bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rising behind NO: cGMP-dependent protein kinases
Authors: Hofmann Franz , Ammendola Aldo , Schlossmann Jens 
Published: April 2021

ABSTRACT
Over the past few years, a wealth of biochemical and functional data has been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGMP kinase proteins generated by molecular biology techniques have yielded important biochemical knowledge, such as the function of the N-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, the substrate specificity of the enzymes and structural details of the catalytic center. Genetic approaches have proved to be especially useful for the analysis of the biological function of cGKs. Recently, some of the in vivo targets and mechanisms leading to smooth muscle relaxation have been identified. In vivo targets are the myosin-binding subunit of myosin phosphatase (PP1M), a member of the protein phosphatase 1, the calcium-activated maxi K+ channel and a new protein named IRAG that forms a complex with the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor and cGKI. Phosphorylation of PP1M by cGKIα activates myosin phosphatase, whereas phosphorylation of IRAG by cGKIβ decreases Ins(1,4,5)P3-induced calcium release. cGKII regulates in vivo intestinal fluid secretion by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), bone growth and renal renin secretion by phosphorylation of unknown proteins.
The G2 DNA damage checkpoint targets both Wee1 and Cdc25
Authors: Raleigh Jeanette M. , O’Connell Matthew J. 
Published: April 2021

ABSTRACT
The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G2 activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p is the endpoint of this checkpoint pathway. We have previously reported that overexpression of Chk1p causes a wee1+-dependent G2 arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggested that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1+ are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14-3-3 proteins the cytoplasmic retention of the 14-3-3/Cdc25p complexes. However, we show here that the G2 DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1+ and cdc25+ abolishes checkpoint control. We also show that activation of the G2 DNA damage checkpoint induces a transient increase in Wee1p levels. We conclude that the G2 DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability.
Dietary fat and reduced levels of TGFβ1 act synergistically to promote activation of the vascular endothelium and formation of lipid lesions
Authors: Grainger David J. , Mosedale David E. , Metcalfe James C. , Böttinger Erwin P. 
Published: April 2021

ABSTRACT
Transforming growth factor-β (TGFβ) has a wide range of activities on vascular cells and inflammatory cells, suggesting it may have different functions during various stages of atherogenesis. We report that mice heterozygous for the deletion of the tgfb1 gene (tgfb1+/− mice) have reduced levels of TGFβ1 in the artery wall until at least 8 weeks of age. On a normal mouse chow diet, the vascular endothelium of tgfb1+/− mice is indistinguishable from wild-type littermates, assessed by morphology and intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. In contrast, levels of the smooth muscle isoforms of actin and myosin in medial smooth muscle cells of tgfb1+/− mice are significantly reduced. Following feeding a cholesterolenriched diet for 12 weeks, high levels of ICAM-1 and VCAM-1 were detected in the vascular endothelial cells of tgfb1+/− mice, but not wild-type mice. Furthermore, marked deposition of lipid into the artery wall was only observed in the tgfb1+/− mice on the cholesterol-enriched diet. These vascular lipid lesions were accompanied by local invasion of macrophages. We conclude that deletion of a single allele of the tgfb1 gene results in a reduced level of TGFβ1 antigen in the aorta together with reduced smooth muscle cell differentiation, whereas the addition of a high fat dietary challenge is required to activate the vascular endothelium and to promote the formation of fatty streaks resembling early atherosclerosis in humans.
Human junction adhesion molecule regulates tight junction resealing in epithelia
Authors: Liu Yuan , Nusrat Asma , Schnell Frederick J. , Reaves Titus A. , Walsh Shaun , Pochet Mildred , Parkos Charles A. 
Published: April 2021

ABSTRACT
Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.
In mouse myoblasts nuclear prosomes are associated with the nuclear matrix and accumulate preferentially in the perinucleolar areas
Authors: Conto Flora De , Pilotti Elisabetta , Razin Sergey V. , Ferraglia Francesca , Géraud Gerard , Arcangeletti Cristina , Scherrer Klaus 
Published: April 2021

