the marginal band by a monoclonal antibody, called 13H9 (Birgbauer and Solomon (1989). J. Cell Biol. 109, 1609-1620; Goslin et al. (1989). J. Cell Biol. 109, 1621-1631). mAb 13H9 also binds to the leading edges of fibroblasts and to neuronal growth cones and recognizes the cytoskeletal protein ezrin. In recent years, two proteins with a high degree of homology to ezrin were identified: moesin and radixin, together comprising the ERM protein family. We now show that the contiguous epitope sufficient for mAb 13H9 binding is a sequence present in each of the ERM proteins, as well as the product of the gene associated with neurofibromatosis 2, merlin or schwannomin. We used biochemical and immunological techniques, as well as PCR to characterize the expression and localization of the ERM proteins in chicken erythrocytes. The results demonstrate that radixin is the major ERM protein associated with the cytoskeleton. Both ezrin and radixin localize to the position of the marginal band. Our results suggest that the ERM proteins play functionally conserved roles in quite diverse organelles.
Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to actin filaments
Authors: Fujimoto Toyoshi , Miyawaki Atsushi , Mikoshiba Katsuhiko 
Published: April 2021

ABSTRACT
We reported that a plasmalemmal inositol 1,4,5-trisphos-phate receptor-like protein (PM InsP3R-L) is localized in caveolae of various non-neuronal cells in vivo (Fujimoto et al. (1992)J. Cell Biol. 119, 1507-1513). In the present study, we investigated the distribution of PM InsP3R-L in cultured cells. In mouse epidermal keratinocytes (Pam 212) cultured in standard Ca2+ (1.8 mM), PM InsP3R-L was dis-tributed densely in the vicinity of cell-to-cell contacts. In contrast, when Pam cells were cultured in low Ca2+ (0.06 mM) without making cell-to-cell contacts, PM InsP3R-L was observed randomly; by restoring the Ca2+ concentra-tion, the circumferential actin filaments became obvious and the density of PM InsP3R-L increased in the contact region. Treatment of Pam cells with cytochalasin D caused aggregation of caveolae where PM InsP3R-L as well as F-actin and fodrin were localized. In bovine aortic endothelial cells, PM InsP3R-L was aligned along actin filaments crossing the cytoplasm in various directions. PM InsP3R-L of Pam cells was hardly extracted by treatment with 0.5% Triton X-100 or 60 mM octyl-glucoside in a cytoskeleton-stabilizing buffer for 15 minutes at 4°C.
The results show that the distribution of caveolae bearing PM InsP3R-L changes when the actin cytoskeleton is modified. They also indicate that the association of PM InsP3R-L with actin filaments may mediate the redistribution of caveolae. Since caveolae are thought to be related to signal transduction, their location defined by the actin cytoskeleton may affect the site where cellular reaction is to occur in response to various stimuli.