bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fgf inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules
Authors: Williams Emma J. , Mittal Bina , Walsh Frank S. , Doherty Patrick 
Published: April 2021

ABSTRACT
We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules.
Loss of mdck cell α2β1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis
Authors: Saelman Edwin U. M. , Keely Patricia J. , Santoro Samuel A. 
Published: April 2021

ABSTRACT
Cellular interactions with collagen in a model of kidney tubulogenesis were investigated using Madin-Darby canine kidney (MDCK) cells in an in vitro morphogenetic system. MDCK cells adhered to collagen types I and IV in a Mg2+-dependent manner, typical of the α2β1 integrin. Collagen-Sepharose affinity chromatography and immunoblotting demonstrated the presence and collagen binding activity of the α2β1 integrin on MDCK cells. To assess the function of α2β1 integrin, MDCK cells were transfected with a plasmid pRSVα2′ which allowed the expression of α2-integrin subunit antisense RNA. Three G418-resistant clones showing reduced adhesion to collagen, stable genomic integration of the antisense construct, decreased α2-integrin subunit mRNA and decreased α2-integrin subunit protein expression were selected for analysis in morphogenetic experiments. MDCK cells and plasmid-only control transfectants, cultured in three-dimensional collagen type I gels, showed normal cyst formation, whereas the antisense RNA transfectants showed increased apoptosis and formed small rudimentary cysts. Stimulation with hepatocyte growth factor/scatter factor-containing 3T3 fibroblast-conditioned medium or recombinant hepatocyte growth factor/scatter factor resulted in extensive branching of the preformed control cysts whereas the surviving small cysts formed by antisense expressing cells increased in size but failed to elongate and branch upon stimulation. We conclude that α2β1 integrin collagen interactions play a crucial role in the hepatocyte growth factor/scatter factor-induced tubulogenesis and branching morphogenesis of MDCK cells in collagen gels as well as an important role in cell survival.
Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts
Authors: Lindsay H. D. , Whitaker M. J. , Ford C. C. 
Published: April 2021

ABSTRACT
Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translational modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500 nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.
Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (ph) domain and enhances protein kinase c activity
Authors: Dellambra Elena , Patrone Mauro , Sparatore Bianca , Negri Armando , Ceciliani Fabrizio , Bondanza Sergio , Molina Francesco , Cancedda Fiorella Descalzi , Luca Michele De 
Published: April 2021

ABSTRACT
The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully under-stood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous σ (stratifin) and ζ isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracel-lular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 ζ) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.
Neither erk nor jnk/sapk map kinase subtypes are essential for histone h3/hmg-14 phosphorylation or c-fos and c-jun induction
Authors: Cano Eva , Hazzalin Catherine A. , Kardalinou Efterpi , Buckle Robin S. , Mahadevan Louis C. 
Published: April 2021

ABSTRACT
The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and JNK/SAPK MAP kinase cascades have been compared with their ability to elicit histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10TG cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of c-fos and c-jun, showing that neither cascade is absolutely essential for these responses. We then analysed the relationship between ERKs, JNK/SAPKs and the transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and c-jun, respec-tively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, par-ticularly ERK-2, to GST-Elk1307-428; there is no cross-specificity of binding. Further, GST-Elk1307-428 binds preferentially to active rather than inactive ERK-2. In vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1307-428, whereas ERKs phosphorylate GST-Elk1307-428 but not GST-cJun1-79. Thus, neither ERKs nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and c-jun induction. The data suggest either that activation of a single MAP kinase subtype is sufficient to elicit a complete nuclear response, or that other uncharac-terised routes exist.
The distribution of Na+,K+-atpase and 5a11 antigen in apical microvilli of the retinal pigment epithelium is unrelated to α-spectrin
Authors: Rizzolo Lawrence J. , Zhou Shaoming 
Published: April 2021