ABSTRACT
Prosomes are the core of 26S proteasomes, although they were originally observed as 20S particles associated with cytoplasmic mRNPs. Here we show for the first time that prosomes are also genuine constituents of the nuclear matrix, chromatin and the nuclear RNP networks. Using mouse myoblasts we tested three monoclonal antibodies recognising the prosomal subunits p23K, p27K and p30K, and found that the corresponding prosome subclasses are characterised by a variable distribution pattern within the nuclei.
Their presence on the nuclear matrix, and most abundantly in the perinucleolar area, is of particular importance. When myoblasts fuse into myotubes, the distribution pattern of certain types of prosomes on the nuclear matrix changes drastically. Surprisingly, DNA strongly interferes with the detection of prosomal antigens by immunofluorescence methods, whereas RNA, histones and other proteins soluble in 2 M NaCl have no such effect. This ‘masking’ of prosomes can be completely overcome by extensive or even mild digestion with DNase I or restriction enzymes. Many nuclear prosomes can be solubilized by combined treatment with 0.5% Triton X-100 and 2 M NaCl, and others can be released by digestion of DNA and/or RNA, and about 10-20% of nuclear prosomes remain tightly bound to the proteinbased nuclear matrix.
Impaired wound healing in embryonic and adult mice lacking vimentin
Authors: Eckes Beate , Guyon Emma Colucci- , Smola Hans , Nodder Sue , Babinet Charles , Krieg Thomas , Martin Paul 
Published: April 2021

ABSTRACT
It is generally assumed that the vimentin intermediate filament network present in most mesenchymallyderived cells is in part responsible for the strength and integrity of these cells, and necessary for any tissue movements that require the generation of significant tractional forces. Surprisingly, we have shown that transgenic KO mice deficient for vimentin are apparently able to undergo embryonic development absolutely normally and go onto develop into adulthood and breed without showing any obvious phenotype. However, fibroblasts derived from these mice are mechanically weak and severely disabled in their capacity to migrate and to contract a 3-D collagen network. To assess whether these functions are necessary for more challenging tissue movements such as those driving in vivo tissue repair processes, we have analysed wound healing ability in wildtype versus vimentindeficient embryos and adult mice. Wounds in vimentindeficient adult animals showed delayed migration of fibroblasts into the wound site and subsequently retarded contraction that correlated with a delayed appearance of myofibroblasts at the wound site. Wounds made to vimentindeficient embryos also failed to heal during the 24 hour culture period it takes for wildtype embryos to fully heal an equivalent wound. By DiI marking the wound mesenchyme and following its fate during the healing process we showed that this impaired healing is almost entirely due to a failure of mesenchymal contraction at the embryonic wound site. These observations reveal an in vivo phenotype for the vimentindeficient mouse, and challenge the dogma that key morphogenetic events occurring during development require generation of significant tractional forces by mesenchymal cells.
Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G proteincoupled receptors
Authors: Santini Francesca , Penn Raymond B. , Gagnon Alison W. , Benovic Jeffrey L. , Keen James H. 
Published: April 2021

ABSTRACT
Nonvisual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G proteincoupled receptors. In vitro experiments have shown that both nonvisual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonistpromoted internalization of many receptors has been found to be stimulated by exogenous overexpression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When β2-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G proteincoupled receptors to a specific nonvisual arrestin in an in vivo setting.
Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex
Authors: Hack Andrew A. , Lam Man-Yee J. , Cordier Laurence , Shoturma Daria I. , Ly Chantal T. , Hadhazy Melissa A. , Hadhazy Michele R. , Sweeney H. Lee , McNally Elizabeth M. 
Published: April 2021