ABSTRACT
The retinal pigment epithelium was used to study the relationship between the cortical cytoskeleton and two plasma membrane proteins that associate with it. These proteins were the Na+,K+-ATPase, an ion pump, and the 5A11 antigen, a member of the immunoglobulin super-family of receptor proteins. The cytoskeleton was marked by two of its constituents, α-spectrin and ankyrin. Ankyrin links the Na+,K+-ATPase to spectrin in many cells. The RPE is of interest, because unlike most epithelia it distributes the Na+,K+-ATPase to the apical membrane. The development of polarity was studied during chick embryogenesis. On embryonic day 6 (E6), each of these proteins was observed in the apical and lateral plasma membranes. As development proceeded, only the Na+,K+-ATPase was removed from the lateral membranes. Beginning on E12, ankyrin, spectrin and 5A11 appeared together in patches along the basal plasma membrane. By E16, these patches coalesced into a uniform distribution along the basal membrane. At the apical pole, α-spectrin appeared near the base of the microvilli, but was undetected in the microvilli them-selves. This distribution resembled the distribution of α-spectrin in the intestine and proximal kidney tubule. By contrast, a pool of ankyrin and 5A11 and nearly all the Na+,K+-ATPase appeared in the microvilli. Despite its segregation from α-spectrin, the Na+,K+-ATPase appeared to associate with a macromolecular complex, as judged by extraction with Triton X-100. Changes in spectrin distribution could not be related to changes in isoform expression, as only one isoform of β-spectrin was detected by co-immunoprecipitation with α-spectrin. By contrast, multiple ankyrin-like peptides could be identified by immunoblotting. These data illustrate some of the unique properties of RPE microvilli. These properties prevent the Na+,K+-ATPase from complexing with the α-spectrin-based cytoskeleton by sequestering the enzyme into the compartment where its activity is required.
Elucidation of nucleus-cytoplasm interaction: change in ability of the nucleus to express sexuality according to clonal age in Paramecium
Authors: Haga Nobuyuki 
Published: April 2021

ABSTRACT
The expression of sexual activity in Paramecium caudatum is repressed for about 50 fissions after conjugation. The ability of the macronucleus to affect the expression of sexual activity according to the age of clonal development was investigated using the macronuclear fusionreorganization method. When a mature macronucleus was transplanted into an immature cell and fused with a macronucleus in the immature cell, the clones derived from the recipient showed sexual immaturity. In a reverse experiment, an immature macronucleus was transplanted into a mature cell, and the clones also showed sexual immaturity. The ability of the macronucleus to transform mature cells to immature cells was clonal agedependent. The characteristics of the immaturemature hybrid macronucleus indicate that the immature macronucleus is dominant over the mature macronucleus with respect to the ability to express sexual activity. On the other hand, in cells of the early immaturity period, the micronucleus, known as the germ nucleus, shows the ability to undergo meiosis and eventually to produce progeny under control of the mature macronucleus. The expression of sexual activity is thought to be governed by the clonal age of the macronucleus and not by the clonal age of the micronucleus or cytoplasm. The macronucleus seems to determine the ability to express sexual activity by counting postconjugation divisions and keeping track of the age of the clone.
A 182 bp fragment of the mouse proα1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice
Authors: Zhou Guang , Garofalo Silvio , Mukhopadhyay Krish , Lefebvre Véronique , Smith Chad N. , Eberspaecher Heidi , Crombrugghe Benoit de 
Published: April 2021

ABSTRACT
Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse proα1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a β-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of β-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted β-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no β-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal β-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer highlevel, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.
Matrigel induces thymosin β4 gene in differentiating endothelial cells
Authors: Grant D. S. , Kinsella J. L. , Kibbey M. C. , LaFlamme S. , Burbelo P. D. , Goldstein A. L. , Kleinman H. K. 
Published: April 2021

ABSTRACT
We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillarylike tubes. The mRNA for one clone, thymosin β4, was increased 5-fold. Immunostaining localized thymosin β4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin β4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin β4 inhibited tube formation on Matrigel. The results suggest that thymosin β4 is induced and likely involved in differentiating endothelial cells. Thymosin β4 may play a role in vessel formation in vivo.
Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains
Authors: Ruppert Christian , Godel Jasminca , Müller Rainer T. , Kroschewski Ruth , Reinhard Jutta , Bähler Martin 
Published: April 2021

ABSTRACT
Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin I. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In sub-cellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density.
The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are suf-ficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper sub-cellular localization.
Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo
Authors: Schuler Frank , Sorokin Lydia M. 
Published: April 2021

ABSTRACT
The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin α2 chain-specific antibodies react with mouse tissues, so rat mono-clonal antibodies were raised against the mouse laminin α2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin β1 and γ1 chains and form a complex with a 380 kDa α chain identified as laminin α2 by immunofluorescence, immunoprecipitation and PCR.
PCR from C2C12 myoblasts and myotubes for the laminin α2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin α1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14-and 17-day-old mouse embryos, while the laminin α2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin α2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin α2 in basement membranes of basal ker-atinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis.
In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was sig-nificantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myo-genesis.
Receptor-mediated adhesive and anti-adhesive functions of chondroitin sulfate proteoglycan preparations from embryonic chicken brain
Authors: Ernst Heidemarie , Zanin Mary K. B. , Everman David , Hoffman Stanley 
Published: April 2021

ABSTRACT
Chondroitin sulfate proteoglycans inhibit the adhesion of cells to extracellular matrix proteins that otherwise permit adhesion. Although proteoglycans are widely assumed to act by masking the other protein in a mixed substrate, recent studies suggest that proteoglycans inhibit adhesion through mechanisms initiated by their binding to specific cell surface receptors. To explore this issue, we developed a purification scheme to isolate proteoglycan aggregates, monomers, and core proteins. Two distinct adhesion assays were used to study the interaction of these proteoglycan preparations with human foreskin fibroblasts: the gravity assay in which cell attachment is stabilized by cell spreading, and the centrifugation assay in which spreading does not play a role. All proteoglycan preparations mediate adhesion in the centrifugation assay but not in the gravity assay. In the centrifugation assay, proteoglycan aggregates and monomers are considerably more active than other extracellular matrix proteins while proteoglycan core proteins are at least as active as other extracellular matrix proteins. Proteoglycan core proteins bind to cell-associated hyaluronic acid, but not to integrins. Using mixed sub-strates in the gravity assay, all proteoglycan preparations inhibited cell attachment to fibronectin and vitronectin but not to collagen I and laminin. Although proteoglycan aggregates and monomers are more active than core proteins in inhibiting adhesion in the gravity assay, core proteins are still clearly active. A variety of control exper-iments suggest that the inhibition of cell attachment by pro-teoglycans is mediated through the specific interactions of proteoglycans with cell surface receptors, resulting in the inhibition of cell spreading. These results suggest at least two molecular mechanisms for proteoglycan-fibroblast interactions, one involving the chondroitin sulfate on the proteoglycan and an as yet unidentified receptor, the other involving the proteoglycan core protein and cell-associated hyaluronic acid.
Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation
Authors: Castle Anna M. , Schwarzbauer Jean E. , Wright Rhonda L. , Castle J. David 
Published: April 2021

ABSTRACT
In pituitary-derived AtT-20 cells, recombinant fibronectin containing the N-terminal matrix assembly domain and the C-terminal half of fibronectin does not follow the regulated secretory pathway but instead concentrates in distinct organelles prior to secretion. These organelles are larger than the dense-core granules and localize to the cell body at sites that differ from lysosomes, endosomes and endo-plasmic reticulum. Unlike the dense-core granules, their discharge is not stimulated by 8-bromo-cyclic-AMP or phorbol esters. The kinetics of intracellular transport and secretion of the recombinant fibronectin suggest that it is present in a post-Golgi pool that turns over more slowly than constitutive vesicles. Indeed, the fibronectin-contain-ing organelles disappear with a half-time of 3 hours after inhibiting protein synthesis. Presence of the organelles correlates with intracellular aggregation of dimeric fibronectin polypeptides. The organelles are absent in cells expressing monomeric recombinant fibronectin (lacking C-terminal dimerization sites) or the C-terminal half of fibronectin (which dimerizes but lacks the N-terminal matrix assembly domain), both of which aggregate less efficiently than dimeric fibronectin. Instead, the latter polypeptides enter the dense-core granules. Thus while the formation of the fibronectin-containing organelles may require efficient aggregation, it may not require a specific structural signal. Moreover, efficient aggregation is not necessarily a prerequisite for following the regulated pathway.