ABSTRACT
Sarcoglycan is a multimeric, integral membrane glycoprotein complex that associates with dystrophin. Mutations in individual sarcoglycan subunits have been identified in inherited forms of muscular dystrophy. To evaluate the contributions of sarcoglycan and dystrophin to muscle membrane stability and muscular dystrophy, we compared muscle lacking specific sarcoglycans or dystrophin. Here we report that mice lacking δ-sarcoglycan developed muscular dystrophy and cardiomyopathy similar to mice lacking γ-sarcoglycan. However, unlike muscle lacking γ-sarcoglycan, δ-sarcoglycan-deficient muscle was sensitive to eccentric contraction-induced disruption of the plasma membrane. In the absence of δ-sarcoglycan, α-, β- and γ-sarcoglycan were undetectable, while dystrophin was expressed at normal levels. In contrast, without γ-sarcoglycan, reduced levels of α-, β- and δ-sarcoglycan were expressed, glycosylated and formed a complex with each other. Thus, the elimination of γ- and δ-sarcoglycan had different molecular consequences for the assembly and function of the dystrophin-glycoprotein complex. Furthermore, these molecular differences were associated with different mechanical consequences for the muscle plasma membrane. Through this in vivo analysis, a model for sarcoglycan assembly is proposed.
Protein kinase Cα actively downregulates through caveolae-dependent traffic to an endosomal compartment
Authors: Prevostel Corinne , Alice Vallentin , Joubert Dominique , Parker Peter J. 
Published: April 2021

ABSTRACT
Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC α from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC α protein. It is established that PKC α is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC α downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC α delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC α is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC α traffic and down regulation. Consistent with this, the endosomes where PKC α is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC α involves active delivery to endosomes via a caveolae mediated process.
High resolution analysis of interphase chromosome domains
Authors: Visser Astrid E. , Jaunin Françoise , Fakan Stanislav , Aten Jacob A. 
Published: April 2021

ABSTRACT
Chromosome territories need to be well defined at high resolution before functional aspects of chromosome organization in interphase can be explored. To visualize chromosomes by electron microscopy (EM), the DNA of Chinese hamster fibroblasts was labeled in vivo with thymidine analogue BrdU. Labeled chromosomes were then segregated during several cell cycles to obtain nuclei containing only 2 to 3 labeled chromosomes. Subsequent immunocytochemical detection of BrdU allowed analysis by EM of chromosome territories and subchromosomal domains in well preserved nuclei. Our results provide the first high resolution visualization of chromosomes in interphase nuclei. We show that chromosome domains are either separated from one another by interchromatin space or are in close contact with no or little intermingling of their DNA. This demonstrates that, while chromosomes form discrete territories, chromatin of adjacent chromosomes may be in contact in limited regions, thus implying chromosome-chromosome interactions. Chromosomes are organized as condensed chromatin with dispersed chromatin extending into the interchromatin space that is largely devoid of DNA. The interchromatin space, which is known to be involved in various nuclear functions, forms interconnecting channels running through and around chromosome territories. Functional implications of this organization are discussed.
CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair
Authors: Ridgway Patricia , Almouzni Geneviève 
Published: April 2021

ABSTRACT
Chromatin is no longer considered to be a static structural framework for packaging DNA within the nucleus but is instead believed to be an interactive component of DNA metabolism. The ordered assembly of chromatin produces a nucleoprotein template capable of epigenetically regulating the expression and maintenance of the genome. Factors have been isolated from cell extracts that stimulate early steps in chromatin assembly in vitro. The function of one such factor, chromatin-assembly factor 1 (CAF-1), might extend beyond simply facilitating the progression through an individual assembly reaction to its active participation in a marking system. This marking system could be exploited at the crossroads of DNA replication and repair to monitor genome integrity and to define particular epigenetic states.
Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein
Authors: Sugaya Kimihiko , Vigneron Marc , Cook Peter R. 
Published: April 2021

ABSTRACT
RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.
Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells
Authors: Sawada Ken , Konishi Yuko , Tominaga Mari , Watanabe Yoshiya , Hirano Jun , Inoue Shimpei , Kageyama Ryoichiro , Blum Martin , Tominaga Akira 
Published: April 2021

ABSTRACT
In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid- mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.
Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils
Authors: Anderson Stephen I. , Hotchin Neil A. , Nash Gerard B. 
Published: April 2021

ABSTRACT
When rolling adherent neutrophils are stimulated, they rapidly immobilise through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin- polymerising agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca2+ seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn2+. Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3- transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.